Tandem repeat-based identification of x-chromosome inactivation (TRIXI)

Abstract

The invention relates to a method and a kit for detecting skewed X-chromosome inactivation (XCI) in a female subject utilizing at least two repeat-containing regions of at least two loci located on the X-chromosome, wherein the loci are ARHGAP6, ZIC3, CNKSR2, TCAC1, or RP2.

Description

[0001] P7621PC00

[0002] Tandem Repeat-based Identification of X-chromosome Inactivation (TRiXi) Technical field

[0003] The present invention relates to methods for detecting skewed X-chromosome inactivation (XCI) via multiplex PCR.

[0004] Background

[0005] To prevent the negative effect of expressing a double dose of X-linked genes, one X-chromosome is inactivated in all female somatic cells during early embryonic development. As the process of X-chromosome inactivation (XCI) occurs randomly in each individual cell of the developing embryo, females are ‘mosaic’ for X-inactivation; different cells in the same individual will have inactivated the paternal X-chromosome (Xp) or maternal X-chromosome (Xm). Consequently, women are expected to exhibit a ratio of ~1 :1 of cells having inactivated the Xp or Xm. Skewed X-inactivation (sXCI) in females is a natural phenomenon that results in silencing of either the Xm or Xp in most cells in one or more tissues (>2:1). Skewed XCI can have serious clinical implications for carriers of X-linked mutations, resulting in penetrance of potentially fatal X-linked disorders in females such as Duchenne Muscular dystrophy and Fragile X Syndrome.

[0006] Skewed XCI may arise due to variants that directly affect the process of XCI on one of the two X-chromosomes (primary selection) or indirectly due to variants that result in a growth advantage in a particular cell or clone (secondary selection). Indeed, it is assumed that much of the sXCI observed in adult females results from clonal hematopoiesis (CH), and skewed XCI was initially employed to identify women with CH. However, extreme skewing of XCI may also be a common and constitutional genetic characteristic in the human population, present at birth in up to 1-5% of females. Constitutional skewing would have a more profound and early effect on the penetrance and expressivity of X-linked traits in female carriers.

[0007] As women are mosaics for X-inactivation, direct determination of skewed XCI in bulk samples such as blood is complex. Measurement of sXCI requires the use of techniques that combine an informative genetic variant with an additional layer of molecular information such as DNA methylation or transcription. The HUMARA assay involves methyl-sensitive restriction digest (only the unmethylated active X is cut) ofP7621PC00

[0008] one highly polymorphic short tandem repeat (STR) in the androgen receptor (AR) gene followed by PCR amplification to reveal XCI skewing; only the methylated, uncut inactive X allele will be amplified. However, the HUMARA assay has a sensitivity of just 80%, with approximately 20% of females homozygous at the AR locus. Moreover, the assay is affected by locus specific alterations in DNA methylation that do not reflect the activation status of the rest of the X-chromosome and the presence of genetic variants in the target sequence.

[0009] Summary

[0010] The present invention provides a method for detecting skewed X-chromosome inactivation (XCI) with improved accuracy and efficiency. Unlike conventional assays that rely on a single locus, the method utilizes a set of up to five loci: ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2, to assess XCI patterns, significantly enhancing sensitivity from approximately 80% in the HUMARA assay to 99.8% without requiring additional experimental steps, equipment, or costs. This improved sensitivity can ensure more reliable detection of skewed XCI, making the method particularly advantageous for clinical and research applications.

[0011] Another advantage of the invention is the exclusion of the AR (androgen receptor) locus from the assay, in adherence to ethical principles of genetic testing. The AR locus is associated with Kennedy’s disease, a neurodegenerative disorder. By omitting AR from the assay, the risk of incidental findings is mitigated.

[0012] The selected five loci share key properties that make them well-suited for detecting skewed XCI. Each locus contains repeat-containing regions with high heterozygosity in the population. Additionally, these regions are subject to X-chromosome methylation, making them compatible with methylation-sensitive restriction enzyme digestion as a means of distinguishing active from inactive X-chromosomes. Furthermore, the design of the primers ensures that the resulting PCR products differ in length, allowing for straightforward separation via a single capillary electrophoresis step without additional processing.

[0013] The primers are specifically designed to enable multiplex PCR amplification of all five loci simultaneously, significantly increasing the efficiency of the assay. This reduces processing time and costs, making the method both rapid and cost-effective whileP7621PC00

[0014] maintaining high sensitivity and specificity. By leveraging a carefully selected panel of loci and an optimized assay design, the present invention can provide a superior alternative to existing XCI detection methods.

[0015] An aspect of the invention relates to a method for detecting skewed X-chromosome inactivation (XCI) in a female subject, comprising:

[0016] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;

[0017] b. digesting the nucleic acid sample with a methylation-sensitive restriction enzyme thereby obtaining a treated nucleic acid sample; c. amplifying said treated nucleic acid sample via polymerase chain reaction (PCR) using a primer set comprising primers specific for at least one repeat-containing region of at least one locus located on the X-chromosome, wherein the locus is ARHGAP6, thereby obtaining at least one PCR product comprising a repeat-containing region of one of said loci;

[0018] d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis; and

[0019] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the maternal and paternal X-chromosomes indicates skewed XCI. Another aspect of the invention relates to a method for detecting skewed

[0020] X-chromosome inactivation (XCI) in a female subject, comprising:

[0021] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;

[0022] b. digesting the nucleic acid sample with a methylation-sensitive restriction enzyme thereby obtaining a treated nucleic acid sample; c. amplifying said treated nucleic acid sample via multiplex polymerase chain reaction (PCR) using a primer set comprising primers specific for at least two repeat-containing region of at least two loci located on the X-chromosome, wherein the loci are

[0023] i. ARHGAP6; andP7621PC00

[0024] ii. ZIC3, CNKSR2, TCAC1 , or RP2, thereby obtaining at least two PCR product comprising a repeat-containing region of two of said loci;

[0025] d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis; and

[0026] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the maternal and paternal X-chromosomes indicates skewed XCI.

[0027] Another aspect of the invention relates to a kit comprising: at least one, such as at least two, such as at least three, such as at least four, such as five primer sets, wherein the primer sets comprise a forward primer consisting of SEQ ID NO: 1 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 2 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof; and

[0028] i. a forward primer consisting of SEQ ID NO: 3 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 4 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or ii. a forward primer consisting of SEQ ID NO: 5 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 6 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or iii. a forward primer consisting of SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or iv. a forward primer consisting of SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof; and instructions for detecting skewed XCI.P7621PC00

[0029] Another aspect of the invention relates to a kit for detecting skewed XCI comprising a. At least one, such as at least two, such as at least three, such as at least four, such as five primer sets, wherein the primer sets comprise a forward primer and reverse primer, wherein the forward primer binds upstream of position 11427654 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 11427890 in the X-chromosome of the human reference genome GRCh38.p13; and

[0030] i. a forward primer and reverse primer, wherein the forward primer binds upstream of position 46836203 in the X- chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 46836692 in the X-chromosome of the human reference genome GRCh38.p13; and / or

[0031] ii. a forward primer and reverse primer, wherein the forward primer binds upstream of position 137568039 in the X- chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 137568606 in the X-chromosome of the human reference genome GRCh38.p13; and / or iii. a forward primer and reverse primer, wherein the forward primer binds upstream of position 137545307 in the X- chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 137545689 in the X-chromosome of the human reference genome GRCh38.p13; and / or iv. a forward primer and reverse primer, wherein the forward primer binds upstream of position 21374408 in the X- chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 21374708 in the X-chromosome of the human reference genome GRCh38.p13; and

[0032] b. instructions for use.P7621PC00

[0033] Another aspect of the invention related to a method for diagnosing a disorder associated with skewed X-chromosome inactivation (XCI) in a female subject, comprising:

[0034] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;

[0035] b. contacting said nucleic acid sample with a methylation-sensitive restriction enzyme to obtain a treated nucleic acid sample; c. amplifying said treated nucleic acid sample via PCR using a primer set comprising primers specific for a least one repeatcontaining region of at least one locus located on the X- chromosome, wherein the locus is

[0036] v. ARHGAP6; and

[0037] vi. ZIC3, CNKSR2, TCAC1 , or RP2, thereby obtaining at least one PCR product comprising a repeat-containing region of one of said loci;

[0038] d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis;

[0039] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the two X-chromosomes indicates skewed XCI; and

[0040] f. correlating the presence of skewed XCI with an increased risk of or presence of an X-linked disorder.

[0041] Description of Drawings

[0042] Figure 1. TriXi as a novel method for the detection of skewed X-inactivation in humans. (A) Idiogram of the human X-chromosome showing the location of the five loci employed in the TriXi assay (upper panel). Example results of three females showing no skewing (lower left panel), and complete skewing (lower middle and right panels). Star indicates a homozygous repeat. (B) The results of HUMARA and TriXi are highly similar. (C & D) 10 of the 50 samples were uninformative using the HUMARA assay due to homozygosity for the repeat length at the AR allele. Skewing for all samples wasP7621PC00

[0043] determined successfully using TriXi. (E) Constitutional (at birth) skewing is a common feature of the human female population.

[0044] Figure 2. Clustering of ONT reads by repeat length at all five repeat loci in the ABIS 1 sample. Grey gradient indicates CpG DNA methylation level. Arrows indicate CCGG sites.

[0045] Figure 3. Allelic-resolution analysis reveals widespread, multi-organ skewing in adults. (A,B) XCI status determined by TRiXi in 1 ,027 neonatal blood samples (a) and by median non-PAR X-linked allelic expression across 4,571 tissues in 264 female donors from the GTEx dataset. (C) Density plot of skewing in whole blood as determined by TRiXi in newborns and allelic expression in adults from GTEx. (D) bar plot showing adults grouped by the number of skewed tissues per individual. (E) bar plot of fraction of extremely skewed (skewing >79%) samples per tissue (top) and ridge plot showing the distribution of skewing in all 45 tissues examined (bottom).

[0046] Detailed description

[0047] Definitions

[0048] As used herein, the term "skewed X-chromosome inactivation (XCI)" refers to a condition in which the inactivation of one X-chromosome, either the maternal X-chromosome or the paternal X-chromosome, occurs at a significantly higher frequency than the other within a population of cells. This may be detected by assessing the relative abundance of methylated and unmethylated alleles at one or more loci on the X-chromosome following digestion with a methylation-sensitive restriction enzyme and subsequent amplification. A deviation from an approximately equal representation of alleles derived from the maternal and paternal X-chromosomes may indicate skewed XCI, which can be associated with various physiological or pathological conditions.

[0049] As used herein, the term "female subject" refers to an individual who may possess two X-chromosomes and can be biologically classified as female. The term may encompass humans as well as non-human animals, including mammals. A female subject may be assessed for X-chromosome inactivation (XCI) status using the methods described herein, which may involve analyzing nucleic acid samples derived from cells containing both a maternal X-chromosome and a paternal X-chromosome.P7621PC00

[0050] As used herein, the term "plurality of cells" refers to a population of cells obtained from a female subject, wherein each cell may contain a maternal X-chromosome and a paternal X-chromosome, and, in the absence of skewed X-chromosome inactivation (XCI), the inactivation of either X-chromosome may occur in approximately equal proportions. Due to the stochastic nature of XCI, analyzing only a very limited number of cells, such as fewer than 10, may result in a high probability that the same X-chromosome is inactivated in all or most of those cells by chance, leading to an inaccurate assessment of XCI skewing. To provide statistically meaningful results and reliably determine whether skewed XCI is present, the analysis may require at least 100 cells.

[0051] As used herein, the term "methylation-sensitive restriction enzyme" refers to an enzyme that may selectively digest DNA at specific recognition sites depending on the methylation status of the DNA. Certain methylation-sensitive restriction enzymes may cleave DNA only when the recognition site is unmethylated, while others may digest DNA only when the site is methylated. By utilizing such enzymes, it may be possible to differentiate between methylated and unmethylated DNA regions, allowing for the assessment of epigenetic modifications such as X-chromosome inactivation (XCI).

[0052] An example of a methylation-sensitive restriction enzyme is Hpall, which recognizes the 5'-CCGG-3' sequence and cleaves the DNA only when the internal cytosine is unmethylated. In contrast, if the cytosine within the recognition site is methylated, Hpall is unable to cut the DNA, thereby preserving the methylated fragment. This property can enable the detection of methylation patterns by comparing digestion products before and after enzymatic treatment, often followed by amplification-based techniques such as polymerase chain reaction (PCR).

[0053] As used herein, the term "multiplex PCR" refers to a polymerase chain reaction (PCR) approach that can amplify multiple target sequences simultaneously within a single reaction, allowing for increased efficiency and reduced reagent use.

[0054] As used herein, the term "repeat-containing region" refers to a segment of nucleic acid that includes a sequence motif that may be repeated multiple times, which can facilitate differentiation between alleles based on fragment length. The repeated sequence may be present at least twice, such as at least three times, at least five times, such as atP7621PC00

[0055] least six times, at least ten times, such as at least fifteen times, such as at least 20 times, such as at least 40 times or at least 50 times. Examples of repeat motifs that may be present in such regions include AAAG, ATCT, TCAC, GAG, or GAG repeats. Repeat-containing regions may exhibit high heterozygosity within a population, meaning that a significant proportion of individuals may carry alleles with different repeat lengths on their two homologous chromosomes. This property can enhance the utility of such regions for assessing X-chromosome inactivation (XCI) because distinct allele sizes allow for clear differentiation between the maternal and paternal X-chromosomes following amplification and separation, such as by electrophoresis. The presence of differentially sized alleles may improve assay resolution and reduce ambiguity in interpreting XCI patterns.

[0056] As used herein, the term "locus" refers to a specific position on a chromosome that may be occupied by a particular gene, genetic marker, or nucleotide sequence. A locus may encompass a single nucleotide position or a larger genomic region and can be characterized by its sequence composition, function, or genetic variation. In the context of X-chromosome inactivation (XCI) analysis, a locus may include a repeat-containing region that can be assessed for differences in methylation status or fragment length to distinguish between the maternal and paternal X-chromosomes. The heterozygosity of a locus, such as variation in repeat number between alleles, may facilitate its use in detecting skewed XCI by enabling clear differentiation of alleles following amplification and electrophoretic separation.

[0057] As used herein, the term "relative abundance" refers to the proportion of one nucleic acid sequence, allele, or biomolecule in comparison to another within a given sample. In the context of X-chromosome inactivation (XCI) analysis, relative abundance may describe the ratio of amplified PCR products corresponding to the maternal and paternal X-chromosomes, which can reflect differences in methylation status or inactivation patterns. A deviation from an approximately equal relative abundance of maternal and paternal alleles may indicate skewed XCI, wherein one X-chromosome is preferentially inactivated in a majority of cells.

[0058] Approximately equal may refer to values that fall within the range of statistical variation and the accuracy of the method used for measurement. Due to natural variability and technical limitations, a slight deviation in relative abundance, such as up to 45:55, such as up to 40:60, such as up to 70:30, may be considered non-skewed XCI. RelativeP7621PC00

[0059] abundance may be assessed using techniques such as capillary electrophoresis, where fragment peak intensities or areas can be quantitatively compared.

[0060] As used herein, the term "ARHGAP6" refers to a gene located on the X-chromosome that encodes a Rho GTPase-activating protein (RhoGAP), which may play a role in cytoskeletal organization and cell signalling. The ARHGAP6 locus contains a repeatcontaining region, which may exhibit heterozygosity within the population, with a heterozygosity rate of approximately 80%, allowing for differentiation between maternal and paternal alleles based on fragment length following amplification. The repeatcontaining region can comprise GAG repeats.

[0061] As used herein, the term "ZIC3" refers to a gene located on the X-chromosome that encodes a zinc finger transcription factor, which may be involved in early embryonic development and the regulation of gene expression. The ZIC3 locus contains a repeatcontaining region, which may exhibit heterozygosity within the population, with a heterozygosity rate of approximately 71%, allowing for differentiation between maternal and paternal alleles based on fragment length following amplification. The repeatcontaining region can comprise ATCT repeats.

[0062] As used herein, the term "CNKSR2" refers to a gene located on the X-chromosome that encodes a connector enhancer of kinase suppressor of Ras 2 (CNKSR2), which may play a role in signal transduction pathways involved in neuronal function and development. The CNKSR2 locus contains a repeat-containing region, which may exhibit heterozygosity within the population, with a heterozygosity rate of approximately 64%, allowing for differentiation between maternal and paternal alleles based on fragment length following amplification. The repeat-containing region can comprise GAG repeats.

