Method for making genetic determinations based on hair root samples - Patents.com

Abstract

The present invention relates to a method for carrying out genetic determinations based on hair root samples, to a kit suitable for carrying out said method, and to the use of hair root samples obtained from test individuals for carrying out genetic determinations.

Description

[Technical field]

[0001] The present invention relates to a method for carrying out genetic determinations based on hair root samples, to a kit suitable for carrying out said method, and to the use of hair root samples obtained from test individuals for carrying out genetic determinations.

[0002] The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to a method for making genetic determinations based on a biological sample. [Background technology]

[0003] Biological samples (e.g., patient samples) are routinely used to provide diagnostic, prognostic or other information related to genetic phenomena. Typically, the nucleic acids contained in the biological sample are utilized to provide these information or measurements.

[0004] When using the genome or DNA of a test individual, problems arise in that post-transcriptional phenomena cannot be detected or cannot be detected sufficiently, resulting in incorrect test results and making the significance of the test relative.

[0005] The use of RNA in so-called gene expression analysis can address these issues, since it allows the detection of alterations in transcriptional and post-transcriptional processes. However, the expression pattern depends on the type of tissue contained in the biological sample and the cells that compose this tissue. Cells usually express only a very limited set of genes, i.e. only those that are required for each of the cellular functions. Therefore, the genetic profile of an entire organism or of disease-related genomic alterations, which for example only affect certain genes that are not expressed in the biological sample analyzed, can only yield very limited or no conclusions using RNA-based methods.

[0006] Moreover, the quality of DNA and RNA in commonly used blood samples deteriorates rapidly, necessitating the use of stabilization systems such as PAXgene®, which adds additional costs and organizational efforts. Moreover, blood samples contain a very high amount of globin mRNA, which reduces the sequencing yield of the genes of interest.

[0007] Furthermore, current methods for obtaining tissue for gene expression analysis are invasive, involving venipuncture or surgical biopsy, and carry risks to subjects undergoing such procedures, including trauma, hematoma, infection, and death.

[0008] WO2005 / 121374 discloses a method for measuring gene expression, in which RNA is isolated from hair follicles. In this known method, the RNA obtained from hair follicles is analyzed using a microarray. However, this known method only quantifies RNA, and does not perform any nucleic acid analysis test, which may be necessary to detect genetic changes or rare genetic variants.

[0009] The object of the present invention is therefore to provide a method for carrying out genetic determinations from biological samples, which makes it possible to avoid or at least reduce the drawbacks of the prior art, in particular, for example, to provide a method which reduces the stress of the subject, is as harmless as possible to the subject and makes it possible to give reliable reports on genetic phenomena such as predisposition to rare genetic diseases or the development of cancer.

[0010] The present invention fulfills these and other needs. Summary of the Invention [Means for solving the problem]

[0011] The problem underlying the present invention is a method for carrying out a genetic determination, comprising the steps of: a) obtaining a hair root sample from a test individual; b) isolating nucleic acids from said hair root sample; c) determining the nucleotide sequence of the nucleic acid to obtain a test nucleotide sequence; d) comparing the test nucleotide sequence to a reference nucleotide sequence; and e) making a genetic determination based on a comparison of the test nucleotide sequence with a reference nucleotide sequence. The problem is solved by providing a method comprising the steps of:

[0012] The problem of the present invention is also solved by the use of a hair root sample from a test individual for carrying out a genetic determination based on nucleotide sequencing of nucleic acid isolated from the hair root sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] According to the present invention, a "hair root sample" is a biological sample that contains a hair root or contains keratinocytes contained in a hair root or keratinocytes derived from a hair root. In one embodiment of the present invention, the hair root sample is hair or body hair obtained from a test individual, such as a human or an animal. In a further embodiment, one hair or one hair root is sufficient, and preferably two, three, four, five, six, seven, eight, nine or ten hairs or two, three, four, five, six, seven, eight, nine or ten hair roots obtained from a test individual. Advantageously, the hair or hair root can be obtained from the test individual by a non-invasive method, for example by plucking the hair. Hair root samples can also be obtained by flash freezing and then thawing or cooling a hair root sample, for example hair root samples stored in a biobank. Hair root samples, unlike other tissue samples such as blood samples, are very stable and can be stored for long periods even at room temperature, can be easily frozen and / or can be stored in biobanks for long periods, and due to their very small size compared to other biological samples, hair root samples are very space efficient and can be stored more cheaply and easily.

[0014] According to the present invention, "nucleic acid" includes any kind of nucleic acid material, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), etc. Nucleic acids are isolated from hair root samples by methods known to those skilled in the art.

[0015] The term "determining the nucleotide sequence" in step (c) of the method of the present invention, or equivalently "sequencing", refers to elucidating the nucleotide sequence of an isolated nucleic acid, specifically the nucleotide sequence of the entire isolated nucleic acid (e.g., the whole genome or the whole transcriptome), or a part thereof (i.e., the sequence of interest; for DNA, e.g., the whole exome or a panel of any number of genes obtained by oligohybridization or amplicon-based methods; for RNA, e.g., a part of the transcriptome, such as polyA mRNA, microRNA, total RNA), or the entire isolated nucleic acid. Sequencing is performed by methods well known in the art, including long-read sequencing of DNA and RNA (e.g., sequencing using PacBio's SMRT or ONT's Nanopore systems), and short-read sequencing (i.e., sequencing using sequencing-by-synthesis systems such as Illumina's NovaSeq). Furthermore, the method of the present invention is the first to report that high-throughput sequencing of RNA and DNA can be utilized and even performed simultaneously on a single sample.

[0016] The nucleotide sequence obtained from the test individual's hair root sample by sequencing or nucleotide sequence determination is referred to as the "test nucleotide sequence."

[0017] The "comparison" of a test nucleotide sequence with a reference sequence performed in step (d) of the method of the present invention refers to a general alignment operation performed to detect nucleotide matches and mismatches between aligned sequences.

