Treating bone mineral density loss with kringle-containing transmembrane protein 1 (KREMEN1) inhibitors

Abstract

The present disclosure provides methods of treating subjects having or at risk of developing bone mineral density loss, and methods of identifying subjects having an increased risk of developing bone mineral density loss.

Description

[Technical field]

[0001] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically as a text file of 196 kilobytes in size under the title 18923808102SEQ, created on June 29, 2022. This Sequence Listing is incorporated herein by reference.

[0002] The present disclosure relates broadly to the treatment of subjects having or at risk of developing bone mineral density loss with kringle-containing transmembrane protein 1 (KREMEN1) inhibitors, and to methods of identifying subjects at high risk for developing bone mineral density loss. [Background technology]

[0003] Degenerative conditions of bone can predispose an individual to fractures, bone pain, and other problems. Two significant degenerative conditions of bone are osteopenia and osteoporosis. Bone mineral density loss (osteopenia) is a bone condition that is a precursor to osteoporosis and is characterized by a decrease in bone mass due to bone loss at a faster rate than new bone growth. Osteopenia is manifested in bones with a mineral density lower than normal peak bone mineral density, but not as low as that seen in osteoporosis. Osteopenia can result from reduced muscle activity, which may occur as a result of fractures, bed rest, fracture fixation, joint reconstruction, arthritis, and the like. Osteoporosis is a progressive disease characterized by the gradual weakening of bones due to bone demineralization. Osteoporosis manifests in thin, brittle bones, making them more susceptible to fracture. Hormonal deficiency associated with menopause in women and aging in both sexes contribute to degenerative conditions of bone. In addition, inadequate dietary intake of minerals essential for bone growth and maintenance is a major cause of bone loss.

[0004] By replicating some of the effects of muscle use on bone, the effects of osteopenia can be slowed, stopped, and even reversed. This typically involves some application or simulation of the effects of mechanical stress on bone. Compounds for treating osteopenia or osteoporosis include pharmaceutical preparations that induce bone growth or slow bone demineralization, or mineral complexes that supplement the diet to replenish lost bone minerals. Low levels of estrogen in women and low levels of androgens in men are the main hormone deficiencies that cause osteoporosis in each gender. Other hormones, such as thyroid hormone, progesterone, and testosterone, also contribute to bone health. Thus, the aforementioned hormone compounds have been synthetically developed or extracted from non-mammalian sources and formulated into therapeutics for treating osteoporosis. Mineral supplement preparations containing iodine, zinc, manganese, boron, strontium, vitamin D3, calcium, magnesium, vitamin K, phosphorus, and copper have also been used to supplement the deficiency of dietary intake of such minerals. However, long-term hormone therapy has undesirable side effects, such as increased risk of cancer. Moreover, many synthetic or non-mammalian hormone-based therapies have additional undesirable side effects, such as increased risk of cardiovascular disorders, neurological disorders, or exacerbation of pre-existing conditions.

[0005] Kringle-containing transmembrane protein 1 (KREMEN1) is a cell surface molecule that regulates WNT signaling by binding to DKK and LRP5 / 6, thereby promoting the uptake of this complex via clathrin-mediated endocytosis (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Mao et al., Nature, 2002, 417, 664-667 Summary of the Invention

[0007] The present disclosure provides a method of treating a subject having or at risk of developing bone mineral density loss, the method comprising administering to the subject a KREMEN1 inhibitor.

[0008] The present disclosure also provides a method of treating a subject having or at risk of developing osteopenia, the method comprising administering to the subject a KREMEN1 inhibitor. The present disclosure also provides a method of treating a subject having or at risk of developing type I osteoporosis, the method comprising administering to the subject a KREMEN1 inhibitor.

[0009] The present disclosure also provides a method of treating a subject having or at risk of developing type II osteoporosis, the method comprising administering to the subject KREMEN1. The present disclosure also provides a method of treating a subject having or at risk of developing secondary osteoporosis, the method comprising administering to the subject a KREMEN1 inhibitor.