[0063] As used herein, the term "TCAC1" refers to a locus located on the X-chromosome that contains a repeat-containing region, which may exhibit heterozygosity within the population, with a heterozygosity rate of approximately 57%, allowing for differentiation between maternal and paternal alleles based on fragment length following amplification. The repeat-containing region can comprise TCAC repeats.P7621PC00

[0064] As used herein, the term "RP2" refers to a gene located on the X-chromosome that encodes a protein involved in retinal function and intracellular trafficking, which may play a role in photoreceptor maintenance. The RP2 locus contains a repeat-containing region, which may exhibit heterozygosity within the population, with a heterozygosity rate of approximately 79%, allowing for differentiation between maternal and paternal alleles based on fragment length following amplification. The repeat-containing region can comprise AAAG repeats.

[0065] As used herein, the term "sample" refers to a biological specimen obtained from a subject that may contain nucleic acids for analysis. A sample may be derived from a human tissue sample, such as blood, saliva, urine, hair, tissue or biopsy samples. In the context of X-chromosome inactivation (XCI) analysis, a sample may comprise a plurality of cells, wherein each cell contains a maternal X-chromosome and a paternal X-chromosome.

[0066] As used herein, the articles “a” and “an” are intended to mean “one or more” unless the context clearly dictates otherwise.

[0067] As used herein, the terms “comprising” and “comprises” are intended to be open-ended and mean “including, but not limited to.” These terms do not exclude the presence of additional elements, components, or steps.

[0068] As used herein, the terms “consisting of” and “consisting” are intended to be closed terms, meaning that the element or composition is limited to the stated features only, and excludes additional features, components, or steps not expressly recited.

[0069] Methods for detecting skewed X-chromosome inactivation (XCI)

[0070] The method for detecting X-chromosome inactivation (XCI) may comprise obtaining a sample from a female subject, wherein the sample contains a plurality of cells, each carrying a maternal and a paternal X-chromosome. The extracted nucleic acids may then be treated with a methylation-sensitive restriction enzyme, such as Hpall, which selectively digests the unmethylated DNA while leaving the methylated (inactive) X-chromosome intact. Following digestion, a multiplex polymerase chain reaction (PCR) may be performed using a primer set specific for up to five loci (ARHGAP6, ZIC3, CNKSR2, TCAC1 , RP2), ensuring amplification occurs only from the methylated,P7621PC00

[0071] undigested X-chromosome. The resulting PCR products may be subjected to capillary electrophoresis, allowing for the separation of fragments based on their repeatcontaining regions, which may differ in length between alleles. The relative abundance of PCR products derived from the maternal and paternal X-chromosomes may then be determined, with a deviation from an approximately equal ratio indicating skewed XCI. Even if a specific locus has identical repeat lengths on both X-chromosomes, analysis across multiple loci may provide sufficient heterozygosity for clear differentiation of XCI patterns.

[0072] The present invention relates to a method for detecting skewed X-chromosome inactivation (XCI) in a female subject, comprising:

[0073] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;

[0074] b. digesting the nucleic acid sample with a methylation-sensitive restriction enzyme thereby obtaining a treated nucleic acid sample; c. amplifying said treated nucleic acid sample via polymerase chain reaction (PCR) using a primer set comprising primers specific for at least one repeat-containing region of at least one locus located on the X-chromosome, wherein the locus is ARHGAP6, ZIC3, CNKSR2, TCAC1 , or RP2, thereby obtaining at least one PCR product comprising a repeat-containing region of one of said loci;

[0075] d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis; and

[0076] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the maternal and paternal X-chromosomes indicates skewed XCI.

[0077] In another aspect, the present invention relates to a method for detecting skewed X-chromosome inactivation (XCI) in a female subject, comprising:

[0078] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;P7621PC00

[0079] b. digesting the nucleic acid sample with a methylation-sensitive restriction enzyme thereby obtaining a treated nucleic acid sample; c. amplifying said treated nucleic acid sample via multiplex polymerase chain reaction (PCR) using a primer set comprising primers specific for at least two repeat-containing region of at least two loci located on the X-chromosome, wherein the loci are ARHGAP6, ZIC3, CNKSR2, TCAC1 , or RP2, thereby obtaining at least two PCR product comprising a repeat-containing region of two of said loci;

[0080] d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis; and

[0081] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the maternal and paternal X-chromosomes indicates skewed XCI.

[0082] ARHGAP6 contains a repeat-containing region that exhibits a high degree of polymorphism, making it likely to be heterozygous and thereby informative in a high proportion of female individuals. In addition, the ARHGAP6 locus exhibits methylation patterns that permit clear distinction between the methylated (inactive) and unmethylated (active) alleles following digestion with a methylation-sensitive restriction enzyme. These characteristics make ARHGAP6 a robust and reliable locus for XCI detection.

[0083] Because of its high informativity and technical reliability, in some embodiments of the invention, ARHGAP6 is the only locus analyzed following methylation-sensitive digestion and PCR amplification. In other embodiments, ARHGAP6 is analyzed together with at least one additional polymorphic locus located on the X-chromosome, such as ZIC3, CNKSR2, TCAC1 , or RP2. Including ARHGAP6 in combination with one further locus increases the likelihood that at least one of the loci is heterozygous in any given subject and allows confirmatory analysis across multiple independent loci. Multiplex PCR amplification of ARHGAP6 together with an additional locus further provides complementary information and enhances assay robustness without considerably increasing procedural complexity.

[0084] In another aspect, the present invention relates to a method for detecting skewed X-chromosome inactivation (XCI) in a female subject, comprising:P7621PC00

[0085] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;

[0086] b. digesting the nucleic acid sample with a methylation-sensitive restriction enzyme thereby obtaining a treated nucleic acid sample; c. amplifying said treated nucleic acid sample via polymerase chain reaction (PCR) using a primer set comprising primers specific for at least one repeat-containing region of at least one locus located on the X-chromosome, wherein the locus is ARHGAP6, thereby obtaining at least one PCR product comprising a repeat-containing region of one of said loci;

[0087] d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis; and

[0088] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the maternal and paternal X-chromosomes indicates skewed XCI.

[0089] In another aspect, the present invention relates to a method for detecting skewed X-chromosome inactivation (XCI) in a female subject, comprising:

[0090] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;

[0091] b. digesting the nucleic acid sample with a methylation-sensitive restriction enzyme thereby obtaining a treated nucleic acid sample; c. amplifying said treated nucleic acid sample via multiplex polymerase chain reaction (PCR) using a primer set comprising primers specific for at least two repeat-containing region of at least two loci located on the X-chromosome, wherein the loci are

[0092] i. ARHGAP6; and

[0093] ii. ZIC3, CNKSR2, TCAC1 , or RP2,

[0094] thereby obtaining at least two PCR product comprising a repeat-containing region of two of said loci;

[0095] d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis; andP7621PC00

[0096] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the maternal and paternal X-chromosomes indicates skewed XCI.

[0097] As used herein, the term “skewed X-chromosome inactivation (XCI)” refers to a deviation from an approximately equal inactivation of the maternal and paternal X-chromosomes across a population of cells. The presence of skewed XCI may be determined based on the relative abundance of amplified PCR products corresponding to the same locus derived from the maternal and paternal X-chromosomes. A variation from an approximately equal amplification ratio may indicate that one X-chromosome is inactivated in a greater proportion of cells than the other.

[0098] Minor deviations from an equal amplification ratio may still be considered within the range of non-skewed XCI, while more pronounced deviations may indicate skewed XCI. The extent of deviation may correspond to the proportion of cells in which each X-chromosome is inactivated, with progressively larger differences suggesting increasingly skewed XCI.

[0099] In some embodiments, the presence of skewed X-chromosome inactivation (XCI) is determined based on the relative abundance of the amplified PCR products, wherein a deviation from an approximately equal amplification ratio indicates skewed XCI, wherein an approximately equal amplification ratio is a ratio of 0.8:1.2 to 1.2:0.8, such as a ratio of between 0.9:1.1 to 1.1 :0.9, such as a ratio of 1 :1 of the same locus derived from the maternal and paternal X-chromosomes. The ratio 0.9:1.1 means that one X-chromosome is producing 90% of the expected PCR product, while the other is producing 110% of the expected PCR product relative to an equal (1 :1) baseline. This does not correspond to a 90:10 X-inactivation ratio, but rather to a 45:55 distribution (where 55% of cells inactivate one X-chromosome, and 45% inactivate the other X-chromosome). The ratio 0.8:1.2 means that one X-chromosome is producing 80% of the expected PCR product, while the other is producing 120% of the expected PCR product relative to an equal (1 :1) baseline. This does not correspond to a 80:20 X-inactivation ratio, but rather to a 40:60 distribution (where 40% of the cells inactivate one X-chromosome, and 60% of the cells inactivate the other X-chromosome). The ratio 0.7:1.3 means that one X-chromosome is producing 70% of the expected PCRP7621PC00

[0100] product, while the other is producing 30% of the expected PCR product relative to an equal (1 :1) baseline. This does correspond to a 70:30 X-inactivation ratio (where 30% of the cells inactivate one X-chromosome, and 70% of the cells inactivate the other X-chromosome).

[0101] Thus, in some embodiments, the presence of skewed XCI is determined based on the relative abundance of the amplified PCT products, wherein a deviation from an approximately equal amplification ratio of the same locus derived from the maternal and paternal X-chromosomes indicates XCI, wherein an approximately equal amplification ratio is observed when 50% of the cells inactivate one X-chromosome and 50% of the cells inactivate the other, such as 49% of the cells inactivate one X-chromosome and 51% of the cells inactivate the other, such as 48% of the cells inactivate one X-chromosome and 52% of the cells inactivate the other, such as 47% of the cells inactivate one X-chromosome and 53% of the cells inactivate the other, such as 46% of the cells inactivate one X-chromosome and 54% of the cells inactivate the other, such as 45% of the cells inactivate one X-chromosome and 55% of the cells inactivate the other, such as 44% of the cells inactivate one X-chromosome and 56% of the cells inactivate the other, such as 43% of the cells inactivate one X-chromosome and 57% of the cells inactivate the other, such as 42% of the cells inactivate one X-chromosome and 58% of the cells inactivate the other, such as 41% of the cells inactivate one X-chromosome and 59% of the cells inactivate the other, such as 40% of the cells inactivate one X-chromosome and 60% of the cells inactivate the other X-chromosome.

[0102] In some embodiments, the presence of skewed X-chromosome inactivation (XCI) is determined based on the relative abundance of the amplified PCR products, wherein a deviation from an approximately equal amplification ratio indicates skewed XCI, wherein a deviation from an approximately equal amplification ratio is a ratio of between 0.7:1.3 to 1.3:0.7, such as a ratio of between 0.6:1.4 to 1.4:0.6, such as a ratio of between 0.5:1.5 to 1.5:0.5, such as a ratio of between 0.4:1.6 to 1.6:0.4 such as a ratio of between 0.3:1.7 to 1.7:0.3, such as a ratio of between 0.2:1.8 to 1.8:0.2, such as a ratio of between 0.1 :1.9 to 1.9:0.1 , such as a ratio of between 0.0:2 to 2:0.0 of the same locus derived from the maternal and paternal X-chromosomes.P7621PC00

[0103] Thus, in some embodiments, the presence of skewed XCI is determined based on the relative abundance of the amplified PCT products, wherein a deviation from an approximately equal amplification ratio of the same locus derived from the maternal and paternal X-chromosomes indicates XCI, wherein an deviation from an approximately equal amplification ratio is observed when 100% of the cells inactivate one X-chromosome and 0% of the cells inactivate the other, such as that 95% of the cells inactivate one X-chromosome and 5% of the cells inactivate the other, such as that 90% of the cells inactivate one X-chromosome and 10% of the cells inactivate the other, such as that 85% of the cells inactivate one X-chromosome and 15% of the cells inactivate the other, such as that 80% of the cells inactivate one X-chromosome and 20% of the cells inactivate the other, such as that 75% of the cells inactivate one X-chromosome and 25% of the cells inactivate the other, such as that 78% of the cells inactivate one X-chromosome and 27% of the cells inactivate the other, such as that 30% of the cells inactivate one X-chromosome and 70% of the cells inactivate the other, such as that 32% of the cells inactivate one X-chromosome and 68% of the cells inactivate the other, such as that 35% of the cells inactivate one X-chromosome and 65% of the cells inactivate the other, such as that 38% of the cells inactivate one X-chromosome and 62% of the cells inactivate the other.

[0104] In some embodiments, skewed XCI corresponds to inactivation of one X-chromosome of at least 70%, such as of at least 71%, such as of at least 72%, such as of at least 73%, such as of at least 74%, such as of at least 75%, such as of at least 76%, such as of at least 77%, such as of at least 78%, such as of at least 79%, such as of at least 80%, such as of at least 85%, such as of at least 90%, such as of at least 95%.

[0105] In some embodiments, extremely skewed XCI corresponds to inactivation of one X-chromosome of at least 79%, such as of at least 80%, such as of at least 82%, such as of at least 84%, such as of at least 86%, such as of at least 88%, such as of at least 90%, such as of at least 92%, such as of at least 95%, such as of at least 97%.

[0106] Repeat containing regions

[0107] Heterozygosity in repeat-containing regions arises from variations in the number of tandem repeat units present at a given locus on the maternal and paternal X-chromosomes. These repeat regions may consist of short sequence motifs, such as AAAG, ATCT, TCAC, GAG, or CAG, which may be repeated a variable number ofP7621PC00

[0108] times among individuals. During meiotic recombination, repeat expansion or contraction may occur due to slippage events in DNA replication or unequal crossing-over, leading to allelic differences in repeat length. As a result, a high percentage of individuals in a population may exhibit heterozygosity at these loci, meaning that their maternal and paternal X-chromosomes contain differently sized alleles.

[0109] In some embodiments, in step c) repeat-containing regions of at least two, such as at least three, such as at least four, such as five of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci are amplified via multiplex PCR.

[0110] In some embodiments, in step c) repeat-containing regions at least two, such as at least three, such as at least four, such as five of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci are amplified sequentially.

[0111] As used herein, the term "human reference genome GRCh38.p13" refers to a version of the human genome assembly that may serve as a standardized reference for genomic analyses. GRCh38.p13 represents the thirteenth patch update of the Genome Reference Consortium Human Build 38 (GRCh38), which may provide a comprehensive and highly curated sequence of the human genome, including both autosomes and sex chromosomes.

[0112] In some embodiments, the PCR product starts at position 46836203 and ends at position 46836692 of the X-chromosome of the human reference genome GRCh38.p13 (RP2).

[0113] In some embodiments, the PCR product starts at position 137568039 and ends at position 137568606 of the X-chromosome of the human reference genome GRCh38.p13 (ZIC3).

[0114] In some embodiments, the PCR product starts at position 137545307 and ends at position 137545689 of the X-chromosome of the human reference genome GRCh38.p13 (TCAC1).P7621PC00

[0115] In some embodiments, the PCR product starts at position 11427654 and ends at position 11427890 of the X-chromosome of the human reference genome GRCh38.p13 (ARHGAP6).

[0116] In some embodiments, the PCR product starts at position 21374408 and ends at position 21374708 of the X-chromosome of the human reference genome GRCh38.p13 (CNKSR2).

[0117] In some embodiments, the PCR product comprises nucleotide repeats, such as AAAG, ATCT, TCAC, GAG or CAG repeats.

[0118] In some embodiments, the PCR products comprise different numbers of nucleotide repeats in the maternal and paternal X-chromosome.