[0018] "Reference nucleotide sequence" is a nucleotide sequence that is not derived from the nucleic acid isolated from hair root sample, but from other sources.For example, in one embodiment, "reference nucleotide sequence" is a nucleotide sequence derived from nucleic acid obtained from one or more other individuals, such as an individual suffering from a genetic disease or a healthy reference individual, a parent of a child, or an individual who is the child of a putative father, and the like.In another embodiment, "reference nucleotide sequence" is a nucleotide sequence derived from a human reference genome, such as GRCh38, available from the Genome Reference Consortium.

[0019] According to the present invention, the "genetic determination" described in step (e) of the method of the present invention means providing results useful for prognosis, specifically including diagnosis, prognosis, predisposition information (particularly information regarding disease predisposition), paternal information, etc.

[0020] The problem underlying the present invention can be completely solved by the disclosure herein.

[0021] The inventors have found that hair root samples are ideal sources of genetic material for reliable genetic determinations, such as the diagnosis of genetic diseases. The inventors have found that a large number of disease-related genes are expressed in hair root samples or keratinocytes derived therefrom, in contrast to other samples, such as blood samples or saliva samples. As found by the inventors, about 25% more disease-related genes are expressed in hair root samples or keratinocytes derived therefrom than in blood samples. About 5% of known disease-related genes are expressed only in hair root samples or keratinocytes derived therefrom, but not in blood cells or fibroblasts. Thus, the method according to the present invention allows for the diagnosis of hundreds of genes that are not expressed in, for example, peripheral blood mononuclear cells (PBMCs) or fibroblasts used in the prior art.

[0022] Moreover, hair root samples can be easily obtained in a non-invasive manner, for example by plucking a hair, without the need for blood sampling or skin punching. Furthermore, the method according to the present invention is suitable for direct use by consumers themselves, since it can be performed simply by taking a hair sample, and does not require a qualified physician. This feature allows tests that previously required a physician to take a sample to be performed in daily life.

[0023] Furthermore, hair root samples are smaller than other samples, such as blood samples, which makes them advantageous for storage in biobanks.

[0024] Furthermore, hair root samples can be stable for long periods of time during transportation even under adverse conditions, do not require cryopreservation, and are not as sensitive to shock as, for example, blood samples.

[0025] Furthermore, hair root samples allow for propagation of sample material using in vitro keratinocyte cultures (multiple passages are possible).

[0026] In one embodiment of the invention, the nucleic acid is deoxyribonucleic acid (DNA).

[0027] By using DNA, genetic alterations in the genome can be detected. Moreover, this approach has the advantage of using highly stable nucleic acids. Another advantage is that it allows the use of widely established methods for nucleic acid isolation and sequencing. Compared to DNA obtained from other samples, such as nasal and buccal swabs, FFPE material, and archived blood samples, DNA isolated from hair root samples is of significantly higher quality.

[0028] In one embodiment of the invention, the nucleic acid is ribonucleic acid (RNA).

[0029] By using RNA, it is possible to detect diseases that cannot be detected by classical DNA-based diagnostic methods. This is based on the fact that in many diseases, genetic changes cannot be observed or reliably interpreted at the DNA level, but appear at the RNA level during or after transcription. Currently, genetic changes causing rare genetic diseases can only be detected in less than 40% of cases (varies depending on the type of disease) by DNA-based diagnostic methods alone. On the other hand, this embodiment can detect various disease-causing changes that were previously difficult to detect in patients, such as splicing and regulatory variants, loss or gain of gene expression, allelic imbalance and nonsense-mediated decay. Furthermore, by using RNA, it is possible to evaluate the effect of complex structural variants and deep intronic variants that can rarely cause splice abnormalities but are difficult to interpret at the DNA level.

[0030] In another embodiment of the method of the present invention, said nucleic acid is RNA and DNA.

[0031] By identifying genetic alterations in the genome or DNA of a test individual, DNA sequencing can be combined with RNA sequencing to further detect splicing and dysregulation, monoallelic gene expression (i.e., loss of expression of one of the two alleles of a gene), "loss of function" (i.e., complete or severe loss of gene expression), or overexpression (e.g., due to local duplications or even chromosomal trisomies). By performing RNA sequencing in parallel, genetic alterations found on the DNA, such as complex structural variants (e.g., variants identified by short or long reads detected by nanopore sequencing) and deep intronic variants, can be evaluated and more deeply interpreted. It is estimated that by utilizing this embodiment, detectable cases can be increased by up to 20%, which corresponds to 30 million patients worldwide and 3 million patients in the European Union.

[0032] In yet another embodiment, in step (c) of the method of the invention, RNA sequencing and / or DNA sequencing is performed.

[0033] This approach has the advantage that well-established methods can be used to determine the nucleotide sequence of said nucleic acid, such as high-throughput sequencing, next-generation sequencing (NGS), third-generation sequencing (single-molecule sequencing, long-read sequencing), nanopore sequencing, single-cell sequencing, etc.

[0034] In one embodiment of the invention, the DNA sequencing comprises epigenomic sequencing, which preferably provides information regarding the methylation of the DNA.

[0035] The epigenome consists of a record of chemical changes in the DNA and histone proteins of an organism, which can be passed on to the organism's descendants via a standard transgenerational epigenome. Changes in the epigenome can result in changes in chromatin structure and in genome function. The epigenome is involved in the regulation of gene expression, development, tissue differentiation, and the suppression of transposable elements. Whereas the genome underlying the epigenome is largely kept static in an individual, the epigenome can change dynamically depending on environmental conditions. Thus, this method according to the invention has the advantage of being able to determine in a test individual genetic changes associated with epigenetic phenomena such as DNA methylation, or acetylation and methylation of histones or accessible open chromatin regions. Epigenome sequencing can be performed using methods well known in the art, including whole genome bisulfite sequencing (WGBS), targeted capture sequencing, methylation capture sequencing, chromatin immunoprecipitation (ChIP) sequencing, DNAsel-seq, ATAC-seq, and the like.