[0010] The disclosure also provides a method of treating a subject with a therapeutic agent for treating or preventing bone mineral density loss, the subject having or at risk of developing bone mineral density loss, the method comprising determining whether the subject has a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 by obtaining or obtaining a biological sample from the subject and performing or performing sequence analysis on the biological sample to determine whether the subject has a genotype that includes the KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1; and i) administering or continuing to administer the therapeutic agent for treating or preventing bone mineral density loss at a standard dosage to the subject who is a KREMEN1 norm. and / or administering a KREMEN1 inhibitor to the subject; ii) administering or continuing to administer to the subject who is heterozygous for the KREMEN1 variant nucleic acid molecule the therapeutic agent for treating or preventing bone mineral density loss at the same or a lower amount than a standard dosage, and / or administering a KREMEN1 inhibitor to the subject; or iii) administering or continuing to administer to the subject who is homozygous for the KREMEN1 variant nucleic acid molecule the therapeutic agent for treating or preventing bone mineral density loss at the same or a lower amount than a standard dosage; the presence of a genotype having the KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 indicates that the subject has a low risk of developing bone mineral density loss.

[0011] The present disclosure also provides a method for identifying a subject having an increased risk of developing bone mineral density loss, the method comprising determining, or having determined, the presence or absence of a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 in a biological sample obtained from the subject; if the subject is a KREMEN1 standard, the subject has an increased risk of developing bone mineral density loss; and if the subject is heterozygous or homozygous for the KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1, the subject has a decreased risk of developing bone mineral density loss.

[0012] The present disclosure also provides a therapeutic agent for treating or preventing bone mineral density loss, the therapeutic agent being for use in treating or preventing bone mineral density loss in a subject having a KREMEN1 mutant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1, a KREMEN1 mutant mRNA molecule encoding a predicted loss-of-function polypeptide of KREMEN1, or a KREMEN1 mutant cDNA molecule encoding a predicted loss-of-function polypeptide of KREMEN1.

[0013] The present disclosure also provides a KREMEN1 inhibitor for use in treating or preventing bone mineral density loss in a subject that a) is reference to a KREMEN1 genomic nucleic acid molecule, a KREMEN1 mRNA molecule, or a KREMEN1 cDNA molecule, or b) is heterozygous for i) a KREMEN1 mutant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1; ii) a KREMEN1 mutant mRNA molecule encoding a predicted loss-of-function polypeptide of KREMEN1; or iii) a KREMEN1 mutant cDNA molecule encoding a predicted loss-of-function polypeptide of KREMEN1.

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several features of the present disclosure. [Brief description of the drawings]