[0119] In some embodiments, the PCR product comprises at least one AAAG nucleotide repeat, such as at least two AAAG nucleotide repeats, such as at least three AAAG nucleotide repeats, such as at least four AAAG nucleotide repeats, such as at least five AAAG nucleotide repeats, such as at least 6 AAAG nucleotide repeats, such as at least 7 AAAG nucleotide repeats, such as at least 8 AAAG nucleotide repeats, such as at least 9 AAAG nucleotide repeats such as at least 10 AAAG nucleotide repeats, such as at least 15 AAAG nucleotide repeats, such as at least 20 AAAG nucleotide repeats, such as at least 25 AAAG nucleotide repeats, such as at least 30 AAAG nucleotide repeats, such as at least 35 AAAG nucleotide repeats, such as at least 40 AAAG nucleotide repeats, such as at least 50 AAAG nucleotide repeats. In some embodiments, the PCR product comprises from 1 to 50 AAAG nucleotide repeats, such as from 2 to 50 AAAG nucleotide repeats, such as from 5 to 50 AAAG nucleotide repeats, such as from 7 to 50 AAAG nucleotide repeats, such as from 10 to 50 AAAG nucleotide repeats, such as from 15 to 50 AAAG nucleotide repeats, such as from 20 to 50 AAAG nucleotide repeats. In some embodiments, the PCR product comprises from 1 to 45 AAAG nucleotide repeats, such as from 1 to 40 AAAG nucleotide repeats, such as from 1 to 37 AAAG nucleotide repeats, such as from 1 to 35 AAAG nucleotide repeats, such as from 1 to 30 AAAG nucleotide repeats, such as from 1 to 25 AAAG nucleotide repeats.P7621PC00

[0120] In some embodiments, the PCR product comprises at least one ATAC nucleotide repeat, such as at least two ATAC nucleotide repeats, such as at least three ATAC nucleotide repeats, such as at least four ATAC nucleotide repeats, such as at least five ATAC nucleotide repeats, such as at least 6 ATAC nucleotide repeats, such as at least 7 ATAC nucleotide repeats, such as at least 8 ATAC nucleotide repeats, such as at least 9 ATAC nucleotide repeats, such as at least 10 ATAC nucleotide repeats, such as at least 15 ATAC nucleotide repeats, such as at least 20 ATAC nucleotide repeats, such as at least 25 ATAC nucleotide repeats, such as at least 30 ATAC nucleotide repeats, such as at least 35 ATAC nucleotide repeats, such as at least 40 ATAC nucleotide repeats, such as at least 50 ATAC nucleotide repeats. In some embodiments, the PCR product comprises from 1 to 50 ATAC nucleotide repeats, such as from 2 to 40 ATAC nucleotide repeats, such as from 5 to 50 ATAC nucleotide repeats, such as from 7 to 50 ATAC nucleotide repeats, such as from 10 to 50 ATAC nucleotide repeats, such as from 15 to 50 ATAC nucleotide repeats, such as from 20 to 50 ATAC nucleotide repeats. In some embodiments, the PCR product comprises from 1 to 45 ATAC nucleotide repeats, such as from 1 to 40 ATAC nucleotide repeats, such as from 1 to 37 ATAC nucleotide repeats, such as from 1 to 35 ATAC nucleotide repeats, such as from 1 to 30 ATAC nucleotide repeats, such as from 1 to 25 ATAC nucleotide repeats.

[0121] In some embodiments, the PCR product comprises at least one TCAC nucleotide repeat, such as at least two TCAC nucleotide repeats, such as at least three TCAC nucleotide repeats, such as at least four TCAC nucleotide repeats, such as at least five TCAC nucleotide repeats, such as at least 6 TCAC nucleotide repeats, such as at least 7 TCAC nucleotide repeats, such as at least 8 TCAC nucleotide repeats, such as at least 9 TCAC nucleotide repeats, such as at least 10 TCAC nucleotide repeats, such as at least 15 TCAC nucleotide repeats, such as at least 20 TCAC nucleotide repeats, such as at least 25 TCAC nucleotide repeats, such as at least 30 TCAC nucleotide repeats, such as at least 35 TCAC nucleotide repeats, such as at least 40 TCAC nucleotide repeats, such as at least 50 TCAC nucleotide repeats. In some embodiments, the PCR product comprises from 1 to 50 TCAC nucleotide repeats, such as from 2 to 40 TCAC nucleotide repeats, such as from 5 to 50 TCAC nucleotide repeats, such as from 7 to 50 TCAC nucleotide repeats, such as from 10 to 50 TCAC nucleotide repeats, such as from 15 to 50 TCAC nucleotide repeats, such as from 20 to 50 TCAC nucleotide repeats. In some embodiments, the PCR product comprises fromP7621PC00

[0122] 1 to 45 TCAC nucleotide repeats, such as from 1 to 40 TCAC nucleotide repeats, such as from 1 to 37 TCAC nucleotide repeats, such as from 1 to 35 TCAC nucleotide repeats, such as from 1 to 30 TCAC nucleotide repeats, such as from 1 to 25 TCAC nucleotide repeats.

[0123] In some embodiments, the PCR product comprises at least one GAG nucleotide repeat, such as at least two GAG nucleotide repeats, such as at least three GAG nucleotide repeats, such as at least four GAG nucleotide repeats, such as at least five GAG nucleotide repeats, such as at least 6 GAG nucleotide repeats, such as at least 7 GAG nucleotide repeats, such as at least 8 GAG nucleotide repeats, such as at least 9 GAG nucleotide repeats, such as at least 10 GAG nucleotide repeats, such as at least 15 GAG nucleotide repeats, such as at least 20 GAG nucleotide repeats, such as at least 25 GAG nucleotide repeats, such as at least 30 GAG nucleotide repeats, such as at least 35 GAG nucleotide repeats, such as at least 40 GAG nucleotide repeats, such as at least 50 GAG nucleotide repeats. In some embodiments, the PCR product comprises from 1 to 50 GAG nucleotide repeats, such as from 2 to 40 GAG nucleotide repeats, such as from 5 to 50 GAG nucleotide repeats, such as from 7 to 50 GAG nucleotide repeats, such as from 10 to 50 GAG nucleotide repeats, such as from 15 to 50 GAG nucleotide repeats, such as from 20 to 50 GAG nucleotide repeats. In some embodiments, the PCR product comprises from 1 to 45 GAG nucleotide repeats, such as from 1 to 40 GAG nucleotide repeats, such as from 1 to 37 GAG nucleotide repeats, such as from 1 to 35 GAG nucleotide repeats, such as from 1 to 30 GAG nucleotide repeats, such as from 1 to 25 GAG nucleotide repeats..

[0124] In some embodiments, the PCR product comprises at least one CAG nucleotide repeat, such as at least two CAG nucleotide repeats, such as at least three CAG nucleotide repeats, such as at least four CAG nucleotide repeats, such as at least five CAG nucleotide repeats, such as at least 6 CAG nucleotide repeats, such as at least 7 CAG nucleotide repeats, such as at least 8 CAG nucleotide repeats, such as at least 9 CAG nucleotide repeats, such as at least 10 CAG nucleotide repeats, such as at least 15 CAG nucleotide repeats, such as at least 20 CAG nucleotide repeats, such as at least 25 CAG nucleotide repeats, such as at least 30 CAG nucleotide repeats, such as at least 35 CAG nucleotide repeats, such as at least 40 CAG nucleotide repeats, such as at least 50 CAG nucleotide repeats. In some embodiments, the PCR product comprises from 1 to 50 CAG nucleotide repeats, such as from 2 to 40 CAG nucleotideP7621PC00

[0125] repeats, such as from 5 to 50 CAG nucleotide repeats, such as from 7 to 50 GAG nucleotide repeats, such as from 10 to 50 CAG nucleotide repeats, such as from 15 to 50 CAG nucleotide repeats, such as from 20 to 50 CAG nucleotide repeats. In some embodiments, the PCR product comprises from 1 to 45 CAG nucleotide repeats, such as from 1 to 40 CAG nucleotide repeats, such as from 1 to 37 CAG nucleotide repeats, such as from 1 to 35 CAG nucleotide repeats, such as from 1 to 30 CAG nucleotide repeats, such as from 1 to 25 CAG nucleotide repeats..

[0126] Sample

[0127] Samples for use in the X-chromosome inactivation (XCI) analysis may be obtained from a female subject and may include biological materials containing nucleic acids from a plurality of cells. Suitable sample sources may include human tissue samples, such as blood, saliva, urine, hair, tissue, or biopsy samples, which may provide sufficient DNA for analysis. The sample may contain both the maternal and paternal X-chromosomes.

[0128] In some embodiments, the nucleic acid sample is genomic DNA extracted from a human tissue sample, such as blood, saliva, urine, hair, tissue or biopsy samples.

[0129] In some embodiments, it may be beneficial to analyse genomic DNA extracted from two or more different tissue samples belonging to the same female subject.

[0130] In some embodiments, the plurality of cells comprises at least 100 cells, such as at least 500 cells, such as at least 1000 cells, such as at least 10000 cells, such as at least 100000 cells.

[0131] In some embodiments, the nucleic acid sample is a DNA sample.

[0132] In some embodiments, the nucleic acid sample is genomic DNA extracted from brain, colon, esophagus, kidney, spleen, nerve, adipose, artery, uterus, pituitary, lung, muscle, heart, thyroid, small intestine, salivary gland, stomach, ovary, vagina, breast, skin, pancreas, liver, blood or adrenal gland tissue.

[0133] In some embodiments, the nucleic acid amount is at least 5 ng, such as at least 7.5 ng, such as at least 10 ng per reaction.P7621PC00

[0134] In some embodiments, the nucleic acid amount is at most 100 ng, such as at most 90 ng, such as at most 80 ng, such as at most 70 ng, such as at most 60 ng, such as at most 50 ng, such as at most 40 ng, such as at most 30 ng, such as at most 20 ng, such as at most 15 ng, such as at most 10 ng per reaction.

[0135] Methylation-sensitive restriction enzyme

[0136] A methylation-sensitive restriction enzyme may be used in the X-chromosome inactivation (XCI) analysis to distinguish between the methylated (inactive) X-chromosome and the unmethylated (active) X-chromosome. These enzymes may selectively digest DNA at specific recognition sites depending on the methylation status of one or more nucleotides within the recognition sequence, such as cytosine or adenine. Some methylation-sensitive restriction enzymes may cleave DNA only when the recognition site is unmethylated, while others may digest DNA only when the site is methylated. An example of a methylation-sensitive restriction enzyme is Hpall, which recognizes the 5'-CCGG-3' sequence and digests the DNA only when the internal cytosine is unmethylated, leaving methylated DNA intact.

[0137] Several other methylation-sensitive restriction enzymes may be used to distinguish between methylated and unmethylated DNA, depending on their specific recognition sequences and cleavage patterns. Other methylation-sensitive restriction enzymes include Notl, which recognizes the 5'-GCGGCCGC-3' sequence and cleaves only if the cytosines are unmethylated, and Smal, which targets the 5'-CCCGGG-3' sequence and cleaves only if the cytosine residues are unmethylated. Additionally, BssHII and Eagl recognize 5'-GCGCGC-3' and 5'-CGGCCG-3', respectively, and cleave only when the cytosines remain unmethylated. In contrast, some restriction enzymes, such as Dpnl, cleave DNA only when adenine is methylated at the 5'-GATC-3' site, whereas Dpnll recognizes the same sequence but cleaves only when adenine is unmethylated. These enzymes may be used individually or in combination to assess methylation patterns in X-chromosome inactivation (XCI) analysis.

[0138] In some embodiments, the methylation-sensitive restriction enzyme is Hpall.

[0139] In some embodiments, the contacting of the nucleic acid sample with the methylation sensitive restriction enzyme in step b) is conducted at least 25°C, such as at least 30°C, such as at least 35°C, preferably 37°C for an incubation period of at least 30 min, such as at least 45 min, such as at least 50 min, preferably 1 hour.P7621PC00

[0140] In some embodiments, the method further comprises a step of inactivating the methylation-sensitive restriction enzyme between step b) and step c).

[0141] In some embodiments, the methylation-sensitive restriction enzyme is inactivated by exposure to a temperature of at least 70°C, such as at least 75°C, preferably at 80°C.

[0142] In some embodiments, the sample may be purified after digestion to remove the methylation-sensitive restriction enzyme and other reaction components before further processing. This purification step may improve the efficiency and specificity of downstream applications, such as multiplex polymerase chain reaction (PCR), by preventing potential interference from residual enzyme activity or reaction byproducts. Purification may be achieved using various techniques, including column-based purification, magnetic bead-based purification, ethanol precipitation, or heat inactivation.

[0143] In some embodiments, the methylation-sensitive restriction enzyme is exposed to a temperature of at least 70°C, such as at least 75°C, preferably at 80°C for at least 10 minutes, such as for at least 15 minutes, preferably for 20 minutes.

[0144] Primer

[0145] In some embodiments, the method for X-chromosome inactivation (XCI) analysis may utilize a primer set designed to specifically amplify repeat-containing regions of one or more loci on the X-chromosome. The primer sequences may be selected to allow for high specificity and efficiency, ensuring robust amplification across samples. In some embodiments, the primers may be designed to enable multiplex PCR, allowing the simultaneous amplification of multiple loci within a single reaction, reducing assay time and reagent consumption. Additionally, the primers may be designed to generate PCR products of different lengths, facilitating clear separation by capillary electrophoresis. The selection of primers targeting highly heterozygous repeat-containing regions may enhance the ability to distinguish between maternal and paternal X-chromosome alleles, increasing the reliability of XCI skewing detection.

[0146] A nucleotide substitution may involve the replacement of a nucleotide at a specific position with a different nucleotide. A nucleotide deletion may refer to the removal of one or more nucleotides from the sequence, which may alter the length and structureP7621PC00

[0147] of the nucleic acid molecule. A nucleotide addition may involve the insertion of one or more additional nucleotides at a specific position, which may extend the sequence. In the context of primers used for polymerase chain reaction (PCR), such modifications may be tolerated at certain positions without significantly affecting binding specificity or amplification efficiency, particularly when limited to one or two nucleotide changes at the 5' or 3' ends of the primer sequence.

[0148] In some embodiments, the primer set comprises a forward primer consisting of SEQ ID NO: 1 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 2 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0149] In some embodiments, the primer set comprises a forward primer consisting of SEQ ID NO: 3 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 4 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0150] In some embodiments, the primer set comprises a forward primer consisting of SEQ ID NO: 5 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 6 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0151] In some embodiments, the primer set comprises a forward primer consisting of SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0152] In some embodiments, the primer set comprises a forward primer consisting of SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0153] In some embodiments, the forward or reverse primer comprises further one or more chemical, structural, or functional modifications, such as fluorophores, quenchers,P7621PC00

[0154] affinity tags, or other chemical modifications that enhance stability, specificity, or detection.

[0155] Such modifications may include fluorophores, which are covalently attached fluorescent dyes such as FAM, HEX, ROX, VIC, or Cy5, that may facilitate real-time PCR detection or fragment analysis via capillary electrophoresis. Additionally, primers may include quenchers, such as BHQ1 , BHQ2, TAMRA, or DABCYL, which may be used in probe-based assays such as TaqMan PCR, enabling fluorescence resonance energy transfer (FRET) for signal detection.

[0156] In some embodiments, primers may also contain affinity tags, such as biotin modifications at the 5' or 3' ends, which may allow for streptavidin-based purification or capture assays. Other modifications may include locked nucleic acids (LNAs), which incorporate nucleotides with a locked ribose conformation to increase binding specificity and thermal stability, improving PCR efficiency, particularly in assays detecting low-abundance targets. Additionally, phosphorothioate linkages may be introduced into the primer backbone, enhancing resistance to exonuclease degradation and increasing stability in longer reaction protocols.

[0157] In some embodiments, the one or two nucleotide substitutions or deletions in the forward primer and / or reverse primer are located in one or two of the first two nucleotides at the 5' end of the primer.

[0158] In some embodiments, the one or two nucleotide substitutions or deletions in the forward primer and / or reverse primer are located in one or two of the first two nucleotides at the 3' end of the primer.

[0159] In some embodiments, the forward primer and / or reverse primer comprises one or two additional nucleotides at the 5' end.

[0160] In some embodiments, the forward primer and / or reverse primer comprises one or two additional nucleotides at the 3' end.

[0161] In some embodiments, the forward primer and / or reverse primer comprises only a single nucleotide substitution, deletion, or addition.P7621PC00

[0162] In some embodiments, the primer set comprises primers specific for at least two distinct repeat-containing regions on at least two of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci, such as on:

[0163] a. ARHGAP6 and ZIC3;

[0164] b. ARHGAP6 and CNKSR2;

[0165] c. ARHGAP6 and TCAC1 ;

[0166] d. ARHGAP6 and RP2;

[0167] e. ZIC3 and CNKSR2;

[0168] f. ZIC3 and TCAC1 ;

[0169] g. ZIC3 and RP2;

[0170] h. CNKSR2 and TCAC1 ;

[0171] i. CNKSR2 and RP2; or

[0172] j. TCAC1 and RP2.