[0036] In one embodiment of the method of the present invention, after step (a) and before step (b), (a') exposing the hair root sample to a liquid that causes lysis of the cells and release of nucleic acids from the cells. Implemented the following: In step (b), nucleic acids are isolated from the liquid.

[0037] This procedure has the advantage of ensuring the release of the nucleic acid for the next isolation step.In this embodiment, a lysis buffer may be used, which is well known to those skilled in the art.

[0038] In yet another embodiment of the method of the present invention, after step (a) and before step (b), a'') Culturing keratinocytes contained in a hair root sample Implemented the following: In step (b), nucleic acid is isolated from the cultured keratinocytes or their progeny.

[0039] This method advantageously allows unlimited growth of sample material for applications where extremely large amounts of DNA or RNA are required, such as nanopore sequencing or PacBio's long-read sequencing. Furthermore, this method allows the detection of various disease-causing alterations, such as splicing and regulatory variants, loss of gene expression, and complex structural variants, which were previously difficult to detect in patients. Furthermore, from one cell material, keratinocyte cultures can be obtained, which can be used to generate induced pluripotent stem cells. These induced pluripotent stem cells can be used, for example, for personalized medicine, for example in individualized clinical trials, regenerative medicine or transplantation.

[0040] In yet another embodiment of the method of the present invention, prior to step (a), a 0 ) obtaining a hair root sample by plucking hair from a test individual; The following will be implemented.

[0041] By this means, hair root samples can be provided and obtained in a simple and non-invasive manner without the need for a doctor or medical professional. Therefore, the method of the present invention is suitable for direct use by consumers themselves, and does not require a qualified doctor. This aspect allows for use in daily life.

[0042] In another embodiment of the method of the invention, after step (a) and / or, if applicable, after step (a″), a''') performing a metabolomic analysis of the hair root sample and / or, if applicable, performing a metabolomic analysis of the cultured keratinocytes. The following will be implemented.

[0043] "Metabolome" refers to any small molecule chemical found in a biological sample, such as a hair root sample used in the present invention. Small molecule chemicals found in the metabolome include endogenous metabolites that are naturally produced by an organism, such as amino acids, organic acids, nucleic acids, fatty acids, amines, sugars, vitamins, cofactors, pigments, antibiotics, and exogenous chemicals that are not naturally produced by an organism, such as drugs, environmental pollutants, food additives, toxins, and other xenobiotics. Thus, the above means have the advantage that, in addition to sequence analysis, metabolome analysis of the test individual can be further performed. In metabolome analysis, metabolic disorders or metabolic diseases can be detected by comparison with the metabolome of a reference individual. Metabolome analysis is performed by operations well known in the art. For example, metabolites can be extracted, which may involve derivatization and the addition of internal standards. Sample analysis involves quantifying metabolites, for example, by liquid or gas chromatography combined with mass spectrometry and / or NMR spectroscopy. The raw output data can be used to extract metabolite features and can be further processed to perform statistical analysis, such as PCA. A large number of bioinformatics tools and software are available that can be used to identify relationships between disease states and outcomes, determine significant correlations, and characterize metabolic signatures based on existing biological knowledge.

[0044] In yet another embodiment of the method of the present invention, after step (b) and before step (c), b') converting said nucleic acids into a form suitable for sequencing techniques by preparing a sequence library with said nucleic acids; Implemented the following: In step (d), the nucleotide sequence of the sequence library is determined to obtain the test nucleotide sequence.

[0045] By this means, the technical requirements for high-throughput sequencing can be achieved in an advantageous manner. In this step, the isolated DNA or RNA can be converted into a form suitable for sequencing, for example, by using common sequencing equipment based on Sanger sequencing, sequencing-by-synthesis, nanopore sequencing, single molecule real-time sequencing, single cell sequencing, or other technologies. This can be done by utilizing various kits provided by companies providing sequencing methods, such as Illumina, NEB, Agilent, Pacific Biosciences, ONT, etc. Technically, depending on the various sequencing technologies, the steps involved in the preparation of a sequencing library vary, but often include quality control of the isolated nucleic acid, shearing of the nucleic acid, reverse transcription (RNA only), adapter ligation, barcoding, and amplification by PCR. DNA and RNA isolated from hair roots or keratinocytes can be used with any currently available library preparation kit.

[0046] In another embodiment of the invention, said reference nucleotide sequence is a nucleotide sequence of a disease-associated gene, such as a cancer-associated gene, and said genetic determination is a diagnosis of a disease or a diagnosis of a predisposition to a disease based on said disease-associated gene.

[0047] By this means, the method of the present invention can be advantageously configured as a diagnostic method capable of diagnosing a genetic disease or genetic disorder, or a predisposition to a disease or cancer.

[0048] In yet another embodiment of the method of the present invention, d') comparing the nucleotide sequence of nucleic acid isolated from a sample other than the hair root of said test individual with said test nucleotide sequence. The following will be implemented.

[0049] According to this embodiment, the "non-hair root sample" is a biological sample obtained from a test individual, which contains DNA and / or RNA, but does not correspond to the aforementioned hair root sample or other hair root samples. In other words, in addition to the hair root sample, another sample from another different tissue is obtained from the test individual. DNA and / or RNA are also isolated from this other sample, and its nucleotide sequence is determined and compared with the reference nucleotide sequence, similar to the nucleotide sequence of the DNA or RNA obtained from the hair root sample. The non-hair root sample is preferably a tumor sample, such as a blood tumor sample, and the blood tumor is more preferably selected from the group consisting of acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML).

[0050] By this means, the method of the present invention can be adapted for cancer diagnosis. Modern tumor diagnosis by next generation sequencing (NGS) requires the comparison of DNA sequencing data of a tumor sample with that of healthy tissue ("normal sample") obtained from the same patient. Sequencing of tumor and normal samples allows the detection of mutations that only affect the tumor (so-called somatic variants), some of which are responsible for tumor growth. In the art, NGS diagnostics for solid tumors usually use blood samples as healthy controls. In contrast, the present invention uses DNA from hair roots to obtain healthy "normal" or control DNA, which is of high quality and easy to obtain. The resulting DNA from hair roots is used as a healthy reference sample to distinguish (tumor-specific) somatic mutations and inherited germline variants in DNA isolated from tumor samples, with the goal of identifying tumor-specific somatic mutations in cancer-related genes that induce tumor growth and suggest treatment options. At the same time, DNA from hair roots can also be used to analyze germline or inherited genetic variants to identify cancer risk variants.