[0015] [Figure 1] Association of rare predicted loss of function (pLoF) and predicted deleterious missense variants in KREMEN1 with higher estimated bone mineral density (eBMD). Association estimates for KREMEN1 pLoF or predicted deleterious missense variant burden with AAF<1% were derived in the United Kingdom Biobank (UKB). Missense variants were predicted to be deleterious by 5 of 5 in silico algorithms (see genotype data for description of in silico algorithms used for characterization of variant deleteriousness). Genotype counts indicate the number of subjects in each of the three genotype categories: RR indicates subjects carrying no rare pLoF or predicted deleterious missense variants in KREMEN1; RA indicates subjects carrying rare pLoF or predicted deleterious missense variants in a single KREMEN1 allele; AA indicates subjects carrying rare pLoF or predicted deleterious missense variants in both KREMEN1 alleles. AAF indicates the alternative allele frequency of the variants included in this analysis, g / cm2 is grams per square centimeter, SD is standard deviation, and CI is confidence interval. [Diagram 2]Association of rare pLoF variants in KREMEN1 with higher eBMD. Association estimates are associated with KREMEN1 pLoF variant burden of AAF<1% and were derived in UKB. Genotype counts indicate the number of subjects in each of the three genotype categories: RR indicates subjects carrying no rare pLoF variants in KREMEN1; RA indicates subjects carrying at least one rare pLoF in a single KREMEN1 allele; AA indicates subjects carrying any rare pLoF variants in both KREMEN1 alleles. AAF is the alternative allele frequency of the variants included in this analysis. g / cm2 is grams per square centimeter, SD is standard deviation, and CI is confidence interval. [Figure 3-1] KREMEN1 pLoF or predicted deleterious missense variants identified by whole exome sequencing (WES) and included in the gene burden association analysis are shown. The genomic coordinates column shows the physical genomic location in base pairs, reference allele, and alternative allele for each variant according to build 38 of the human genome sequence by the Human Genome Reference Consortium. Coding DNA and protein changes are provided according to the Human Genome Variation Society nomenclature and refer to the KREMEN1 transcripts shown in the "Transcript(s)" column. Transcripts were sourced from the Ensembl database (Howe et al., Nuc. Acids Res., 2020, 49(D1), D884-D891). AAF is the alternative allele frequency of the variants included in this analysis, and pLoF is the predicted loss of function. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Diagram 3-4] Same as above. [Figure 3-5] Same as above. [Diagram 3-6] Same as above. [Diagram 3-7] Same as above. [Diagram 3-8] Same as above. [Diagram 3-9] Same as above. [Figure 3-10] Same as above. [Figure 3-11] Same as above. [Figure 3-12] Same as above. [Figure 3-13] Same as above. [Figure 3-14] Same as above. [Figure 3-15] Same as above. [Figure 3-16] Same as above. [Figure 3-17] Same as above. [Figure 3-18] Same as above. [Figure 3-19] Same as above. [Figure 3-20] Same as above. [Figure 3-21] Same as above. [Figure 3-22] Same as above. [Figure 3-23] Same as above. [Figure 3-24] Same as above. [Figure 3-25] Same as above. [Figure 3-26] Same as above. [Figure 3-27] Same as above. [Figure 3-28] Same as above. [Figure 3-29] Same as above. [Figure 3-30] Same as above. [Figure 3-31] Same as above. [Figure 3-32] Same as above. [Figure 3-33] Same as above. [Figure 3-34] Same as above. [Figure 3-35] Same as above. [Figure 3-36] Same as above. [Figure 3-37] Same as above. [Figure 3-38] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Various terms relating to the aspects of the present disclosure are used throughout the specification and claims. Unless otherwise indicated, such terms are to be given their ordinary meaning in the art. Other terms that are specifically defined are to be interpreted in a manner consistent with the definitions set forth herein.

[0017] Unless expressly stated otherwise, any method or embodiment set forth herein is in no way intended to be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim specifically specifies in the claim or description that the steps are to be limited to a particular order, it is in no way intended to dictate order in any respect. This also applies to any possible implicit criteria of interpretation, including logical matters regarding the arrangement of steps or work flow, general meanings derived from grammatical construction or punctuation, or the number or type of embodiments described herein.

[0018] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. As used herein, the term "about" means that a cited numerical value is approximate, and small variations do not significantly affect the practice of the disclosed embodiments. When a numerical value is used, unless otherwise indicated by context, the term "about" means that the numerical value can vary by ±10% and remain within the range of the disclosed embodiments.

[0019] As used herein, the term "comprising" may in certain embodiments be replaced with "consisting" or "consisting essentially of," as desired.

[0020] As used herein, with respect to a nucleic acid molecule or polypeptide, the term "isolated" means that the nucleic acid molecule or polypeptide is in a state other than its native environment, e.g., away from blood and / or animal tissue. In some embodiments, an isolated nucleic acid molecule or polypeptide is substantially free of other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, the nucleic acid molecule or polypeptide can be in a highly purified form, i.e., more than 95% pure or more than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same nucleic acid molecule or polypeptide in alternative physical forms, such as dimers or alternatively phosphorylated or derivatized forms.

[0021] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" can include polymeric forms of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multistranded. A strand of a nucleic acid also refers to its complement.

[0022] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs), pet animals (e.g., dogs, cats), laboratory animals (e.g., mice, rats, rabbits), and non-human primates. In some embodiments, the subject is a human. In some embodiments, the human is a patient under the care of a physician.