[0173] In some embodiments, the primer set comprises primers specific for at least three distinct repeat-containing regions on at least three of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci, such as on:

[0174] a. ARHGAP6, ZIC3, and CNKSR2;

[0175] b. ARHGAP6, ZIC3, and TCAC1 ;

[0176] c. ARHGAP6, ZIC3, and RP2;

[0177] d. ARHGAP6, CNKSR2, and TCAC1 ;

[0178] e. ARHGAP6, CNKSR2, and RP2;

[0179] f. ARHGAP6, TCAC1 , and RP2;

[0180] g. ZIC3, CNKSR2, and TCAC1 ;

[0181] h. ZIC3, CNKSR2, and RP2;

[0182] i. ZIC3, TCAC1, and RP2; or

[0183] j. CNKSR2, TCAC1 , and RP2.

[0184] In some embodiments, the primer set comprises primers specific for at least four distinct repeat-containing regions on at least four of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci, such as on:

[0185] a. ARHGAP6, ZIC3, CNKSR2, and TCAC1 ;

[0186] b. ARHGAP6, ZIC3, CNKSR2, and RP2;

[0187] c. ARHGAP6, ZIC3, TCAC1 , and RP2;

[0188] d. ARHGAP6, CNKSR2, TCAC1 , and RP2; orP7621PC00

[0189] e. ZIC3, CNKSR2, TCAC1 , and RP2.

[0190] In some embodiments, the primer set comprises primers specific for five distinct repeat-containing regions on each of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci.

[0191] One advantage of using a primer set comprising primers specific for the repeatcontaining regions of two or more of the loci of the present disclosure is that the method of detecting skewed XCI has higher sensitivity compared to a method based on the use of a primer set comprising primers specific for the repeat-containing region of a single locus.

[0192] The probability of the loci ARHGAP6 and CNKSR2 being homozygous is 0.07224. Thus, in some embodiments, the primer set comprises primers specific for the repeatcontaining regions of ARHGAP6 and CNKSR2, and the sensitivity of detecting skewed XCI is 92.8%.

[0193] The probability of the loci ARHGAP6 and RP2 being homozygous is 0.0432. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of ARHGAP6 and RP2, and the sensitivity of detecting skewed XCI is 95.7%.

[0194] The probability of the loci ARHGAP6 and TCAC1 being homozygous is 0.0878. Thus, in some embodiments, the primer set comprises primers specific for the repeatcontaining regions of ARHGAP6 and TCAC1 , and the sensitivity of detecting skewed XCI is 91.2%.

[0195] The probability of the loci ARHGAP6 and ZIC3 being homozygous is 0.0589. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of ARHGAP6 and ZIC3, and the sensitivity of detecting skewed XCI is 94.1%.

[0196] The probability of the loci CNKSR2 and RP2 being homozygous is 0.07669. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of CNKSR2 and RP2, and the sensitivity of detecting skewed XCI is 92.3%.P7621PC00

[0197] The probability of the loci CNKSR2 and TCAC1 being homozygous is 0.15588. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of CNKSR2 and TCAC1 , and the sensitivity of detecting skewed XCI is 84.4%.

[0198] The probability of the loci CNKSR2 and ZIC3 being homozygous is 0.10458. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of CNKSR2 and ZIC3, and the sensitivity of detecting skewed XCI is 89.5%.

[0199] The probability of the loci RP2 and TCAC1 being homozygous is 0.09321. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of RP2 and TCAC1 , and the sensitivity of detecting skewed XCI is 90.7%.

[0200] The probability of the loci RP2 and ZIC3 being homozygous is 0.06253. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of RP2 and ZIC3, and the sensitivity of detecting skewed XCI is 93.7%.

[0201] The probability of the loci TCAC1 and ZIC3 being homozygous is 0.1271. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of TCAC1 and ZIC3, and the sensitivity of detecting skewed XCI is 87.3%.

[0202] The probability of the loci ARHGAP6, CNKSR2, and RP2 being homozygous is 0.01547. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, and RP2, and the sensitivity of detecting skewed XCI is 98.5%.

[0203] The probability of the loci ARHGAP6, CNKSR2, and TCAC1 being homozygous is 0.03144. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, and TCAC1 , and the sensitivity of detecting skewed XCI is 96.9%.

[0204] The probability of the loci ARHGAP6, CNKSR2, and ZIC3 being homozygous is 0.0211. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, and ZIC3, wherein the sensitivity of detecting skewed XCI is 97.9%.P7621PC00

[0205] The probability of the loci ARHGAP6, RP2, and TCAC1 being homozygous is 0.0188. Thus, in some embodiments, the primer set comprises primers specific for the repeatcontaining regions of ARHGAP6, RP2, and TCAC1 , and the sensitivity of detecting skewed XCI is 98.1%.

[0206] The probability of the loci ARHGAP6, RP2, and ZIC3 being homozygous is 0.01261. Thus, in some embodiments, the primer set comprises primers specific for the repeatcontaining regions of ARHGAP6, RP2, and ZIC3, and the sensitivity of detecting skewed XCI is 98.7%.

[0207] The probability of the loci ARHGAP6, TCAC1 , and ZIC3 being homozygous is 0.02564. Thus, in some embodiments, the primer set comprises primers specific for the repeatcontaining regions of ARHGAP6, TCAC1 , and ZIC3, and the sensitivity of detecting skewed XCI is 97.4%.

[0208] The probability of the loci CNKSR2, RP2, and TCAC1 being homozygous is 0.03338. Thus, in some embodiments, the primer set comprises primers specific for the repeatcontaining regions of CNKSR2, RP2, and TCAC1 , wherein the sensitivity of detecting skewed XCI is 96.7%.

[0209] The probability of the loci CNKSR2, RP2, and ZIC3 being homozygous is 0.0224. Thus, in some embodiments, the primer set comprises primers specific for the repeatcontaining regions of CNKSR2, RP2, and ZIC3, and the sensitivity of detecting skewed XCI is 97.8%.

[0210] The probability of the loci CNKSR2, TCAC1 , and ZIC3 being homozygous is 0.04552. Thus, in some embodiments, the primer set comprises primers specific for the repeatcontaining regions of CNKSR2, TCAC1 , and ZIC3, and the sensitivity of detecting skewed XCI is 95.4%.

[0211] The probability of the loci RP2, TCAC1 , and ZIC3 being homozygous is 0.02722. Thus, in some embodiments, the primer set comprises primers specific for the repeatcontaining regions of RP2, TCAC1 , and ZIC3, and the sensitivity of detecting skewed XCI is 97.3%.P7621PC00

[0212] The probability of the loci ARHGAP6, CNKSR2, RP2, TCAC1 being homozygous is 0.00673. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, RP2, and TCAC1 , and the sensitivity of detecting skewed XCI is 99.3%.

[0213] The probability of the loci ARHGAP6, CNKSR2, RP2, ZIC3 being homozygous is 0.00452. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, RP2, and ZIC3, and the sensitivity of detecting skewed XCI is 99.5%.

[0214] The probability of the loci ARHGAP6, CNKSR2, TCAC1 , ZIC3 being homozygous is 0.00918. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, TCAC1 , and ZIC3, and the sensitivity of detecting skewed XCI is 99.1%.

[0215] The probability of the loci ARHGAP6, RP2, TCAC1 , ZIC3 being homozygous is 0.00549. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, TCAC1 , and ZIC3, and the sensitivity of detecting skewed XCI is 99.5%.

[0216] The probability of the loci CNKSR2, RP2, TCAC1 , ZIC3 being homozygous is 0.00975. Thus, in some embodiments, the primer set comprises primers specific for the repeatcontaining regions of CNKSR2, RP2, TCAC1 , and ZIC3, and the sensitivity of detecting skewed XCI is 99.0%.

[0217] The probability of the loci ARHGAP6, CNKSR2, RP2, TCAC1 , ZIC3 being homozygous is 0.00197. Thus, in some embodiments, the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, RP2, TCAC1 , and ZIC3, and the sensitivity of detecting skewed XCI is 99.8%.

[0218] In some embodiments, the primer set comprises two forward primer consisting of SEQ ID NO: 1 and SEQ ID NO: 3 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and two reverse primer consisting of SEQ ID NO: 2 and SEQ ID NO: 4 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.P7621PC00

[0219] In some embodiments, the primer set comprises two forward primer consisting of SEQ ID NO: 1 and SEQ ID NO: 5 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and two reverse primer consisting of SEQ ID NO: 2 and SEQ ID NO: 6 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0220] In some embodiments, the primer set comprises two forward primer consisting of SEQ ID NO: 1 and SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and two reverse primer consisting of SEQ ID NO: 2 and SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0221] In some embodiments, the primer set comprises two forward primer consisting of SEQ ID NO: 1 and SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and two reverse primer consisting of SEQ ID NO: 2 and SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0222] In some embodiments, the primer set comprises two forward primer consisting of SEQ ID NO: 3 and SEQ ID NO: 5 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and two reverse primer consisting of SEQ ID NO: 4 and SEQ ID NO: 6 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0223] In some embodiments, the primer set comprises two forward primer consisting of SEQ ID NO: 3 and SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and two reverse primer consisting of SEQ ID NO: 4 and SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0224] In some embodiments, the primer set comprises two forward primer consisting of SEQ ID NO: 3 and SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and two reverse primer consisting of SEQ ID NO: 4 and SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.P7621PC00

[0225] In some embodiments, the primer set comprises two forward primer consisting of SEQ ID NO: 5 and SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and two reverse primer consisting of SEQ ID NO: 6 and SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0226] In some embodiments, the primer set comprises two forward primer consisting of SEQ ID NO: 5 and SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and two reverse primer consisting of SEQ ID NO: 6 and SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0227] In some embodiments, the primer set comprises two forward primer consisting of SEQ ID NO: 7 and SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and two reverse primer consisting of SEQ ID NO: 8 and SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0228] In some embodiments, the primer set comprises three forward primers consisting of SEQ ID NO: 1 , SEQ ID NO: 3, and SEQ ID NO: 5 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and three reverse primers consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0229] In some embodiments, the primer set comprises three forward primers consisting of SEQ ID NO: 1 , SEQ ID NO: 3, and SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and three reverse primers consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0230] In some embodiments, the primer set comprises three forward primers consisting of SEQ ID NO: 1 , SEQ ID NO: 3, and SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and three reverse primers consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.P7621PC00

[0231] In some embodiments, the primer set comprises three forward primers consisting of SEQ ID NO: 1 , SEQ ID NO: 5, and SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and three reverse primers consisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0232] In some embodiments, the primer set comprises three forward primers consisting of SEQ ID NO: 1 , SEQ ID NO: 5, and SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and three reverse primers consisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0233] In some embodiments, the primer set comprises three forward primers consisting of SEQ ID NO: 1 , SEQ ID NO: 7, and SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and three reverse primers consisting of SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0234] In some embodiments, the primer set comprises three forward primers consisting of SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and three reverse primers consisting of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0235] In some embodiments, the primer set comprises three forward primers consisting of SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and three reverse primers consisting of SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0236] In some embodiments, the primer set comprises three forward primers consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and three reverse primers consisting of SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.P7621PC00

[0237] In some embodiments, the primer set comprises four forward primers consisting of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and four reverse primers consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0238] In some embodiments, the primer set comprises four forward primers consisting of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and four reverse primers consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0239] In some embodiments, the primer set comprises four forward primers consisting of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and four reverse primers consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0240] In some embodiments, the primer set comprises four forward primers consisting of SEQ ID NO: 1 , SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and four reverse primers consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0241] In some embodiments, the primer set comprises four forward primers consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and four reverse primers consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.P7621PC00

[0242] In some embodiments, the primer set comprises five forward primers consisting of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and five reverse primers consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0243] Multiplex PCR and separation of PCR products

[0244] In some embodiments, the method for XCI analysis may utilize multiplex PCR to amplify multiple repeat-containing regions from a single reaction mixture, thereby increasing efficiency and reducing assay time. The primer set may comprise two, three, four, or five forward primers and corresponding reverse primers, such as those specific for two or more of ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2, allowing the simultaneous amplification of two or more loci on the X-chromosome. The primers may be designed to generate PCR products of different lengths, enabling clear separation by capillary electrophoresis without the need for additional processing steps.

[0245] In some embodiments, the multiplex polymerase chain reaction of step c) is performed by amplifying all locus regions in one tube.

[0246] In some embodiments, the multiplex polymerase chain reaction of step c) is performed by using a DNA polymerase, such as a high fidelity polymerase. As used herein, the term "high-fidelity polymerase" refers to a DNA polymerase enzyme that may exhibit low error rates during DNA synthesis, ensuring accurate replication of nucleic acid sequences. High-fidelity polymerases may possess proofreading activity, such as 3' to 5' exonuclease activity, which can detect and correct misincorporated nucleotides, reducing the occurrence of mutations in the amplified product.

[0247] In some embodiments, the separation of the PCR products is performed by electrophoresis, such as capillary electrophoresis, conventional slab gel electrophoresis or microfluidic electrophoresis.

[0248] In some embodiments, the multiplex PCR assay is performed using fluorescently labeled primers and the resulting PCR products are detected via fluorescence-based capillary electrophoresis. Such modifications may include fluorophores, which areP7621PC00

[0249] covalently attached fluorescent dyes such as FAM, HEX, ROX, VIC, or Cy5, that may facilitate real-time PCR detection or fragment analysis via capillary electrophoresis. Additionally, primers may include quenchers, such as BHQ1 , BHQ2, TAMRA, or DABCYL, which may be used in probe-based assays such as TaqMan PCR, enabling fluorescence resonance energy transfer (FRET) for signal detection.

[0250] In some embodiments, the capillary length is at least 25 cm, such as at least 30 cm, such as at least 35 cm, such as at least 40 cm, such as at least 45 cm, such as at least 50 cm.

[0251] Kit

[0252] In some embodiments, the method for X-chromosome inactivation (XCI) analysis may be provided as a kit comprising reagents and components necessary for detecting skewed XCI in a sample.

[0253] In another aspect, the present invention relates to a kit comprising:

[0254] a. at least one, such as at least two, such as at least three, such as at least four, such as five primer sets, wherein the primer sets comprise

[0255] i. a forward primer consisting of SEQ ID NO: 1 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 2 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or

[0256] ii. a forward primer consisting of SEQ ID NO: 3 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 4 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or

[0257] iii. a forward primer consisting of SEQ ID NO: 5 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 6 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or

[0258] iv. a forward primer consisting of SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, orP7621PC00

[0259] additions thereof, and a reverse primer consisting of SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or

[0260] v. a forward primer consisting of SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof; and

[0261] b. instructions for detecting skewed XCI.

[0262] In another aspect, the present invention relates to a kit for detecting skewed XCI comprising

[0263] a. at least one, such as at least two, such as at least three, such as at least four, such as five primer sets, wherein the primer sets comprise

[0264] i. a forward primer and reverse primer, wherein the forward primer binds upstream of position 46836203 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 46836692 in the X- chromosome of the human reference genome GRCh38.p13; and / or

[0265] ii. a forward primer and reverse primer, wherein the forward primer binds upstream of position 137568039 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 137568606 in the X- chromosome of the human reference genome GRCh38.p13; and / or

[0266] iii. a forward primer and reverse primer, wherein the forward primer binds upstream of position 137545307 in the X-chromosome and / of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 137545689 in the X-chromosome of the human reference genome GRCh38.p13; and / or

[0267] iv. a forward primer and reverse primer, wherein the forward primer binds upstream of position 11427654 in the X-chromosome of the human reference genome GRCh38.p13 and the reverseP7621PC00

[0268] primer binds downstream of position 11427890 in the X- chromosome of the human reference genome GRCh38.p13; and / or

[0269] v. a forward primer and reverse primer, wherein the forward primer binds upstream of position 21374408 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 21374708 in the X- chromosome of the human reference genome GRCh38.p13; and b. instructions for use.

[0270] In another aspect, the present invention relates to a kit comprising: at least one, such as at least two, such as at least three, such as at least four, such as five primer sets, wherein the primer sets comprise a forward primer consisting of SEQ ID NO: 1 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 2 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof; and

[0271] iii. a forward primer consisting of SEQ ID NO: 3 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 4 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or

[0272] iv. a forward primer consisting of SEQ ID NO: 5 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 6 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or

[0273] v. a forward primer consisting of SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / orP7621PC00

[0274] vi. a forward primer consisting of SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof; and

[0275] g. instructions for detecting skewed XCI.