[0051] In a further embodiment of the method of the invention, said reference nucleotide sequence is a nucleotide sequence of a gene associated with alopecia and said genetic determination is a determination of the cause of alopecia.

[0052] By this means the method of the invention can be advantageously adapted to elucidating the genetic causes of alopecia.

[0053] Another subject of the invention is a kit for carrying out genetic determination, comprising: A container for storing hair root samples; Buffer solutions for nucleic acid sequencing, and Instructions for carrying out the methods of the present invention Including, Cell lysis buffer solution, and Keratinocyte Culture Medium The present invention relates to a kit which may comprise:

[0054] A "kit" is a combination of individual components useful for carrying out the method of the invention, each component being optimized for use together in the method of the invention. The kit of the invention may further include additional reagents, chemicals, buffers, reaction vials, etc., which may be useful for carrying out the method of the invention. Such a kit may integrate all the essential elements required to make the method of the invention work, thus minimizing the risk of introducing errors. Thus, such a kit may allow even semi-skilled laboratory staff to carry out the method of the invention.

[0055] The container for containing the hair root sample can be used for storing and / or transporting the hair root sample, for example for sending to a remote laboratory for further or additional analysis. In one embodiment of the present invention, the container for containing the hair root sample can be realized by the system disclosed in WO2018 / 065514.

[0056] The features, characteristics, advantages and embodiments disclosed for the method of the present invention apply equally to the kit of the present invention.

[0057] The features mentioned above and those to be described below can be used not only in the combinations shown in the respective embodiments, but also in other combinations or alone without departing from the scope of the present invention. [Brief description of the drawings]

[0058] The present invention will now be described and explained in more detail with reference to the following examples and figures, but the present invention is not limited to these examples and figures. [Figure 1] FIG. 1 is a schematic diagram showing culture and DNA / RNA isolation using hair. [Diagram 2] Functional annotation of mapped RNA-seq reads obtained from two keratinocyte cultures from hair is shown. [Diagram 3] Functional annotation of RNA-seq reads obtained and mapped from nine fresh hair root samples is shown. [Figure 4] Expression of known disease genes in hair keratinocyte cultures and fresh hair roots compared to sample types commonly used in clinical diagnosis. [Diagram 5] A comparison of OMIM disease genes expressed at reliably detectable levels (>3 tpm) in various samples for diagnostic analysis is shown. [Figure 6] 1 shows the similarity of gene expression patterns in various tissues. [Figure 7] We show that keratinocytes are the best source of material for transcriptome analysis using nanopore sequencing. [Figure 8] We present an example of the application of hair root-derived RNA in the clinical diagnosis of patients with rare diseases. [Figure 9] 1 is a flow chart showing a variant of the method of the invention for the diagnosis of (rare) genetic diseases. [Figure 10]1 is a flow chart illustrating one embodiment of a method of the present invention for diagnosing hematological cancer and selecting targeted therapy. [Figure 11] 1 is a flow chart illustrating one embodiment of a method of the present invention for identifying specific patterns of hair loss. [Figure 12] FIG. 1 is a flow chart illustrating one embodiment of the method of the present invention including a general workflow for wet-lab library preparation and sequencing. EXAMPLES

[0059] 1. Materials and Methods Isolation and culture of keratinocytes by the classical method according to Aasen et al. Isolation and culture of keratinocytes, which is recognized as the "classical method," was performed based on a modified protocol of the method by Aasen et al. (Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes, Nat. Biotechnol. 2008 Nov;26(11):1276-84). Briefly, hairs with visibly intact outer root sheaths were plucked from human donors using forceps (Fine Science Tools, Germany) and directly placed in high glucose DMEM medium (Thermo Fisher Scientific, USA) + antibiotic-antimycotic (AA) (Thermo Fisher Scientific, USA) for storage until further processing. Next, hair roots with intact outer root sheaths were cut from the hair and placed on T25 flasks (Greiner Bio-One, Germany) coated with Matrigel (Corning, USA) diluted 1:10 with Epilife (Thermo Fisher Scientific, USA) and incubated at 37°C for 60 min. Then, one drop of Matrigel diluted 1:5 was placed on top of the hair roots. After 2 h in a 37°C incubator, isolation medium (high glucose DMEM + 10-15% fetal bovine serum (Thermo Fisher Scientific, USA) + 1% non-essential amino acids (NEAA) + 1% amino acids + 10 ng / ml fibroblast growth factor 2 (FGF2) (Peprotech, USA) + 10 μM Y-27632 (ROCK inhibitor, Ascent Scientific, USA)) was carefully added to the flask and the medium was changed daily until keratinocyte elongation occurred. The medium was then changed to Epilife+HKGS (Thermo Fisher Scientific, USA)+ROCK inhibitor, and the medium was changed daily. Upon reaching confluence, cells were detached with Dispase (Bacillus polymyxa) (Corning, USA) and either reseeded on Synth-A-Freeze (Thermo Fisher Scientific, USA) or cryopreserved in this medium.For reseeding, well plates were coated with type IV collagen (diluted 1:100 in calcium- and magnesium-free phosphate-buffered saline (PBS--), Sigma-Aldrich, USA) for 1 h at 37°C.