[0023] According to the present disclosure, it has been observed that KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1 (regardless of whether these mutations are homozygous or heterozygous in a particular subject) are associated with a reduced risk of developing bone mineral density loss. It is believed that KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1 are not associated with bone mineral density loss in genome-wide or exome-wide association studies. Thus, subjects who are KREMEN1 norm or heterozygous for KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1 can be treated with KREMEN1 inhibitors to prevent bone mineral density loss, alleviate symptoms thereof, and / or inhibit the onset of symptoms. It is also believed that such subjects with bone mineral density loss can be further treated with therapeutic agents that treat or prevent bone mineral density loss.

[0024] For the purposes of this disclosure, any particular subject, such as a human, can be classified as having one of three KREMEN1 genotypes: i) KREMEN1 standard; ii) heterozygous for KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1; or iii) homozygous for KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1. If the subject does not have a copy of KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1, the subject is KREMEN1 standard. If the subject has a single copy of KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1, the subject is heterozygous for KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1. A KREMEN1 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 is any nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) encoding a mutant KREMEN1 polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. A subject having a KREMEN1 polypeptide with partial loss-of-function (or predicted partial loss-of-function) is hypomorphic for KREMEN1. If a subject has two copies (same or different) of a KREMEN1 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1, the subject is homozygous for a KREMEN1 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1.

[0025] For subjects who are genotyped or determined to be KREMEN1-based, such subjects have an increased risk of developing bone mineral density loss, such as osteopenia, type I osteoporosis, type II osteoporosis, and / or secondary osteoporosis. For subjects who are genotyped or determined to be KREMEN1-based or heterozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1, such subjects or subjects can be treated with a KREMEN1 inhibitor.

[0026] In any of the embodiments described herein, the KREMEN1 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 can be any nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) encoding a KREMEN1 mutant polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. In some embodiments, the KREMEN1 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 is associated with a reduced in vitro response to a KREMEN1 ligand compared to a reference KREMEN1. In some embodiments, the KREMEN1 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 is a KREMEN1 mutant that results, or is predicted to result, in premature truncation of the KREMEN1 polypeptide compared to a human reference genome sequence. In some embodiments, the KREMEN1 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 is a mutant predicted to be damaging by an in vitro prediction algorithm, such as Polyphen, SIFT, or a similar algorithm. In some embodiments, the KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 is a variant that causes or is predicted to cause a non-synonymous amino acid substitution in KREMEN1, the allele frequency of which is less than 1 / 100 alleles in the population from which the subject is selected. In some embodiments, the KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 is any rare variant (allele frequency <0.1%; or 1 in 1,000 alleles), or any splice site, stop gain, start loss, stop loss, frameshift, or in-frame indel, or other frameshift KREMEN1 variant.

[0027] In any of the embodiments described herein, the predicted loss-of-function polypeptide of KREMEN1 can be any KREMEN1 polypeptide having a partial loss of function, a complete loss of function, a predicted partial loss of function, or a predicted complete loss of function.

[0028] In any of the embodiments described herein, a KREMEN1 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 may comprise a mutation at position chromosome 22 (SEQ ID NO:1; ENSG00000183762.13; ENST00000327813.9; chr22:29073118-29168333 in the GRCh38 / hg38 human genome assembly; alternatively, chr22:29073035-29168333 or chr22:29073077-29168333) using the nucleotide sequence of a KREMEN1 reference genomic nucleic acid molecule as a reference sequence.

[0029] There are numerous genetic variants in KREMEN1 that result in subsequent changes in the KREMEN1 polypeptide sequence, including but not limited to the variants listed in FIG. 3, Table 2, or listed elsewhere herein.

[0030] Any one or more (i.e., any combination) of KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1 can be used in any of the methods described herein to determine whether a subject has an increased risk of developing bone mineral density loss. A particular variant combination can form a mask that is used to statistically analyze a particular correlation between KREMEN1 and an increased risk of developing bone mineral density loss.