[0276] In another aspect, the present invention relates to a kit for detecting skewed XCI comprising

[0277] a. At least one, such as at least two, such as at least three, such as at least four, such as five primer sets, wherein the primer sets comprise a forward primer and reverse primer, wherein the forward primer binds upstream of position 11427654 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 11427890 in the X-chromosome of the human reference genome GRCh38.p13; and

[0278] i. a forward primer and reverse primer, wherein the forward primer binds upstream of position 46836203 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 46836692 in the X-chromosome of the human reference genome GRCh38.p13; and / or

[0279] ii. a forward primer and reverse primer, wherein the forward primer binds upstream of position 137568039 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 137568606 in the X-chromosome of the human reference genome GRCh38.p13; and / or

[0280] iii. a forward primer and reverse primer, wherein the forward primer binds upstream of position 137545307 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 137545689 in the X-chromosome of the human reference genome GRCh38.p13; and / or

[0281] iv. a forward primer and reverse primer, wherein the forward primer binds upstream of position 21374408 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstreamP7621PC00

[0282] of position 21374708 in the X-chromosome of the human reference genome GRCh38.p13; and

[0283] b. instructions for use.

[0284] In some embodiments, the kit comprises further a methylation-sensitive restriction enzyme, such as Hpall. The methylation-sensitive restriction enzyme can be a methylation-sensitive restriction enzyme as described in the chapter “Methylationsensitive restriction enzyme”.

[0285] In some embodiments, the kit comprises further a DNA polymerase, such as a high fidelity polymerase.

[0286] In some embodiments, the kit comprises further digestion buffer, such as a digestion buffer that facilitates enzymatic digestions of DNA by said methylation-sensitive restriction enzyme.

[0287] In some embodiments, the kit further comprises PCR buffer, such as a PCR buffer that facilitates activity of said DNA polymerase.

[0288] In some embodiments, the kit further comprises control sample, such as a sample comprising DNA with skewed XCI.

[0289] In some embodiments, the forward or reverse primer comprises further one or more chemical, structural, or functional modifications, such as fluorophores, quenchers, affinity tags, or other chemical modifications that enhance stability, specificity, or detection. The primer can be a primer as described in the chapter “Primer”.

[0290] Disorders associated with skewed X-chromosome inactivation (XCI)

[0291] Skewed XCI has been implicated in various genetic, developmental, and autoimmune disorders, where an imbalance in XCI may lead to altered gene expression and disease susceptibility. In some cases, skewed XCI may act as a protective mechanism, while in others, it may contribute to disease pathology. For example, X-linked recessive disorders, such as Duchenne muscular dystrophy (DMD), Hemophilia A and B, and X-linked severe combined immunodeficiency (SCID-X1), may manifest in female carriers if the functional X-chromosome is preferentially inactivated. Similarly, Rett syndromeP7621PC00

[0292] (MECP2 mutations) and X-linked intellectual disabilities (e.g., ATRX or ARX mutations) may show variable phenotypic severity due to differences in XCI patterns. Additionally, autoimmune diseases, such as systemic lupus erythematosus (SLE) and scleroderma, have been associated with non-random XCI, suggesting a potential role in immune system dysregulation. Detecting skewed XCI may therefore provide valuable insights into disease risk, carrier status, and potential therapeutic interventions.

[0293] In another aspect, the present invention relates to a method for diagnosing a disorder associated with skewed X-chromosome inactivation (XCI) in a female subject, comprising:

[0294] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;

[0295] b. contacting said nucleic acid sample with a methylation-sensitive restriction enzyme to obtain a treated nucleic acid sample; c. amplifying said treated nucleic acid sample via PCR using a primer set comprising primers specific for a least one repeat-containing region of at least one locus located on the X-chromosome, wherein the locus is ARHGAP6, ZIC3, CNKSR2, TCAC1, and RP2, thereby obtaining at least one PCR product comprising a repeat-containing region of one of said loci;

[0296] d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis;

[0297] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the two X-chromosomes indicates skewed XCI; and

[0298] f. correlating the presence of skewed XCI with an increased risk of or presence of an X-linked disorder.

[0299] In another aspect, the present invention relates to a method for diagnosing a disorder associated with skewed X-chromosome inactivation (XCI) in a female subject, comprising:P7621PC00

[0300] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;

[0301] b. contacting said nucleic acid sample with a methylation-sensitive restriction enzyme to obtain a treated nucleic acid sample; c. amplifying said treated nucleic acid sample via PCR using a primer set comprising primers specific for a least one repeat-containing region of at least one locus located on the X-chromosome, wherein the locus is v. ARHGAP6; and

[0302] vi. ZIC3, CNKSR2, TCAC1 , or RP2, thereby obtaining at least one PCR product comprising a repeat-containing region of one of said loci; d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis;

[0303] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the two X-chromosomes indicates skewed XCI; and

[0304] f. correlating the presence of skewed XCI with an increased risk of or presence of an X-linked disorder.

[0305] In some embodiments, the disorder associated with skewed X-chromosome inactivation (XCI) is Duchenne Muscular Dystrophy (DMD), X-Linked Severe Combined Immunodeficiency (X-SCID), X-Linked Adrenoleukodystrophy (ALD), Hemophilia A & B, Rett Syndrome, Hunter Syndrome (MPS II), Wiskott-Aldrich Syndrome (WAS), Menkes Disease, Lesch-Nyhan Syndrome, Fabry Disease (Severe Form), Kennedy's Disease (SBMA) or Charcot-Marie-Tooth Disease (CMTX1).

[0306] In some embodiments, the female subjects shows symptoms, such as progressive muscle weakness, loss of ambulation, respiratory and cardiac failure, absent immune function, severe infections, failure to thrive, neurological deterioration, loss of motor function, adrenal insufficiency, uncontrolled bleeding, joint damage, internal hemorrhages, severe cognitive & motor impairment, breathing abnormalities, seizures, progressive organ damage, skeletal abnormalities, cognitive decline, immune deficiency, bleeding disorders, eczema, increased cancer risk, copper transport disorder, neurological degeneration, failure to thrive, self-mutilation, severe movementP7621PC00

[0307] disorders, kidney issues, severe pain, kidney failure, stroke risk, cardiac disease, swallowing difficulties, hormonal abnormalities, progressive nerve damage, muscle weakness, foot deformities or sensory loss.

[0308] Examples

[0309] Example 1: Tandem Repeat-based Identification of X-chromosome Inactivation (TRiXi)

[0310] Aim:

[0311] To investigate if an assay can predict XCI with high sensitivity and robustness.

[0312] Material and methods:

[0313] PCR primer and product design

[0314] PCR primers were designed to produce PCR products with differing lengths, as fluorescein (FAM) was to be used for detection. The GC% content did vary between PCR products, ranging from 40% to 71%. To ensure no common genetic variations would interfere with the PCR, dbSNP (build 151 ) common variant vcf files were downloaded. Variants with a minor allele frequency > 1 % in 10OOGenomes or TOPMed were included.

[0315] TriXi protocol

[0316] Hpall digestion

[0317] Genomic DNA (20-40 ng) was digested at 372C for 60 min using 1 x rCutsmart buffer and 1 U / pL of methylation-sensitive restriction enzyme Hpall, as per the manufacturer's instructions (New England Biolabs, NEB-R0171M). The enzyme was inactivated at 802C for 20 min after digestion. For each sample an undigested control sample was prepared without the / - / pall enzyme. Additionally, for each run, a blank sample and a sample of known skewing were included as negative and positive controls, respectively.

[0318] PCR amplification

[0319] PCR amplification mix contained 1x PCR buffer (New England Biolabs, NEB-M0530L), 8 nM dNTP mix, 0.6 ul DMSO, and 0.4 U Phusion Polymerase. A 2ul TRiXi primer mixP7621PC00

[0320] comprising primers with SEQ ID NOs: 1 to 10 was added to the master mix. PCR was performed using the entire digested and undigested genomic DNA sample. PCR amplification was performed using the following thermocycler protocol: One cycle of 30 s at 98 °C; 30 cycles of 10 s at 98 °C, followed by 30 s at 63 °C, followed by 40 s at 72 °C; one cycle of 5 min at 72 °C; 4 °C hold.

[0321] Capillary electrophoresis

[0322] For fragment analysis, 1 pl of amplified sample was transferred to a 96-well plate and diluted with 8 pl of a master mix consisting of 8.3 pl Hi- Di Formamide (Thermo Fisher) and 0.2 pl GeneScan™ 600 LIZ™ (Applied Biosystems). Sample plates were denatured at 95°C / 5 min, cooled on ice, and resolved using a 3500 Genetic Analyzer (Thermo Fisher).

[0323] Calculating skewing

[0324] Peak height, area and nucleotide size utilized to calculate skewing and repeat length were obtained using GeneMapper5 and calculations were performed in R. Briefly, the .fsa files obtained from the Genetic Analyzer 3500 were loaded into GeneMapper (v5, Thermo Fisher). Peaks were called using microsatellite analysis (default setting with LIZ600+normalization as ladder). The resulting table with peak information was exported and loaded into R where skewing was calculated. First, the two highest peaks (separated by at least 2.5 bases) per undigested sample were identified and matched to the closest sized peaks in the digested sample, obtaining two peak areas for both undigested and digested sample. Samples where the peaks were not distinguishable from each other in the undigested sample were deemed homozygous and the repeat was excluded from skewing calculations for that sample. Skewing was assessed individually for each repeat and the mean of all informative repeats were used to determine degree of skewing. The skewing calculation itself was performed as described in Roberts et al., 2022:

[0325] allele Ratio Mock Digestion (Rm)= allele 1 peak height / allele 2 peak height

[0326] allele Ratio Hpall Digestion (Rh) = allele 1 peak height / allele 2 peak height normalized Ratio (Rn)=Rh / Rm

[0327] XCI percentage = [Rn / (Rn + 1)] * 100P7621PC00

[0328] For each sample, the mean XCI percentage (ranging from 0 to 100%) was calculated, with 50% representing perfectly balanced XCI. XCI values were collapsed to a 50-100% range (as the direction of deviation from 50% is uninformative). Finally, the standard deviation and mean of the total data set was used to determine thresholds for skewing and extreme skewing:

[0329] random XCI: XCI value <p+o

[0330] skewed XCkXCI value > p+u

[0331]

[0332] extremely skewed XCI :XCI value >p+2o

[0333] Where / / is the population mean and a is the population standard deviation.

[0334] Consequently, skewed XCI equated to values > 71.09% and extremely skewed XCI to values > 79.76% (Figure 2X). XCI skew values above either threshold was classified as skewed, or extremely skewed, respectively.

[0335] Results:

[0336] Five (i) highly polymorphic tri- and tetra-nucleotide repeat sequences, (ii) flanked by at least one Hpall site, and (iii) characterized by 50% methylation were identified, indicating that the locus is subject to XCI. Primer pairs were designed for each the five loci, ARHGAP6, RP2, CNKSR2, TCAC1 and ZIC3, to result in amplified products of non-overlapping size range, allowing separation of all possible alleles on a single lane of capillary electrophoresis (Figure 1 A). Direct comparison of TRiXi with HUMARA on 50 randomly selected samples showed good agreement between the methods (rho = 0.66, p-value = 7.4 x 10“6, Figure 1 B). However, as expected, HUMARA assay was uninformative in 11 of the 50 samples tested (Figures 1C & 1 D). Having validated the sensitivity and robustness of TriXi, levels of constitutional skewing in umbilical cord blood of 360 healthy newborns were assessed.

[0337] Conclusion:

[0338] The inventors of the present disclosure have shown that the assay offers a vast improvement in sensitivity over the HUMARA assay for detection of skewed X-inactivation in humans, while maintaining the simplicity and low sample requirements of the HUMARA assay. Levels of constitutional skewing in umbilical cord blood of 360 healthy newborns show that 10.24% of females were highly skewed (mean +1 SD, >71.09% skewing) for XCI at birth with 5.17% exhibiting extreme skewing (mean +2 SD, >79.76% skewing).P7621PC00

[0339] Example 2: Oxford Nanopore Technologies (ONT) long-read sequencing

[0340] Aim: To validate sensitivity and robustness of TriXi

[0341] Material and Methods:

[0342] Oxford Nanopore long-read sequencing

[0343] 483 1.5 g of genomic DNA was sheared to approximately 10 kb using Covaris g-TUBEs (Covaris, 500291) in an Eppendorf 5415 R centrifuge at 5,000 RPM for 1 minute at room temperature. Library preparation was performed using the Ligation Sequencing Kit V14 (ONT, SQK-LSK114) according to the manufacturer’s protocol (GDE_9161_v114_revY, 30 January 2025). Briefly, DNA repair and end-preparation were conducted using the NEBNext FFPE DNA Repair Mix and Ultra™ II End Prep Enzyme Mix (NEB, E7672S) followed by adapter ligation. The final library was eluted and quantified using the Qubit 1X dsDNA High Sensitivity Assay (Thermo Fisher Scientific, Q33231). A total of 50 fmol of the prepared library was loaded onto a PromethlON Flow Cell (ONT, FLO-PRO114) and sequenced on the PromethlON 2 integrated device for 72 hours. The sequencing run generated 20.4 million reads at ~46X coverage with a basecalled N50 read length of approximately 10kbp.

[0344] Determining XCI skew from the ONT long-read data was performed as previously described by Gocuk et al., 2024. POD5 files were basecalled and aligned to the human genome build 38 (hg38, GCA_000001405.15_GRCh38_no_alt_analysis_set.fna) using dorado vO.7.4 (https: / / github.com / nanoporetech / dorado, which utilizes minimap257 for mapping) and the dna_r10.4.1_e8.2_400bps_sup@v4.3.0 model. For modified basecalling, 5mC and 5hmC were called in a GC context. To allow phasing, high-quality variants were called X-linked variants were called using DeepVariant58 v1.8.0 using the model ‘ONT_R104’ and subsequent filtering with BCFtools41 (only ‘PASS’ variants were kept). Phasing of the VCF and subsequent haplotag assignment was performed using LongPhase59 v1.7.3. Reads assigned to incorrect haplotype blocks were identified in Integrative Genomics Viewer60 (v. 2.19.4) and subsequently corrected. Each finalized modbam file (with haplotags) was then processed individually in R using NanoMethViz61 v3.2.0. Methylation probabilities for each CCGG site in the TRiXi 1-5 PCR products were extracted per read, using the ‘query_methy’ function. Methylation probabilities were classified as low (and thus, associated with the active X (Xa) if below 0.1 and high (and associated with the Xi) if above 0.9. Within each repeat, the number of haplotype 1 / Xa, haplotype 2 / Xi, haplotype 1 / Xi, and haplotype 2 / XaP7621PC00

[0345] reads were counted to estimate the repeat-wise skew. To assess XCI skew, the mean of all repeat-wise skew values was used.

[0346] Results:

[0347] To further validate TriXi, whole genome Oxford Nanopore Technologies (ONT) long-read sequencing on one highly skewed sample (97% sXCI) was performed. Following phasing of reads based on repeat length, ONT long-read sequencing confirmed the extreme skewing observed in this sample (Fig. 2); see also Goldmann et al. 2025. Further, using a recently published method for calculating skewing from X-chromosome ONT data, resulted in a global X-chromosome skewing of 88.1%, again highly concordant with that obtained with TRiXi (97%) (Gocuk et al. 2024).

[0348] Conclusion:

[0349] The inventors of the present disclosure have shown that the assay of the present invention, TriXi, is sensitive and robust.

[0350] Example 3: Evidence of multi-organ constitutional sXCI in adult females

[0351] Aim: To investigate skewed X-inactivation across tissues in the same individual.

[0352] Material and methods:

[0353] The GTEx dataset, which contains RNA-sequencing data for 4,571 individual tissues across 264 female donors was used, skewed X-inactivation levels for each tissue were determined by measuring the allele-specific expression of reads arising from genes 188 that undergo XCI, as recently described by Gylemo et al., 2025.

[0354] Results:

[0355] Whereas the average skewing across tissues per individual was highly similar to that observed at birth; with 12.89% of females highly skewed (median +1 SD, >71.4% skewing) for XCI and 6.25% exhibiting extreme skewing (median +2 SD, >79% skewing) (Fig. 3a, b), whole blood exhibited significantly higher levels of sXCI in older adults (Fig. 3c), consistent with the selective effects of clonal hematopoiesis. Notably, across all adults tested, constitutional skewing was the likely origin of 23.4% of extremely skewed females and in individuals under 50 years of age, constitutional skewing was still the dominant cause of extreme sXCI (Fig. 3c). Unexpectedly, skewedP7621PC00

[0356] X-inactivation was also highly prevalent across non-hematopoietic tissues, with 70% of females (181 / 256) having at least one extremely skewed tissue (Fig. 3d). In total, 13.6% (622 / 4571 , ~1 in 7) of tissues examined exhibited extreme skewed X-inactivation with adrenal gland, whole blood, liver and pancreas among the most frequently skewed (Fig. 3e).