[0060] RNA / DNA isolation from cultured keratinocytes To isolate DNA / RNA, keratinocytes were isolated from hair roots obtained from human donors using the method described by Aasen et al. and cultured in Epilife+HKGS in well plates coated with type IV collagen until confluent. The keratinocyte medium was then removed and the cells were washed once with PBS--. Next, 500 μl of TrypLE (Thermo Fisher Scientific, USA) was added to each well and the cells were placed in a 37°C incubator for 8-20 min. Once the cells were confirmed to be completely detached, PBS-- was added to terminate the incubation. Next, the cells were collected in a 15 ml conical tube, centrifuged at 1500 rpm for 2 min, and the supernatant was discarded. If RNA / DNA was not to be isolated directly, the cell pellet was frozen and stored at -80°C. For direct RNA / DNA isolation, DNA was isolated using the DNeasy Blood & Tissue kit (Qiagen, Germany) and RNA was isolated using the RNeasy mini kit (Qiagen, Germany). Each kit was used according to the manufacturer's protocol as detailed below. For DNA extraction, cell pellets were incubated for 10 min at 56°C in 280 μl ATL buffer + 28 μl proteinase K (both provided in the DNeasy kit). After incubation, 28 μl RNase I (stock concentration: 10 μg / ml) was added to each sample and incubated for 3 min at room temperature. Final DNA was eluted with 100 μl AE buffer provided in the kit. For RNA isolation, 700 μl RLT buffer (provided in the RNeasy mini kit) was added to the cell pellet. The solution was then transferred to a QiaShredder column (Qiagen, Germany) and centrifuged at 13,000 rpm for 1 min. DNAse digestion was performed in this column according to the manufacturer's protocol. The final RNA was obtained by eluting twice with 30 μl of the same AE buffer (provided in the DNeasy micro kit).

[0061] Direct isolation of RNA / DNA from hair Hairs with intact outer root sheaths for RNA / DNA extraction were plucked with forceps from three human donors. For RNA isolation, five roots were used per sample, and for DNA isolation, 10 roots were used per sample. To prevent drying, hairs were placed directly into a Petri dish containing high glucose DMEM. The roots were then cut from the hair shaft and placed into a 1.5 ml reaction tube filled with PBS-- (calcium / magnesium free, Thermo Fisher Scientific, USA). Isolation was performed using the DNeasy Blood & Tissue kit (for DNA, Qiagen, Germany) or the RNeasy micro kit (for RNA, Qiagen, Germany). Each kit was used according to the manufacturer's protocol and as detailed below. For DNA extraction, the PBS-- was removed and replaced with 180 μl ATL buffer + 20 μl proteinase K (both provided in the DNeasy kit). Samples were incubated at 56°C for 1 h. During incubation, samples were vortexed every 20 min. After incubation, the supernatants (without hair) were placed in new 1.5 ml reaction tubes and 20 μl of RNase I (stock concentration: 10 μg / ml) was added to each sample and incubated at room temperature for 3 min. Final DNA was obtained by eluting twice with 20 μl of the same AE buffer provided in the kit. For RNA isolation, PBS-- was removed and replaced with 700 μl of RLT buffer (provided in the RNeasy micro kit) and the tubes were vortexed for 30 s. The supernatants were transferred to a QiaShredder column (Qiagen, Germany) and centrifuged at 13,000 rpm for 1 min. DNA digestion was performed in this column according to the manufacturer's protocol. Final RNA was obtained by eluting twice with 16 μl of the same RNAse-free water (provided in the RNeasy micro kit).

[0062] Short-read RNA sequencing RNA quality was determined by measuring the 260 / 280 nm and 230 / 260 nm absorbance ratios using a spectrophotometer (Nanodrop ND-1000; Peqlab), RNA concentration using the Qubit Fluorometric Quantitation and RNA Broad-Range Assay (Thermo Fisher Scientific), and RNA Integrity Number (RIN) using the Fragment Analyzer 5300 and Fragment Analyzer RNA Kit (Agilent Technologies). To prepare libraries, the mRNA fraction was enriched by capturing polyA from 100 ng of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). Then, mRNA libraries were prepared using the NEB Next Ultra II Directional RNA Library Prep Kit for Illumina (NEB) according to the manufacturer's instructions. Library molar concentrations were determined by measuring library size (~400 bp) using a Fragment Analyzer 5300 and Fragment Analyzer DNA HS NGS fragment kit (Agilent Technologies) and library concentration (>0.5 ng / μl) using the Qubit Fluorometric Quantitation and dsDNA High Sensitivity assay (Thermo Fisher Scientific). In the first experiment, prepared libraries were denatured according to the manufacturer's instructions, diluted to 270 pM, and sequenced on an Illumina NovaSeq 6000 (Illumina) with paired-end reads of 2 × 100 bp and a sequencing depth of >25 million clusters per sample.

[0063] Long-read RNA sequencing RNA extraction and RNA quality control were performed in the same manner as for short-read RNA sequencing. Blood samples in PAXgene Blood RNA tubes (16 × 100 mm / 2.5 mL) were mixed gently 10 times, left at room temperature overnight, and then frozen and stored. Total RNA was extracted with the PAXgene Blood RNA kit using the QIAsymphony RNA kit (Qiagen). To process the samples in the PAXgene Blood RNA tubes, cDNA was prepared using 700 ng of RNA treated with the NEBNext® Globin & rRNA Depletion kit (human / mouse / rat). To prepare libraries for all types of RNA samples, the cDNA-PCR Sequencing kit (SQK-PCS109) was used according to the manufacturer's instructions. 50 ng of total RNA was annealed, strand-switching reactions were performed, and reverse transcribed with Maxima H Minus RT (Thermo Fisher Scientific). The resulting cDNA was amplified using LongAmp Ta Master Mix (NEB). Library molarities were determined by measuring library size using a Fragment Analyzer 5300 with the Fragment Analyzer DNA HS NGS fragment kit (Agilent Technologies) and library concentration (>5ng / μl) using the Qubit Fluorometric Quantitation and dsDNA High Sensitivity assay (Thermo Fisher Scientific). Rapid adapters were added to the amplified cDNA, and the resulting library, in an amount of less than 100 fmol, was loaded onto a PromethION flow cell (FLO-PRO002).