[0031] In any of the embodiments described herein, the bone mineral density loss is osteopenia, type I osteoporosis, type II osteoporosis, and / or secondary osteoporosis. In some embodiments, the bone mineral density loss is osteopenia. In some embodiments, the bone mineral density loss is type I osteoporosis. In some embodiments, the bone mineral density loss is type II osteoporosis. In some embodiments, the bone mineral density loss is secondary osteoporosis.

[0032] Symptoms of reduced bone mineral density include, but are not limited to, increased bone fragility (manifesting as a fracture as a result of mild to moderate trauma), decreased bone density, localized bone pain and weakness in the area of ​​the fracture, loss of height or changes in posture (such as stooping), high levels of serum calcium or alkaline phosphatase in a blood test, Vitamin D deficiency, and joint or muscle pain, or any combination thereof.

[0033] The present disclosure provides a method of treating a subject having or at risk of developing bone mineral density loss, the method comprising administering to the subject a KREMEN1 inhibitor.

[0034] The present disclosure also provides a method of treating a subject having or at risk of developing osteopenia, the method comprising administering to the subject a KREMEN1 inhibitor. The present disclosure also provides a method of treating a subject having or at risk of developing type I osteoporosis, the method comprising administering to the subject a KREMEN1 inhibitor.

[0035] The present disclosure also provides a method of treating a subject having or at risk of developing type II osteoporosis, the method comprising administering to the subject KREMEN1. The present disclosure also provides a method of treating a subject having or at risk of developing secondary osteoporosis, the method comprising administering to the subject a KREMEN1 inhibitor.

[0036] In some embodiments, the KREMEN1 inhibitor comprises an inhibitory nucleic acid molecule. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNA (siRNA), and short hairpin RNA (shRNA). Such inhibitory nucleic acid molecules can be designed to target any region of the KREMEN1 nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes with a sequence in a KREMEN1 genomic nucleic acid molecule or mRNA molecule and reduces the expression of a KREMEN1 polypeptide in a cell of a subject. In some embodiments, the KREMEN1 inhibitor comprises an antisense molecule that hybridizes with a KREMEN1 genomic nucleic acid molecule or mRNA molecule and reduces the expression of a KREMEN1 polypeptide in a cell of a subject. In some embodiments, the KREMEN1 inhibitor comprises an siRNA that hybridizes with a KREMEN1 genomic nucleic acid molecule or mRNA molecule and reduces the expression of a KREMEN1 polypeptide in a cell of a subject. In some embodiments, KREMEN1 inhibitors include shRNAs that hybridize with KREMEN1 genomic nucleic acid molecules or mRNA molecules and reduce expression of KREMEN1 polypeptide in cells of a subject.

[0037] The inhibitory nucleic acid molecule can comprise RNA, DNA, or both RNA and DNA. The inhibitory nucleic acid molecule can also be linked or fused to a heterologous nucleic acid sequence, for example in a vector, or a heterologous label. For example, the inhibitory nucleic acid molecule can be present as an exogenous donor sequence in or containing a vector that contains the inhibitory nucleic acid molecule and the heterologous nucleic acid sequence. The inhibitory nucleic acid molecule can also be linked or fused to a heterologous label. The label can be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, an enzyme, or a fluorophore quencher). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme, whereby an enzyme-dependent secondary generation of a signal occurs. The term "label" can also refer to a "tag" or hapten that can be selectively attached to a binding molecule such that the binding molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag together with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and probed using a colorimetric (e.g., tetramethylbenzidine (TMB)) or fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3xFLAG, 6xHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their colorimetric, fluorescent and chemiluminescent substrates, as well as other labels.

[0038] Inhibitory nucleic acid molecules can include, for example, nucleotides, or non-natural or modified nucleotides, such as, for example, nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or nucleotides that incorporate non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides.

[0039] The inhibitory nucleic acid molecule can also include one or more nucleotide analogs or nucleotide substitutes. A nucleotide analog is a nucleotide that contains a modification to either the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, as well as various purine or pyrimidine bases, such as, for example, pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (cytosine), and thymine. Examples of uracils and cytosines include, but are not limited to, 4-isopropyl uracil, 4-isopropyl uracil, 8 ...