[0357] Conclusion These results indicate that multi-tissue constitutive skewing is a common phenomenon in the normal human population.

[0358] Sequences

[0359] SEQ ID NO: 1 : ARHGAP6 F

[0360] 5’-GAGTTCCCCGCACTTCACT-3’

[0361] SEQ ID NO: 2: ARHGAP6 R

[0362] 5’-GCCCATCGCATCAGACTC-3’

[0363] SEQ ID NO: 3: CNKSR2 F

[0364] 5’-GAGGCCCCTCGTCATTTC-3’

[0365] SEQ ID NO: 4: CNKSR2 R

[0366] 5’-ACAGGGAGGTAAACGTGCAG-3’

[0367] SEQ ID NO: 5: TCAC1 F

[0368] 5’-AAAGCACCTCAGGACATCCC-3’

[0369] SEQ ID NO: 6: TCAC1 R

[0370] 5’-GTCTTCAAAGGGCGAGGTCA-3’

[0371] SEQ ID NO: 7: RP2 F

[0372] 5’-AGCTACTCAGGAAGGAGGCT-3’

[0373] SEQ ID NO: 8: RP2 R

[0374] 5’-TGGTGGGTTCTCTAGCTGG-3’

[0375] SEQ ID NO: 9: ZIC3 FP7621PC00

[0376] 5’-CTGGGTTACCGTGAAGGGAC-3’

[0377] SEQ ID NO: 10: ZIC3 R

[0378] 5’-TTGTGTTTCCACCCTACCCG-3’

[0379] References

[0380] A. L. Roberts etal., Age acquired skewed X-chromosome inactivation is associated with adverse health outcomes in humans. Elife 11 , (2022).

[0381] Gocuk, S.A. et al. Measuring X-Chromosome inactivation skew for X-linked diseases with adaptive nanopore sequencing. Genome Res 34, 1954-1965 (2024)

[0382] Gylemo, B., Bensberg, M. & Nestor, G.E. A whole-organism landscape of X-inactivation in humans. Elife 14 (2025).

[0383] Goldmann D. et al. TRiXi: A Multi-Target Tandem Repeat-Based Method for Accurate Detection of X-lnactivation Skewing in Humans PrePrint doi:

[0384]

[0385] Items

[0386] 1. A method for detecting skewed X-chromosome inactivation (XCI) in a female subject, comprising:

[0387] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;

[0388] b. digesting the nucleic acid sample with a methylation-sensitive restriction enzyme thereby obtaining a treated nucleic acid sample; c. amplifying said treated nucleic acid sample via polymerase chain reaction (PGR) using a primer set comprising primers specific for at least one repeat-containing region of at least one locus located on the X-chromosome, wherein the locus is ARHGAP6, ZIC3, CNKSR2, TCAC1 , or RP2, thereby obtaining at least one PGR product comprising a repeat-containing region of one of said loci;

[0389] d. separating the resulting PGR products by electrophoresis, such as capillary electrophoresis; andP7621PC00

[0390] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the maternal and paternal X-chromosomes indicates skewed XCI.

[0391] 2. A method for detecting skewed X-chromosome inactivation (XCI) in a female subject, comprising:

[0392] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;

[0393] b. digesting the nucleic acid sample with a methylation-sensitive restriction enzyme thereby obtaining a treated nucleic acid sample;

[0394] c. amplifying said treated nucleic acid sample via polymerase chain reaction (PCR) using a primer set comprising primers specific for at least one repeat-containing region of at least one locus located on the X-chromosome, wherein the locus is ARHGAP6, thereby obtaining at least one PCR product comprising a repeat-containing region of one of said loci;

[0395] d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis; and

[0396] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the maternal and paternal X-chromosomes indicates skewed XCI.

[0397] 3. A method for detecting skewed X-chromosome inactivation (XCI) in a female subject, comprising:

[0398] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;

[0399] b. digesting the nucleic acid sample with a methylation-sensitive restriction enzyme thereby obtaining a treated nucleic acid sample;

[0400] c. amplifying said treated nucleic acid sample via multiplex polymerase chain reaction (PCR) using a primer set comprising primers specific for at least two repeat-containing region of at least two loci located on theP7621PC00

[0401] X-chromosome, wherein the loci are ARHGAP6, ZIC3, CNKSR2, TCAC1 , or RP2, thereby obtaining at least two PGR product comprising a repeat-containing region of two of said loci;

[0402] d. separating the resulting PGR products by electrophoresis, such as capillary electrophoresis; and

[0403] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PGR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the maternal and paternal X-chromosomes indicates skewed XCI.

[0404] 4. A method for detecting skewed X-chromosome inactivation (XCI) in a female subject, comprising:

[0405] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;

[0406] b. digesting the nucleic acid sample with a methylation-sensitive restriction enzyme thereby obtaining a treated nucleic acid sample;

[0407] c. amplifying said treated nucleic acid sample via multiplex polymerase chain reaction (PCR) using a primer set comprising primers specific for at least two repeat-containing region of at least two loci located on the X-chromosome, wherein the loci are

[0408] i. ARHGAP6; and

[0409] ii. ZIC3, CNKSR2, TCAC1 , or RP2,

[0410] thereby obtaining at least two PCR product comprising a repeat-containing region of two of said loci;

[0411] d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis; and

[0412] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the maternal and paternal X-chromosomes indicates skewed XCI.

[0413] 5. The method according to any one of items 1 to 2, wherein in step c) repeatcontaining regions of at least two, such as at least three, such as at least four,P7621PC00

[0414] such as five of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci are amplified via multiplex PGR.

[0415] 6. The method according to any one of items 1 to 2, wherein in step c) repeatcontaining regions at least two, such as at least three, such as at least four, such as five of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci are amplified sequentially.

[0416] 7. The method according to any one of the preceding items, wherein the nucleic acid sample is genomic DNA extracted from a human tissue sample, such as blood, saliva, urine, hair, tissue or biopsy samples.

[0417] 8. The method according to any one of the preceding items, wherein the nucleic acid sample is genomic DNA extracted from brain, colon, esophagus, kidney, spleen, nerve, adipose, artery, uterus, pituitary, lung, muscle, heart, thyroid, small intestine, salivary gland, stomach, ovary, vagina, breast, skin, pancreas, liver, blood or adrenal gland tissue.

[0418] 9. The method according to any one of the preceding items, wherein the plurality of cells comprises at least 100 cells, such as at least 500 cells, such as at least 1000 cells, such as at least 10000 cells, such as at least 100000 cells.

[0419] 10. The method according to any one of the preceding items, wherein the nucleic acid sample is a DNA sample.

[0420] 11. The method according to any one of the preceding items, wherein the methylation-sensitive restriction enzyme is Hpall.

[0421] 12. The method according to any one of the preceding items, wherein the nucleic acid amount is at least 5 ng, such as at least 7.5 ng, such as at least 10 ng per reaction.

[0422] 13. The method according to any one of the preceding items, wherein the nucleic acid amount is at most 100 ng, such as at most 90 ng, such as at most 80 ng, such as at most 70 ng, such as at most 60 ng, such as at most 50 ng, such asP7621PC00

[0423] at most 40 ng, such as at most 30 ng, such as at most 20 ng, such as at most 15 ng, such as at most 10 ng per reaction.

[0424] 14. The method according to any one of the preceding items, wherein the contacting of the nucleic acid sample with the methylation-sensitive restriction enzyme in step b) is conducted at least 25°C, such as at least 30°C, such as at least 35°C, preferably 37°C for an incubation period of at least 30 min, such as at least 45 min, such as at least 50 min, preferably 1 hour.

[0425] 15. The method according to any one of the preceding items, wherein the method further comprises a step of inactivating the methylation-sensitive restriction enzyme between step b) and step c).

[0426] 16. The method according to item 15, wherein the methylation-sensitive restriction enzyme is inactivated by exposure to a temperature of at least 70°C, such as at least 75°C, preferably at 80°C.

[0427] 17. The method according to any one of items 15 to 16, wherein the methylationsensitive restriction enzyme is exposed to a temperature of at least 70°C, such as at least 75°C, preferably at 80°C for at least 10 minutes, such as for at least 15 minutes, preferably for 20 minutes.

[0428] 18. The method according to any one of the preceding items, wherein the primer set comprises a forward primer consisting of SEQ ID NO: 1 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 2 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0429] 19. The method according to any one of the preceding items, wherein the primer set comprises a forward primer consisting of SEQ ID NO: 3 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 4 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.P7621PC00

[0430] 20. The method according to any one of the preceding items, wherein the primer set comprises a forward primer consisting of SEQ ID NO: 5 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 6 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0431] 21. The method according to any one of the preceding items, wherein the primer set comprises a forward primer consisting of SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0432] 22. The method according to any one of the preceding items, wherein the primer set comprises a forward primer consisting of SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

[0433] 23. The method according to any one of the items 18 to 22, wherein the forward or reverse primer comprises further one or more chemical, structural, or functional modifications, such as fluorophores, quenchers, affinity tags, or other chemical modifications that enhance stability, specificity, or detection.

[0434] 24. The method according to any one of the items 18 to 23, wherein the one or two nucleotide substitutions or deletions in the forward primer and / or reverse primer are located in one or two of the first two nucleotides at the 5' end of the primer.

[0435] 25. The method according to any one of the items 18 to 24, wherein the one or two nucleotide substitutions or deletions in the forward primer and / or reverse primer are located in one or two of the first two nucleotides at the 3' end of the primer.

[0436] 26. The method according to any one of the items 18 to 25, wherein the forward primer and / or reverse primer comprises one or two additional nucleotides at the 5' end.P7621PC00

[0437] 27. The method according to any one of the items 18 to 25, wherein the forward primer and / or reverse primer comprises one or two additional nucleotides at the 3' end.

[0438] 28. The method according to any one of the items 18 to 27, wherein the forward primer and / or reverse primer comprises only a single nucleotide substitution, deletion, or addition.

[0439] 29. The method according to any one of the preceding items, wherein the primer set comprises primers specific for at least two distinct repeat-containing regions on at least two of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci, such as on:

[0440] a. ARHGAP6 and ZIC3;

[0441] b. ARHGAP6 and CNKSR2;

[0442] c. ARHGAP6 and TCAC1 ;

[0443] d. ARHGAP6 and RP2;

[0444] e. ZIC3 and CNKSR2;

[0445] f. ZIC3 and TCAC1 ;

[0446] g. ZIC3 and RP2;

[0447] h. CNKSR2 and TCAC1 ;

[0448] i. CNKSR2 and RP2; or

[0449] j. TCAC1 and RP2.

[0450] 30. The method according to any one of the preceding items, wherein the primer set comprises primers specific for at least three distinct repeat-containing regions on at least three of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci, such as on:

[0451] a. ARHGAP6, ZIC3, and CNKSR2;

[0452] b. ARHGAP6, ZIC3, and TCAC1 ;

[0453] c. ARHGAP6, ZIC3, and RP2;

[0454] d. ARHGAP6, CNKSR2, and TCAC1 ;

[0455] e. ARHGAP6, CNKSR2, and RP2;

[0456] f. ARHGAP6, TCAC1 , and RP2;

[0457] g. ZIC3, CNKSR2, and TCAC1 ;

[0458] h. ZIC3, CNKSR2, and RP2;P7621PC00

[0459] i. ZIC3, TCAC1 , and RP2; or

[0460] j. CNKSR2, TCAC1 , and RP2.

[0461] 31. The method according to any one of the preceding items, wherein the primer set comprises primers specific for at least four distinct repeat-containing regions on at least four of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci, such as on:

[0462] a. ARHGAP6, ZIC3, CNKSR2, and TCAC1 ;

[0463] b. ARHGAP6, ZIC3, CNKSR2, and RP2;

[0464] c. ARHGAP6, ZIC3, TCAC1 , and RP2;

[0465] d. ARHGAP6, CNKSR2, TCAC1 , and RP2; or

[0466] e. ZIC3, CNKSR2, TCAC1 , and RP2.

[0467] 32. The method according to any one of the preceding items, wherein the primer set comprises primers specific for five distinct repeat-containing regions on each of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci.

[0468] 33. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6 and CNKSR2, and wherein the sensitivity of detecting skewed XCI is 92.8%.

[0469] 34. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6 and RP2, and wherein the sensitivity of detecting skewed XCI is 95.7%.

[0470] 35. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6 and TCAC1 , and wherein the sensitivity of detecting skewed XCI is 91.2%.

[0471] 36. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6 and ZIC3, and wherein the sensitivity of detecting skewed XCI is 94.1%.P7621PC00

[0472] 37. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of CNKSR2 and RP2, and wherein the sensitivity of detecting skewed XCI is 92.3%.

[0473] 38. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of CNKSR2 and TCAC1 , and wherein the sensitivity of detecting skewed XCI is 84.4%.

[0474] 39. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of CNKSR2 and ZIC3, and wherein the sensitivity of detecting skewed XCI is 89.5%.

[0475] 40. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of RP2 and TCAC1 , and wherein the sensitivity of detecting skewed XCI is 90.7%.

[0476] 41. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of RP2 and ZIC3, and wherein the sensitivity of detecting skewed XCI is 93.7%.

[0477] 42. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of TCAC1 and ZIC3, and wherein the sensitivity of detecting skewed XCI is 87.3%.

[0478] 43. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, and RP2, and wherein the sensitivity of detecting skewed XCI is 98.5%.

[0479] 44. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, and TCAC1 , and wherein the sensitivity of detecting skewed XCI is 96.9%.P7621PC00

[0480] 45. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, and ZIC3, and wherein the sensitivity of detecting skewed XCI is 97.9%.

[0481] 46. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, RP2, and TCAC1 , and wherein the sensitivity of detecting skewed XCI is 98.1%.

[0482] 47. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, RP2, and ZIC3, and wherein the sensitivity of detecting skewed XCI is 98.7%.

[0483] 48. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, TCAC1 , and ZIC3, and wherein the sensitivity of detecting skewed XCI is 97.4%.

[0484] 49. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of CNKSR2, RP2, and wherein TCAC1 , and the sensitivity of detecting skewed XCI is 96.7%.

[0485] 50. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of CNKSR2, RP2, and ZIC3, and wherein the sensitivity of detecting skewed XCI is 97.8%.

[0486] 51. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of CNKSR2, TCAC1 , and ZIC3, and wherein the sensitivity of detecting skewed XCI is 95.4%.

[0487] 52. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of RP2,P7621PC00

[0488] TCAC1 , and ZIC3, and wherein the sensitivity of detecting skewed XCI is 97.3%.

[0489] 53. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, RP2, and TCAC1 , and wherein the sensitivity of detecting skewed XCI is 99.3%.

[0490] 54. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, RP2, and ZIC3, and wherein the sensitivity of detecting skewed XCI is 99.5%.

[0491] 55. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, TCAC1 , and ZIC3, and wherein the sensitivity of detecting skewed XCI is 99.1%.

[0492] 56. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, RP2, TCAC1 , and ZIC3, and wherein the sensitivity of detecting skewed XCI is 99.5%.

[0493] 57. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of CNKSR2, RP2, TCAC1 , and ZIC3, and wherein the sensitivity of detecting skewed XCI is 99.0%.

[0494] 58. The method according to any one of the preceding items, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, RP2, TCAC1 , and ZIC3, and wherein the sensitivity of detecting skewed XCI is 99.8%.P7621PC00

[0495] 59. The method according to any one of the preceding items, wherein the PCR product starts at position 46836203 and ends at position 46836692 of the X- chromosome of the human reference genome GRCh38.p13 (RP2).

[0496] 60. The method according to any one of the preceding items, wherein the PCR product starts at position 137568039 and ends at position 137568606 of the X- chromosome of the human reference genome GRCh38.p13 (ZIC3).

[0497] 61. The method according to any one of the preceding items, wherein the PCR product starts at position 137545307 and ends at position 137545689 of the X- chromosome of the human reference genome GRCh38.p13 (TCAC1).

[0498] 62. The method according to any one of the preceding items, wherein the PCR product starts at position 11427654 and ends at position 11427890 of the X- chromosome of the human reference genome GRCh38.p13 (ARHGAP6).