[0064] Short-read DNA sequencing For short-read sequencing, DNA isolated from cultured keratinocyte cells was used. DNA quality was determined by measuring the 260 / 280 nm and 230 / 260 nm absorbance ratios in a spectrophotometer (Nanodrop ND-1000; Peqlab). DNA was quantified with the Qubit Fluorometric Quantitation and DNA High-Sensitivity Assay (Thermo Fisher Scientific). Genomic integrity was assessed by pulsed-field capillary electrophoresis using a FemtoPulse instrument and the Genomic DNA 165 kb kit (Agilent). Libraries were prepared starting from 350 ng of DNA using the Illumina DNA PCR-Free Prep tagmentation protocol according to the manufacturer's instructions (Illumina) on a Biomek i5 automated liquid-handling workstation (Beckman Coulter). The final pool at a molar concentration of 600 pM was loaded onto a NovaSeq S4 flow cell (300 cycles) and sequenced in paired-end mode.

[0065] Long-read DNA sequencing DNA isolated from cultured keratinocyte cells was used for long-read sequencing. DNA quality was determined by measuring the absorbance ratios at 260 / 280 nm and 230 / 260 nm in a spectrophotometer (Nanodrop ND-1000; Peqlab). DNA was quantified with the Qubit Fluorometric Quantitation and DNA High-Sensitivity Assay (Thermo Fisher Scientific). Genomic integrity was assessed by pulsed-field capillary electrophoresis using a FemtoPulse instrument and the Genomic DNA 165 kb Kit (Agilent). After size selection from 2.5 μg of genomic DNA and cleanup with SPRI beads at a ratio of 0.7 if necessary, 1D libraries were prepared using 1.5 μg of DNA of selected sizes according to the Oxford Nanopore Technologies (ONT) protocol for ligation sequencing (SQK-LSK109). Duplicate library preparations were performed for each sample, and 500-600 ng of each library was loaded onto a single flow cell (FLO-PRO002) in a PromethION instrument (Oxford Nanopore Technologies (ONT)).

[0066] Analysis of short-read RNA-seq data Short-read RNA-seq data from hair keratinocyte cultures, fresh hair roots, blood and fibroblasts were used to evaluate the similarity of gene expression profiles in various types of tissues for clinical diagnosis. Repetitive sequences with low average quality resulting from sequencing cycles, adapter contamination or PCR amplification were identified by assessing the quality of reads in the RNA-seq data contained in the fastq files using ngs-bits (v.2020_06). Reads were aligned to the human reference genome GRCh38 using STAR v2.7.3a. The quality of the alignments was analyzed using ngs-bits (v.2020_06) and visually inspected with Integrative Genome Viewer (v2.7.2). Reads that overlapped known genes were annotated as reads originating from CDS exons, 5'UTR exons, 3'UTR exons, introns or intergenic regions, or around the transcription start site (TS) or transcription end site (TES). The percentage of reads aligning to exonic regions is used to compare RNA-seq quality between samples.

[0067] Normalized read counts for all genes were obtained using Subread (v2.0.0) and edgeR (v3.30.3). Genes that did not show a minimum gene expression value of 1 cpm (counts per million) in at least two samples were excluded. Log-normalized cpm (log 2 The distribution of expression values ​​(cpm) allows an assessment of similarity across all samples.

[0068] Analysis of sample similarity and differential gene expression Using filtered normalized expression data. Similarity between each pair was measured by calculating Spearman rank correlation coefficient for each pair of samples. Hierarchical clustering was performed on the resulting similarity values. Differential gene expression analysis was performed using edgeR. Statistical models incorporating group characteristics of samples were tested by fitting a negative binomial distribution using the generalized linear model (GLM) method. Fold change in gene expression (log 2 Fold change (FDR) was calculated and statistical tests were performed to assess significance. Statistical significance was presented as raw p-values ​​and adjusted p-values ​​(FDR).

[0069] Comparison of disease genes expressed in four tissues Gene expression values ​​were compared for 4237 disease genes annotated in Online Mendelian Inheritance in Men (OMIM; an online catalog of human genes and genetic diseases, https: / / www.omim.org) between three types of tissue that can be non-invasively obtained (and cultured) from patients (blood, fresh hair root, and keratinocyte cultures from hair) as well as fibroblasts cultured from skin punch biopsy samples (often used to diagnose rare diseases). Gene expression values ​​were labeled as low expression (<0.5 cpm), moderate expression (0.5–3 cpm) or high expression (>3 cpm). Venn diagrams were used to compare and visualize the number of OMIM disease genes that reached a given level (e.g., high expression) in the four types of samples mentioned above. Genes that showed at least 3 cpm were defined as genes that could be diagnosed using a particular type of tissue. Based on known associations between disease phenotype descriptions (Human Phenotype Ontology) and disease genes (OMIM or HGMD), we developed a tool to select the best tissue type (or tissue combination) to sample for clinical diagnosis of a disease. The tool simply calculates the number of known disease genes of a disease that show high expression values ​​in a given tissue or combination of tissues.

[0070] Analysis of long-read RNA-seq data Long-read RNA from blood, fresh hair, or hair-based keratinocyte cultures was analyzed using a laboratory-made pipeline consisting of quality control, minimapped splice alignment, and transcript isoform detection using the ONT PromethION. Long reads were classified as "perfect splice match (FSM)" if they completely overlapped with known transcript isoforms, as "incomplete splice match (ISM)" if they partially overlapped with known isoforms but did not show alternative splicing, as "novel in catalog (NIC)" if they represented novel isoforms of known genes (a new combination of known and novel splice sites), and as "other" if they did not overlap with known exons. Assuming that the detection of novel isoforms or novel genes is a rare event, the quality of long-read RNA-seq in the compared tissues was assessed as the ratio of FSM reads to ISM reads.

[0071] 2.Results Schematic diagram showing hair culture and DNA / RNA isolation FIG. 1 shows a diagram illustrating culture using hair and isolation of DNA / RNA. a) Patient DNA and RNA can be obtained by i) keratinocyte culture from hair, ii) direct isolation from fresh hair roots, or iii) isolation from fresh frozen hair roots. b) Keratinocyte culture on hair using Matrigel droplets. c) Keratinocyte culture on hair using a system for preserving hair samples, for example as described in WO2018 / 065514. d) Direct extraction of DNA and RNA from fresh hairy roots.

[0072] Functional annotation of mapped RNA-seq reads from two hair-based keratinocyte cultures Figure 2 shows the functional annotation results of the mapped RNA-seq reads obtained from two hair keratinocyte cultures. For the purpose of benchmarking, a large percentage of reads aligned to exonic regions was considered good, whereas a large percentage of intronic or intergenic reads was indicative of a possible problematic wet-lab operation. More than 93% of reads aligned to known genes and more than 90% of reads originated from exonic regions indicated a very high quality of sampling and sequencing operation.

[0073] Functional annotation of RNA-seq reads obtained and mapped from nine fresh hairy root samples Functional annotation of the mapped RNA-seq reads from nine fresh hair root samples is shown in Figure 3. More than 90% of the reads aligned to known genes and more than 85% were derived from exonic regions, indicating that the sampling and sequencing procedures were of very high quality. The quality measures for fresh hair were only slightly lower than those for keratinocyte cultures from hair, which may reflect either less starting material or a greater complexity of cell types composing hair roots than in cultures.

[0074] Expression of known disease genes in hair-based keratinocyte cultures and fresh hair roots compared to sample types commonly used in clinical diagnosis Figure 4 shows the analysis of expression of known disease genes in hair keratinocyte cultures and fresh hair roots compared to sample types commonly used for clinical diagnosis (blood and fibroblast cultures from skin punch biopsies). Expression values ​​are measured as normalized read counts (TPM = transcripts per million sequenced reads). Gene expression is reliably detectable above 0.5 tpm (yellow). Detection of splice variants and allele-specific expression requires high expression values ​​above 3 tpm (green). Blood had the highest number of nondiagnostic genes (red, tpm < 0.5), whereas fresh hair had the lowest number of nondetectable genes. 877 (21%) of 4237 OMIM disease genes can be reliably measured in keratinocytes or hair roots (green, tpm > 3), whereas such measurement is not possible in blood.

[0075] Comparison of reliably expressed OMIM disease genes at detectable expression levels for diagnostic analysis For diagnostic analysis, OMIM disease genes that were expressed at reliably detectable levels (>3 tpm) in blood, fibroblasts, fresh hair roots, and hair-derived keratinocytes were compared. The results are shown in Figure 5. A total of 4237 OMIM disease genes were examined, of which 1109 could not be reliably detected in any tissue. Of the remaining 3129 genes, expression of 248 genes (8%) could be reliably measured only in hair roots (fresh or cultured). 877 genes (28%) could not be detected in blood but could be detected in hair roots. Of the remaining genes, 174 (5.5%) could be reliably measured only in fibroblasts. Diagnostic testing of 94.5% of all detectable disease genes is possible in combination with non-invasive blood and hair sampling.

[0076] Similarity of gene expression patterns in various tissues The similarity of gene expression patterns in various tissues was analyzed. The results are shown in Figure 6. Gene expression in fresh hair roots and keratinocytes is more similar to that of fibroblasts than to that of blood. This result indicates that hair roots can be collected non-invasively as an alternative to fibroblasts, which are usually collected by skin punch biopsy.

[0077] Keratinocytes are the best source of tissue for transcriptome analysis using nanopore sequencing We investigated which sample material is most suitable for transcriptome analysis using nanopore sequencing. The results are shown in Figure 7. The results shown in the figure indicate that keratinocytes are the best sample to be collected. By simply using known splice sites (green) or novel splice sites, up to 70% of the sequenced transcripts map to known genes. In comparison, when blood samples are used, only 30% of the sequenced transcripts map to known genes.

[0078] Application of hair root-derived RNA in clinical diagnosis of patients with rare diseases Figure 8 shows one embodiment of the method of the present invention in the clinical diagnosis of patients with rare diseases. RNA for diagnostic sequencing can be obtained from fresh hair roots or keratinocyte cultures obtained from subjects suspected of having a rare disease (1, 2A, 2B), followed by RNA extraction (3A) and DNA extraction (3B), preparation of a sequencing library (4), sequencing (5), and computational analysis of disease gene expression (5).

[0079] Variations In Figures 9 to 12 different variants of the method of the invention are shown. DNA-based sequencing methods (genomic or epigenomic sequencing) are shown in blue. RNA-based sequencing methods (gene expression analysis) are shown in yellow. Metabolite analysis is shown in red. Diagnostic results are shown in green.

[0080] Figure 9 shows a flow chart of a variant of the method of the present invention for the diagnosis of (rare) genetic diseases. Rare genetic diseases are usually Mendelian diseases and are caused by a single variant affecting a disease gene. Diagnosis involves the analysis of DNA (genome) to identify a single causative variant or multiple candidate variants. Candidate causative variants (e.g., spliceopathy, allele-specific expression, loss of function causing nonsense-dependent decay or deletion, copy number gain, and regulatory variants causing loss or gain of gene expression) can then be evaluated by analyzing RNA-seq (transcriptome).

[0081] A complete diagnosis can be made using DNA and RNA extracted from fresh hair roots or keratinocyte cultures. Hair samples offer less of an advantage for DNA sequencing compared to blood samples, but are almost always a better option for RNA sequencing and RNA analysis, for the following reasons: 1) The quality of RNA extracted from hair roots and keratinocytes is much better than that from blood. 2) RNA from blood contains a very high percentage of hemoglobin (up to 70%), so it is necessary to remove the hemoglobin (which further reduces the quality of the RNA) or lose up to 70% of the sequencing yield. 3) Many disease genes are expressed in keratinocytes but not in blood. The inventors estimate that of the approximately 4000 known disease genes, 800 are measurable in keratinocytes but not in blood. 4) Hair roots can be preserved for much longer periods than blood samples.