[0499] 63. The method according to any one of the preceding items, wherein the PCR product starts at position 21374408 and ends at position 21374708 of the X- chromosome of the human reference genome GRCh38.p13 (CNKSR2).

[0500] 64. The method according to any one of the preceding items, wherein the PCR product comprises nucleotide repeats, such as AAAG, ATCT, TCAC, GAG or CAG repeats.

[0501] 65. The method according to item 64, wherein the PCR products comprise different numbers of nucleotide repeats in the maternal and paternal X-chromosome.

[0502] 66. The method according to any one of items 64 to 65, wherein the PCR product comprises at least one AAAG nucleotide repeat, such as at least two AAAG nucleotide repeats, such as at least five AAAG nucleotide repeats, such as at least 10 AAAG nucleotide repeats, such as at least 50 AAAG nucleotide repeats.

[0503] 67. The method according to any one of items 64 to 65, wherein the PCR product comprises at least one ATAC nucleotide repeat, such as at least two ATACP7621PC00

[0504] nucleotide repeats, such as at least five ATAC nucleotide repeats, such as at least 10 ATAC nucleotide repeats, such as at least 50 ATAC nucleotide repeats.

[0505] 68. The method according to any one of items 64 to 65, wherein the PCR product comprises at least one TCAC nucleotide repeat, such as at least two TCAC nucleotide repeats, such as at least five TCAC nucleotide repeats, such as at least 10 TCAC nucleotide repeats, such as at least 50 TCAC nucleotide repeats.

[0506] 69. The method according to any one of items 64 to 65, wherein the PCR product comprises at least one GAG nucleotide repeat, such as at least two GAG nucleotide repeats, such as at least five GAG nucleotide repeats, such as at least 10 GAG nucleotide repeats, such as at least 50 GAG nucleotide repeats.

[0507] 70. The method according to any one of items 64 to 65, wherein the PCR product comprises at least one CAG nucleotide repeat, such as at least two CAG nucleotide repeats, such as at least five CAG nucleotide repeats, such as at least 10 CAG nucleotide repeats, such as at least 50 CAG nucleotide repeats.

[0508] 71. The method according to any one of the preceding items, wherein the multiplex polymerase chain reaction of step c) is performed by amplifying all locus regions in one tube.

[0509] 72. The method according to any one of the preceding items, wherein the multiplex polymerase chain reaction of step c) is performed by using a DNA polymerase, such as a high fidelity Polymerase.

[0510] 73. The method according to any one of the preceding items, wherein the separation of the PCR products is performed by electrophoresis, such as capillary electrophoresis, conventional slab gel electrophoresis or microfluidic electrophoresis.

[0511] 74. The method according to any one of the preceding items, wherein the multiplex PCR assay is performed using fluorescently labeled primers and the resulting PCR products are detected via fluorescence-based capillary electrophoresis.P7621PC00

[0512] 75. The method according to any one of the preceding items, wherein the capillary length is at least 25 cm, such as at least 30 cm, such as at least 35 cm, such as at least 40 cm, such as at least 45 cm, such as at least 50 cm.

[0513] 76. The method according to any one of the preceding items, wherein an approximately equal amplification ratio of the same locus derived from the maternal and paternal X-chromosomes is a ratio of between 0.7:1.3 to 1.3:0.7, such as a ratio of between 0.8:1.2 to 1.2:0.8, such as ratio of between 0.9:1.1 to 1.1 :0.9, such as a ratio of 0.8:1.2, or 1.2:0.8, such as a ratio of 0.7:1.3, or 1.3:0.7.

[0514] 77. The method according to any one of the preceding items, wherein an approximately equal amplification ratio of the same locus derived from the maternal and paternal X-chromosomes is observed when 40 to 60% of the cells inactivate one X-chromosome and the corresponding 60 to 40% of the cells inactivate the other X-chromosome.

[0515] 78. The method according to any one of the preceding items, wherein a deviation from an approximately equal amplification ratio is a ratio of between 0.7:1.3 to 1.3:0.7, such as a ratio of between 0.6:1.4 to 1.4:0.6, such as a ratio of between 0.5:1.5 to 1.5:0.5, such as a ratio of between 0.4:1.6 to 1.6:0.4 such as a ratio of between 0.3:1.7 to 1.7:0.3, such as a ratio of between 0.2:1.8 to 1.8:0.2, such as a ratio of between 0.1 :1.9 to 1.9:0.1 , such as a ratio of between 0.0:2 to 2:0.0 of the same locus derived from the maternal and paternal X- chromosomes.

[0516] 79. The method according to any one of the preceding items, wherein a deviation from an approximately equal amplification ratio is observed when 100% of the cells inactivate one X-chromosome and 0% of the cells inactivate the other, such as that 95% of the cells inactivate one X-chromosome and 5% of the cells inactivate the other X-chromosome, such as that 90% of the cells inactivate one X-chromosome and 10% of the cells inactivate the other X-chromosome, such as that 85% of the cells inactivate one X-chromosome and 15% of the cells inactivate the other X-chromosome, such as that 80% of the cells inactivate one X-chromosome and 20% of the cells inactivate the other X-chromosome, suchP7621PC00

[0517] as that 75% of the cells inactivate one X-chromosome and 25% of the cells inactivate the other X-chromosome, such as that 78% of the cells inactivate one X-chromosome and 27% of the cells inactivate the other X-chromosome, such as that 30% of the cells inactivate one X-chromosome and 70% of the cells inactivate the other X-chromosome, such as that 32% of the cells inactivate one X-chromosome and 68% of the cells inactivate the other X-chromosome.

[0518] 80. A kit comprising: at least one, such as at least two, such as at least three, such as at least four, such as five primer sets, wherein the primer sets comprise i. a forward primer consisting of SEQ ID NO: 1 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 2 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or

[0519] ii. a forward primer consisting of SEQ ID NO: 3 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 4 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or

[0520] iii. a forward primer consisting of SEQ ID NO: 5 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 6 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or

[0521] iv. a forward primer consisting of SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or

[0522] v. a forward primer consisting of SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof; and

[0523] b. instructions for detecting skewed XCI.P7621PC00

[0524] 81. A kit comprising: at least one, such as at least two, such as at least three, such as at least four, such as five primer sets, wherein the primer sets comprise a forward primer consisting of SEQ ID NO: 1 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 2 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof; and

[0525] i. a forward primer consisting of SEQ ID NO: 3 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 4 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or

[0526] ii. a forward primer consisting of SEQ ID NO: 5 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 6 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or

[0527] iii. a forward primer consisting of SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / or

[0528] iv. a forward primer consisting of SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof; and

[0529] b. instructions for detecting skewed XCI.

[0530] 82. A kit for detecting skewed XCI comprising

[0531] a. At least one, such as at least two, such as at least three, such as at least four, such as five primer sets, wherein the primer sets comprise i. a forward primer and reverse primer, wherein the forward primer binds upstream of position 46836203 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 46836692 in the X-P7621PC00

[0532] chromosome of the human reference genome GRCh38.p13; and / or

[0533] ii. a forward primer and reverse primer, wherein the forward primer binds upstream of position 137568039 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 137568606 in the X- chromosome of the human reference genome GRCh38.p13; and / or

[0534] iii. a forward primer and reverse primer, wherein the forward primer binds upstream of position 137545307 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 137545689 in the X- chromosome of the human reference genome GRCh38.p13; and / or

[0535] iv. a forward primer and reverse primer, wherein the forward primer binds upstream of position 11427654 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 11427890 in the X- chromosome of the human reference genome GRCh38.p13; and / or

[0536] v. a forward primer and reverse primer, wherein the forward primer binds upstream of position 21374408 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 21374708 in the X- chromosome of the human reference genome GRCh38.p13; and b. instructions for use.

[0537] 83. A kit for detecting skewed XCI comprising

[0538] a. At least one, such as at least two, such as at least three, such as at least four, such as five primer sets, wherein the primer sets comprise a forward primer and reverse primer, wherein the forward primer binds upstream of position 11427654 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 11427890 in the X-chromosome of the human reference genome GRCh38.p13; andP7621PC00

[0539] i. a forward primer and reverse primer, wherein the forward primer binds upstream of position 46836203 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 46836692 in the X- chromosome of the human reference genome GRCh38.p13; and / or

[0540] ii. a forward primer and reverse primer, wherein the forward primer binds upstream of position 137568039 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 137568606 in the X- chromosome of the human reference genome GRCh38.p13; and / or

[0541] iii. a forward primer and reverse primer, wherein the forward primer binds upstream of position 137545307 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 137545689 in the X- chromosome of the human reference genome GRCh38.p13; and / or

[0542] iv. a forward primer and reverse primer, wherein the forward primer binds upstream of position 21374408 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 21374708 in the X- chromosome of the human reference genome GRCh38.p13; and b. instructions for use.

[0543] 84. The kit according to any one of items 77 to 82, wherein the kit comprises further a methylation-sensitive restriction enzyme, such as Hpall.

[0544] 85. The kit according to any one of items 7777 to 84, wherein the kit comprises further a DNA polymerase, such as a high fidelity polymerase.

[0545] 86. The kit according to item 84, wherein the kit comprises further digestion buffer, such as a digestion buffer that facilitates enzymatic digestions of DNA by said methylation-sensitive restriction enzyme.P7621PC00

[0546] 87. The kit according to item 85, wherein the kit further comprises PCR buffer, such as a PCR buffer that facilitates activity of said DNA polymerase.

[0547] 88. The kit according to any one of items 77 to 87, wherein the kit further comprises control sample, such as a sample comprising DNA with skewed XCI.

[0548] 89. The kit according to any one of items 77 to 88, wherein the forward or reverse primer comprises further one or more chemical, structural, or functional modifications, such as fluorophores, quenchers, affinity tags, or other chemical modifications that enhance stability, specificity, or detection.

[0549] 90. A method for diagnosing a disorder associated with skewed X-chromosome inactivation (XCI) in a female subject, comprising:

[0550] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;

[0551] b. contacting said nucleic acid sample with a methylation-sensitive restriction enzyme to obtain a treated nucleic acid sample;

[0552] c. amplifying said treated nucleic acid sample via PCR using a primer set comprising primers specific for a least one repeat-containing region of at least one locus located on the X-chromosome, wherein the locus is ARHGAP6, ZIC3, CNKSR2, TCAC1 , or RP2, thereby obtaining at least one PCR product comprising a repeat-containing region of one of said loci;

[0553] d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis;

[0554] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the two X-chromosomes indicates skewed XCI; and

[0555] f. correlating the presence of skewed XCI with an increased risk of or presence of an X-linked disorder.

[0556] 91. A method for diagnosing a disorder associated with skewed X-chromosome inactivation (XCI) in a female subject, comprising:P7621PC00

[0557] a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;

[0558] b. contacting said nucleic acid sample with a methylation-sensitive restriction enzyme to obtain a treated nucleic acid sample;

[0559] c. amplifying said treated nucleic acid sample via PCR using a primer set comprising primers specific for a least one repeat-containing region of at least one locus located on the X-chromosome, wherein the locus is i. ARHGAP6; and

[0560] ii. ZIC3, CNKSR2, TCAC1 , or RP2, thereby obtaining at least one PCR product comprising a repeat-containing region of one of said loci;

[0561] d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis;

[0562] e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the two X-chromosomes indicates skewed XCI; and

[0563] f. correlating the presence of skewed XCI with an increased risk of or presence of an X-linked disorder.

[0564] 92. The method according to any one of items 90 to 91 , wherein the disorder associated with skewed X-chromosome inactivation (XCI) is Duchenne Muscular Dystrophy (DMD), X-Linked Severe Combined Immunodeficiency (X- SCID), X-Linked Adrenoleukodystrophy (ALD), Hemophilia A & B, Rett Syndrome, Hunter Syndrome (MPS II), Wiskott-Aldrich Syndrome (WAS), Menkes Disease, Lesch-Nyhan Syndrome, Fabry Disease (Severe Form), Kennedy's Disease (SBMA) or Charcot-Marie-Tooth Disease (CMTX1).

[0565] 93. The method according to any one of items 90 to 92, wherein the female subjects shows symptoms, such as progressive muscle weakness, loss of ambulation, respiratory & cardiac failure, absent immune function, severe infections, failure to thrive, neurological deterioration, loss of motor function, adrenal insufficiency, uncontrolled bleeding, joint damage, internal hemorrhages, severe cognitive & motor impairment, breathing abnormalities,P7621PC00

[0566] seizures, progressive organ damage, skeletal abnormalities, cognitive decline, immune deficiency, bleeding disorders, eczema, increased cancer risk, copper transport disorder, neurological degeneration, failure to thrive, self-mutilation, severe movement disorders, kidney issues, severe pain, kidney failure, stroke risk, cardiac disease, swallowing difficulties, hormonal abnormalities, progressive nerve damage, muscle weakness, foot deformities or sensory loss.

Claims

P7621PC00Claims1. A method for detecting skewed X-chromosome inactivation (XCI) in a female subject, comprising:a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;b. digesting the nucleic acid sample with a methylation-sensitive restriction enzyme thereby obtaining a treated nucleic acid sample; c. amplifying said treated nucleic acid sample via polymerase chain reaction (PCR) using a primer set comprising primers specific for at least one repeat-containing region of at least one locus located on the X-chromosome, wherein the locus is ARHGAP6, thereby obtaining at least one PCR product comprising a repeat-containing region of one of said loci;d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis; ande. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the maternal and paternal X-chromosomes indicates skewed XCI.

2. A method for detecting skewed X-chromosome inactivation (XCI) in a female subject, comprising:a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;b. digesting the nucleic acid sample with a methylation-sensitive restriction enzyme thereby obtaining a treated nucleic acid sample; c. amplifying said treated nucleic acid sample via multiplex polymerase chain reaction (PCR) using a primer set comprising primers specific for at least two repeat-containing region of at least two loci located on the X-chromosome, wherein the loci arei. ARHGAP6; andii. ZIC3, CNKSR2, TCAC1 , or RP2,P7621PC00thereby obtaining at least two PCR product comprising a repeat-containing region of two of said loci;d. separating the resulting PCR products by electrophoresis, such as capillary electrophoresis; ande. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PCR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the maternal and paternal X-chromosomes indicates skewed XCI.

3. The method according to claim 1 , wherein in step c) repeat-containing regions of at least two, such as at least three, such as at least four, such as five of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci are amplified via multiplex PCR.

4. The method according to claim 1 , wherein in step c) repeat-containing regions at least two, such as at least three, such as at least four, such as five of the ARHGAP6, ZIC3, CNKSR2, TCAC1, and RP2 loci are amplified sequentially.

5. The method according to any one of the preceding claims, wherein the methylation-sensitive restriction enzyme is Hpall.

6. The method according to any one of the preceding claims, wherein the nucleic acid sample is a DNA sample.

7. The method according to any one of the preceding claims, wherein the nucleic acid sample is genomic DNA extracted from a human tissue sample, such as blood, saliva, urine, hair, tissue or biopsy samples.

8. The method according to any one of the preceding claims, wherein the nucleic acid sample is genomic DNA extracted from brain, colon, esophagus, kidney, spleen, nerve, adipose, artery, uterus, pituitary, lung, muscle, heart, thyroid, small intestine, salivary gland, stomach, ovary, vagina, breast, skin, pancreas, liver, blood or adrenal gland tissue.P7621PC009. The method according to any one of the preceding claims, wherein the plurality of cells comprises at least 100 cells, such as at least 500 cells, such as at least 1000 cells, such as at least 10000 cells, such as at least 100000 cells.

10. The method according to any one of the preceding claims, wherein the nucleic acid sample is a DNA sample.

11. The method according to any one of the preceding claims, wherein the methylation-sensitive restriction enzyme is Hpall.

12. The method according to any one of the preceding claims, wherein the nucleic acid amount is at least 5 ng, such as at least 7.5 ng, such as at least 10 ng per reaction.

13. The method according to any one of the preceding claims, wherein the nucleic acid amount is at most 100 ng, such as at most 90 ng, such as at most 80 ng, such as at most 70 ng, such as at most 60 ng, such as at most 50 ng, such as at most 40 ng, such as at most 30 ng, such as at most 20 ng, such as at most 15 ng, such as at most 10 ng per reaction.