[0082] FIG. 10 shows a flow chart illustrating one embodiment of the method of the present invention for the diagnosis of blood cancer and the selection of targeted therapy. Modern tumor diagnosis by next generation sequencing (NGS) requires the comparison of DNA sequencing data of a tumor sample with that of healthy tissue ("normal sample") obtained from the same patient. Sequencing of tumor and normal samples allows the detection of mutations that only affect the tumor (somatic variants), some of which are responsible for tumor growth. In NGS diagnosis for solid tumors, blood samples are usually used as healthy controls. For the diagnosis of blood cancer, as proposed by the inventors, healthy ("normal") DNA can be obtained from DNA derived from hair roots. At the same time, DNA derived from hair roots can also be used for the analysis of germline (inherited) gene variants to identify cancer risk variants. Note: RNA derived from hair roots is not useful for tumor diagnosis, since gene expression levels in tumor tissue cannot be compared to gene expression levels in hair roots.

[0083] FIG. 11 shows a flow chart of one embodiment of the method of the present invention for identifying hair loss specific patterns. Common diseases such as hair loss are often referred to as complex diseases, and usually involve multiple genes and are also influenced by non-genetic factors. Environmental factors such as nutrition, smoking, alcohol, and sports have direct or indirect effects on DNA epigenome or modification, transcriptome or gene expression, and cellular metabolites. In particular, epigenome such as DNA methylation can be a stepping stone to know the age of tissues, and can know premature aging and accelerated aging of tissues, for example, premature aging and accelerated aging of hair roots caused by genetic predisposition or environmental factors. By combining genome sequencing, epigenome sequencing, and transcriptome sequencing with metabolite analysis, patterns related to common diseases such as hair loss can be identified. Since hair loss, thinning or poor hair quality can be caused not only by genetic predisposition but also by hormones, environmental factors or metabolites, and even by drugs such as chemotherapy drugs, a combined analysis of genome, gene expression, DNA methylation and metabolites in hair roots is a promising method to detect hair loss specific patterns. A small number of fresh hair roots can be used to perform the complete operation. Genomic and epigenomic analysis is performed using extracted DNA. Gene expression analysis is performed using extracted RNA. Metabolites can be extracted from any tissue, and inexpensive analysis methods that do not use sequencing can be used.

[0084] FIG. 12 shows a flow chart of one embodiment of the method of the present invention, including a typical wet-lab library workflow for the applications described herein. This embodiment includes nucleic acid and metabolite extraction, quality control of these extracted biomaterials, preparation of sequencing libraries, and types of sequencing. Library preparation and sequencing protocols and chemistries are available from various vendors, such as Illumina, NEB, Agilent, Twist, IDT, ONT, Pacific Biosciences, etc. Sequencing types include any kind of DNA sequencing (whole genome sequencing, whole exome sequencing, gene panel sequencing, amplicon sequencing), epigenomic sequencing (e.g., bisulfite sequencing for analysis of DNA methylation, ChIP-seq for analysis of DNA-protein binding, or histone modification analysis), RNA sequencing with prior reverse transcription of RNA to cDNA ("cDNA sequencing"), or direct RNA sequencing using nanopore technology. Data analysis differs for each type of sequencing and is described above.

Claims

1. 1. A method for making a genetic determination comprising the steps of: a) obtaining a hair root sample from a test individual; b) isolating nucleic acid from said hair root sample; c) determining the nucleotide sequence of the nucleic acid to obtain a test nucleotide sequence; d) comparing the test nucleotide sequence to a reference nucleotide sequence; and e) making a genetic determination based on a comparison of the test nucleotide sequence with a reference nucleotide sequence. The method includes:

2. The method of claim 1 , wherein the nucleic acid is RNA.

3. The method of claim 1 or 2, wherein the nucleic acid is DNA.

4. 2. The method of any one of the preceding claims, wherein in step (c) RNA sequencing and / or DNA sequencing is performed.

5. The method of claim 4, wherein the DNA sequencing comprises epigenomic sequencing, preferably obtaining information regarding the methylation of the DNA.

6. After the step (a) and before the step (b), (a') exposing said hair root sample to a liquid that causes lysis of cells and release of nucleic acids from said cells. Implemented the following: In the step (b), the nucleic acid is isolated from the liquid.

10. A method according to any one of the preceding claims.

7. After the step (a) and before the step (b), a'') culturing the keratinocytes contained in the hair root sample Implemented the following: In step (b), nucleic acid is isolated from the cultured keratinocytes or their progeny. The method according to any one of claims 1 to 5.

8. Prior to the step (a), a ０ ) obtaining a hair root sample by plucking hair from a test individual; 2. The method of any one of the preceding claims, further comprising:

9. After step (a) and / or after step (a″), a''') performing metabolomic analysis of the hair root sample and / or the cultured keratinocytes 2. The method of any one of the preceding claims, further comprising:

10. After step (b) and before step (c), b') preparing a sequence library using the nucleic acid Implemented the following: In the step (d), determining the nucleotide sequence of the sequence library to obtain the test nucleotide sequence.

10. A method according to any one of the preceding claims.

11. 10. The method of any one of the preceding claims, wherein the reference nucleotide sequence is a nucleotide sequence of a disease-associated gene and the genetic determination is a diagnosis of a disease or a diagnosis of a predisposition to a disease based on the disease-associated gene.

12. 12. The method of claim 11, wherein the reference nucleotide sequence is a nucleotide sequence of tumor DNA of the test individual, preferably a nucleotide sequence of hematological tumor DNA, more preferably, the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML).

13. 10. The method of any one of the preceding claims, wherein the reference nucleotide sequence is a nucleotide sequence of a gene associated with alopecia and the genetic determination is a determination of the cause of alopecia.

14. A kit for carrying out genetic determination, comprising: A container for storing hair root samples; Buffer solutions for nucleic acid sequencing, and Instructions for carrying out the method according to any one of claims 1 to 13. Including, Cell lysis buffer solution, and Keratinocyte Culture Medium The kit may further comprise:

15. Use of a hair root sample from a test individual to make a genetic determination based on nucleotide sequencing of nucleic acid isolated from the hair root sample.

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