14. The method according to any one of the preceding claims, wherein the contacting of the nucleic acid sample with the methylation-sensitive restriction enzyme in step b) is conducted at least 25°C, such as at least 30°C, such as at least 35°C, preferably 37°C for an incubation period of at least 30 min, such as at least 45 min, such as at least 50 min, preferably 1 hour.

15. The method according to any one of the preceding claims, wherein the method further comprises a step of inactivating the methylation-sensitive restriction enzyme between step b) and step c).

16. The method according to claim 15, wherein the methylation-sensitive restriction enzyme is inactivated by exposure to a temperature of at least 70°C, such as at least 75°C, preferably at 80°C.P7621PC0017. The method according to any one of claims 15 to 16, wherein the methylationsensitive restriction enzyme is exposed to a temperature of at least 70°C, such as at least 75°C, preferably at 80°C for at least 10 minutes, such as for at least 15 minutes, preferably for 20 minutes.

18. The method according to any one of the preceding claims, wherein the primer set comprises a forward primer consisting of SEQ ID NO: 1 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 2 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof; and / orwherein the primer set comprises a forward primer consisting of SEQ ID NO: 3 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 4 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof; and / orwherein the primer set comprises a forward primer consisting of SEQ ID NO: 5 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 6 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof; and / orwherein the primer set comprises a forward primer consisting of SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof; and / orwherein the primer set comprises a forward primer consisting of SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof.

19. The method according to claim 18, wherein the forward or reverse primer comprises further one or more chemical, structural, or functional modifications, such as fluorophores, quenchers, affinity tags, or other chemical modifications that enhance stability, specificity, or detection.P7621PC0020. The method according to claim 18, wherein the one or two nucleotide substitutions or deletions in the forward primer and / or reverse primer are located in one or two of the first two nucleotides at the 5' end of the primer.

21. The method according to claim 18, wherein the one or two nucleotide substitutions or deletions in the forward primer and / or reverse primer are located in one or two of the first two nucleotides at the 3' end of the primer.

22. The method according to any one of claims 18 to 21 , wherein the forward primer and / or reverse primer comprises one or two additional nucleotides at the 5' end.

23. The method according to any one of claims 18 to 22, wherein the forward primer and / or reverse primer comprises one or two additional nucleotides at the 3' end.

24. The method according to any one of claims 18 to 23, wherein the forward primer and / or reverse primer comprises only a single nucleotide substitution, deletion, or addition.

25. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for at least two distinct repeat-containing regions on at least two of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci, such as on:a. ARHGAP6 and ZIC3;b. ARHGAP6 and CNKSR2;c. ARHGAP6 and TCAC1 ;d. ARHGAP6 and RP2;e. ZIC3 and CNKSR2;f. ZIC3 and TCAC1 ;g. ZIC3 and RP2;h. CNKSR2 and TCAC1 ;i. CNKSR2 and RP2; orj. TCAC1 and RP2.P7621PC0026. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for at least three distinct repeat-containing regions on at least three of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci, such as on:a. ARHGAP6, ZIC3, and CNKSR2;b. ARHGAP6, ZIC3, and TCAC1 ;c. ARHGAP6, ZIC3, and RP2;d. ARHGAP6, CNKSR2, and TCAC1 ;e. ARHGAP6, CNKSR2, and RP2;f. ARHGAP6, TCAC1 , and RP2;g. ZIC3, CNKSR2, and TCAC1 ;h. ZIC3, CNKSR2, and RP2;i. ZIC3, TCAC1, and RP2; orj. CNKSR2, TCAC1 , and RP2.

27. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for at least four distinct repeat-containing regions on at least four of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci, such as on:a. ARHGAP6, ZIC3, CNKSR2, and TCAC1 ;b. ARHGAP6, ZIC3, CNKSR2, and RP2;c. ARHGAP6, ZIC3, TCAC1 , and RP2;d. ARHGAP6, CNKSR2, TCAC1 , and RP2; ore. ZIC3, CNKSR2, TCAC1 , and RP2.

28. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for five distinct repeat-containing regions on each of the ARHGAP6, ZIC3, CNKSR2, TCAC1 , and RP2 loci.

29. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6 and CNKSR2, and wherein the sensitivity of detecting skewed XCI is 92.8%.P7621PC0030. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6 and RP2, and wherein the sensitivity of detecting skewed XCI is 95.7%.

31. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6 and TCAC1 , and wherein the sensitivity of detecting skewed XCI is 91.2%.

32. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6 and ZIC3, and wherein the sensitivity of detecting skewed XCI is 94.1%.

33. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of CNKSR2 and RP2, and wherein the sensitivity of detecting skewed XCI is 92.3%.

34. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of CNKSR2 and TCAC1 , and wherein the sensitivity of detecting skewed XCI is 84.4%.

35. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of CNKSR2 and ZIC3, and wherein the sensitivity of detecting skewed XCI is 89.5%.

36. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of RP2 and TCAC1 , and wherein the sensitivity of detecting skewed XCI is 90.7%.

37. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of RP2 and ZIC3, and wherein the sensitivity of detecting skewed XCI is 93.7%.

38. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of TCAC1 and ZIC3, and wherein the sensitivity of detecting skewed XCI is 87.3%.P7621PC0039. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, and RP2, and wherein the sensitivity of detecting skewed XCI is 98.5%.

40. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, and TCAC1 , and wherein the sensitivity of detecting skewed XCI is 96.9%.

41. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, and ZIC3, and wherein the sensitivity of detecting skewed XCI is 97.9%.

42. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, RP2, and TCAC1 , and wherein the sensitivity of detecting skewed XCI is 98.1%.

43. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, RP2, and ZIC3, and wherein the sensitivity of detecting skewed XCI is 98.7%.

44. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, TCAC1 , and ZIC3, and wherein the sensitivity of detecting skewed XCI is 97.4%.

45. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of CNKSR2, RP2, and wherein TCAC1 , and the sensitivity of detecting skewed XCI is 96.7%.P7621PC0046. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of CNKSR2, RP2, and ZIC3, and wherein the sensitivity of detecting skewed XCI is 97.8%.

47. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of CNKSR2, TCAC1 , and ZIC3, and wherein the sensitivity of detecting skewed XCI is 95.4%.

48. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of RP2, TCAC1 , and ZIC3, and wherein the sensitivity of detecting skewed XCI is 97.3%.

49. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, RP2, and TCAC1 , and wherein the sensitivity of detecting skewed XCI is 99.3%.

50. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, RP2, and ZIC3, and wherein the sensitivity of detecting skewed XCI is 99.5%.

51. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, TCAC1 , and ZIC3, and wherein the sensitivity of detecting skewed XCI is 99.1%.

52. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, RP2, TCAC1 , and ZIC3, and wherein the sensitivity of detecting skewed XCI is 99.5%.80P7621PC0053. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of CNKSR2, RP2, TCAC1 , and ZIC3, and wherein the sensitivity of detecting skewed XCI is 99.0%.

54. The method according to any one of the preceding claims, wherein the primer set comprises primers specific for the repeat-containing regions of ARHGAP6, CNKSR2, RP2, TCAC1 , and ZIC3, and wherein the sensitivity of detecting skewed XCI is 99.8%.

55. The method according to any one of the preceding claims, wherein the PCR product starts at position 46836203 and ends at position 46836692 of the X- chromosome of the human reference genome GRCh38.p13 (RP2).

56. The method according to any one of the preceding claims, wherein the PCR product starts at position 137568039 and ends at position 137568606 of the X- chromosome of the human reference genome GRCh38.p13 (ZIC3).

57. The method according to any one of the preceding claims, wherein the PCR product starts at position 137545307 and ends at position 137545689 of the X- chromosome of the human reference genome GRCh38.p13 (TCAC1).

58. The method according to any one of the preceding claims, wherein the PCR product starts at position 11427654 and ends at position 11427890 of the X- chromosome of the human reference genome GRCh38.p13 (ARHGAP6).

59. The method according to any one of the preceding claims, wherein the PCR product starts at position 21374408 and ends at position 21374708 of the X- chromosome of the human reference genome GRCh38.p13 (CNKSR2).

60. The method according to any one of the preceding claims, wherein the PCR product comprises nucleotide repeats, such as AAAG, ATCT, TCAC, GAG or CAG repeats, and wherein the PCR products comprise different numbers of nucleotide repeats in the maternal and paternal X-chromosomes.81P7621PC0061. The method according to claim 60, wherein the PCR product comprises at least one AAAG nucleotide repeat, such as at least two AAAG nucleotide repeats, such as at least five AAAG nucleotide repeats, such as at least 10 AAAG nucleotide repeats, such as at least 50 AAAG nucleotide repeats.

62. The method according to any one of claims 60 to 61 , wherein the PCR product comprises at least one ATAC nucleotide repeat, such as at least two ATAC nucleotide repeats, such as at least five ATAC nucleotide repeats, such as at least 10 ATAC nucleotide repeats, such as at least 50 ATAC nucleotide repeats.

63. The method according to any one of claims 60 to 62, wherein the PCR product comprises at least one TCAC nucleotide repeat, such as at least two TCAC nucleotide repeats, such as at least five TCAC nucleotide repeats, such as at least 10 TCAC nucleotide repeats, such as at least 50 TCAC nucleotide repeats.

64. The method according to any one of claims 60 to 63, wherein the PCR product comprises at least one GAG nucleotide repeat, such as at least two GAG nucleotide repeats, such as at least five GAG nucleotide repeats, such as at least 10 GAG nucleotide repeats, such as at least 50 GAG nucleotide repeats.

65. The method according to any one of claims 60 to 64, wherein the PCR product comprises at least one CAG nucleotide repeat, such as at least two CAG nucleotide repeats, such as at least five CAG nucleotide repeats, such as at least 10 CAG nucleotide repeats, such as at least 50 CAG nucleotide repeats.

66. The method according to any one of the preceding claims, wherein the multiplex polymerase chain reaction of step c) is performed by amplifying all locus regions in one tube.

67. The method according to any one of the preceding claims, wherein the multiplex polymerase chain reaction of step c) is performed by using a DNA polymerase, such as a high fidelity Polymerase.82P7621PC0068. The method according to any one of the preceding claims, wherein the separation of the PCR products is performed by electrophoresis, such as capillary electrophoresis, conventional slab gel electrophoresis or microfluidic electrophoresis.

69. The method according to any one of the preceding claims, wherein the multiplex PCR assay is performed using fluorescently labeled primers and the resulting PCR products are detected via fluorescence-based capillary electrophoresis.

70. The method according to any one of the preceding claims, wherein the capillary length is at least 25 cm, such as at least 30 cm, such as at least 35 cm, such as at least 40 cm, such as at least 45 cm, such as at least 50 cm.

71. The method according to any one of the preceding claims, wherein an approximately equal amplification ratio of the same locus derived from the maternal and paternal X-chromosomes is a ratio of between 0.7:1.3 to 1.3:0.7, such as a ratio of between 0.8:1.2 to 1.2:0.8, such as ratio of between 0.9:1.1 to 1.1 :0.9, such as a ratio of 0.8:1.2, or 1.2:0.8, such as a ratio of 0.7:1.3, or 1.3:0.7, such as wherein an approximately equal amplification ratio of the same locus derived from the maternal and paternal X-chromosomes is observed when 40 to 60% of the cells inactivate one X-chromosome and the corresponding 60 to 40% of the cells inactivate the other X-chromosome.

72. The method according to any one of the preceding claims, wherein a deviation from an approximately equal amplification ratio is a ratio of between 0.7:1.3 to 1.3:0.7, such as a ratio of between 0.6:1.4 to 1.4:0.6, such as a ratio of between 0.5:1.5 to 1.5:0.5, such as a ratio of between 0.4:1.6 to 1.6:0.4 such as a ratio of between 0.3:1.7 to 1.7:0.3, such as a ratio of between 0.2:1.8 to 1.8:0.2, such as a ratio of between 0.1 :1.9 to 1.9:0.1 , such as a ratio of between 0.0:2 to 2:0.0 of the same locus derived from the maternal and paternal X- chromosomes; such as wherein a deviation from an approximately equal amplification ratio is observed when 100% of the cells inactivate one X- chromosome and 0% of the cells inactivate the other, such as that 95% of the cells inactivate one X-chromosome and 5% of the cells inactivate the other X-83P7621PC00chromosome, such as that 90% of the cells inactivate one X-chromosome and 10% of the cells inactivate the other X-chromosome, such as that 85% of the cells inactivate one X-chromosome and 15% of the cells inactivate the other X- chromosome, such as that 80% of the cells inactivate one X-chromosome and 20% of the cells inactivate the other X-chromosome, such as that 75% of the cells inactivate one X-chromosome and 25% of the cells inactivate the other X- chromosome, such as that 78% of the cells inactivate one X-chromosome and 27% of the cells inactivate the other X-chromosome, such as that 30% of the cells inactivate one X-chromosome and 70% of the cells inactivate the other X- chromosome, such as that 32% of the cells inactivate one X-chromosome and 68% of the cells inactivate the other X-chromosome.

73. A kit comprising: at least one, such as at least two, such as at least three, such as at least four, such as five primer sets, wherein the primer sets comprise a forward primer consisting of SEQ ID NO: 1 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 2 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof; andi. a forward primer consisting of SEQ ID NO: 3 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 4 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / orii. a forward primer consisting of SEQ ID NO: 5 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 6 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / oriii. a forward primer consisting of SEQ ID NO: 7 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and a reverse primer consisting of SEQ ID NO: 8 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof, and / oriv. a forward primer consisting of SEQ ID NO: 9 or a sequence having one or two nucleotide substitutions, deletions, or84P7621PC00additions thereof, and a reverse primer consisting of SEQ ID NO: 10 or a sequence having one or two nucleotide substitutions, deletions, or additions thereof; andb. instructions for detecting skewed XCI.

74. A kit for detecting skewed XCI comprisinga. At least one, such as at least two, such as at least three, such as at least four, such as five primer sets, wherein the primer sets comprise a forward primer and reverse primer, wherein the forward primer binds upstream of position 11427654 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 11427890 in the X-chromosome of the human reference genome GRCh38.p13; andi. a forward primer and reverse primer, wherein the forward primer binds upstream of position 46836203 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 46836692 in the X- chromosome of the human reference genome GRCh38.p13; and / orii. a forward primer and reverse primer, wherein the forward primer binds upstream of position 137568039 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 137568606 in the X- chromosome of the human reference genome GRCh38.p13; and / oriii. a forward primer and reverse primer, wherein the forward primer binds upstream of position 137545307 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse primer binds downstream of position 137545689 in the X- chromosome of the human reference genome GRCh38.p13; and / oriv. a forward primer and reverse primer, wherein the forward primer binds upstream of position 21374408 in the X-chromosome of the human reference genome GRCh38.p13 and the reverse85P7621PC00primer binds downstream of position 21374708 in the X- chromosome of the human reference genome GRCh38.p13; and b. instructions for use.

75. A method for diagnosing a disorder associated with skewed X-chromosome inactivation (XCI) in a female subject, comprising:a. providing a nucleic acid sample from the female subject, the sample comprising the paternal and maternal X-chromosomes of a female subject of a plurality of cells;b. contacting said nucleic acid sample with a methylation-sensitive restriction enzyme to obtain a treated nucleic acid sample;c. amplifying said treated nucleic acid sample via PGR using a primer set comprising primers specific for a least one repeat-containing region of at least one locus located on the X-chromosome, wherein the locus is i. ARHGAP6; andii. ZIC3, CNKSR2, TCAC1 , or RP2, thereby obtaining at least one PGR product comprising a repeat-containing region of one of said loci;d. separating the resulting PGR products by electrophoresis, such as capillary electrophoresis;e. determining the presence of skewed X-chromosome inactivation based on the relative abundance of the amplified PGR products, wherein a deviation from a 1 :1 amplification ratio of the same locus derived from the two X-chromosomes indicates skewed XCI; andf. correlating the presence of skewed XCI with an increased risk of or presence of an X-linked disorder.

76. The method according to claim 75, wherein the disorder associated with skewed X-chromosome inactivation (XCI) is Duchenne Muscular Dystrophy (DMD), X-Linked Severe Combined Immunodeficiency (X-SCID), X-Linked Adrenoleukodystrophy (ALD), Hemophilia A & B, Rett Syndrome, Hunter Syndrome (MPS II), Wiskott-Aldrich Syndrome (WAS), Menkes Disease, Lesch- Nyhan Syndrome, Fabry Disease (Severe Form), Kennedy's Disease (SBMA) or Charcot-Marie-Tooth Disease (CMTX1).

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