[0040] Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose, as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C-alkyl groups. 1~10 Alkyl or C 2~10 Alkenyl, and C 2~10 Exemplary 2' sugar modifications include -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON [(CH2) n Other modifications at the 2' position include, but are not limited to, C 1~10Examples of suitable substituents include, but are not limited to, alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications may be made at other positions on the sugar, particularly the 3' position of the sugar in the 3' terminal nucleotide or 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Modified sugars can also include those containing modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0041] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those in which the linkage between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. Such phosphate or modified phosphate linkages between two nucleotides can be via 3'-5' or 2'-5' linkages, and the linkages can contain reverse polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNAs).

[0042] In some embodiments, the antisense nucleic acid molecule is a gapmer, whereby the first 1-7 nucleotides of the 5'-end and the 3'-end, respectively, have a 2'-methoxyethyl (2'-MOE) modification. In some embodiments, the first 5 nucleotides of the 5'-end and the 3'-end, respectively, have a 2'-MOE modification. In some embodiments, the first 1-7 nucleotides of the 5'-end and the 3'-end are RNA nucleotides. In some embodiments, the first 5 nucleotides of the 5'-end and the 3'-end are RNA nucleotides. In some embodiments, each of the internucleotide backbone linkages is a phosphorothioate linkage.

[0043] In some embodiments, the siRNA molecule has terminal modification.In some embodiments, the 5'-end of the antisense strand is phosphorylated.In some embodiments, a 5'-phosphate analog that cannot be hydrolyzed, such as 5'-(E)-vinyl-phosphonate, is used.

[0044] In some embodiments, the siRNA molecule has a backbone modification. In some embodiments, modified phosphodiester groups linking consecutive ribose nucleosides have been shown to increase the stability and bioavailability of siRNA in vivo. Non-ester groups (-OH, =O) of the phosphodiester bond can be replaced with sulfur, boron, or acetate to obtain phosphorothioate, boranophosphate, and phosphonoacetate linkages. In addition, the phosphodiester group can be replaced with a phosphotriester to facilitate cellular uptake of the siRNA and retention in serum components by removing its negative charge. In some embodiments, the siRNA molecule has a sugar modification. In some embodiments, the sugar is deprotonated (a reaction catalyzed by exonucleases and endonucleases), allowing the 2'-hydroxyl to act as a nucleophile and attack the adjacent phosphorus of the phosphodiester bond. Such alternatives include 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro modifications.

[0045] In some embodiments, the siRNA molecule has base modifications, in some embodiments, the bases may be replaced with modified bases such as pseudouridine, 5'-methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

[0046] In some embodiments, siRNA molecules are bound to lipid.Lipid can be bound to 5'-end or 3'-end of siRNA, and can improve their bioavailability in vivo by associating with serum lipoprotein.Representative lipids include, but are not limited to, cholesterol and vitamin E, and fatty acids such as palmitic acid and tocopherol.

[0047] In some embodiments, an exemplary siRNA has the following formula: Sense: mN*mN* / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / *mN* / 32FN / Antisense: / 52FN / * / i2FN / *mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN*N*N where "N" is a base; "2F" is a 2'-F modification; "m" is a 2'-O-methyl modification; "I" is an internal base; and "*" is a phosphorothioate backbone linkage.

[0048] The present disclosure also provides a vector comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the vector comprises any one or more of the inhibitory nucleic acid molecules and a heterologous nucleic acid. The vector can be a viral vector or a non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or a cosmid (such as a circular double-stranded DNA to which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector to which additional DNA segments can be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast artificial chromosomes (YACs), Epstein-Barr (EBV) derived episomes, and other expression vectors known in the art.

[0049] The present disclosure also provides compositions comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and / or excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules. The carrier may comprise a buffered salt solution such as PBS, HBSS, and the like.

[0050] Exemplary KREMEN1 inhibitors include, but are not limited to, KREMEN2 (Sumia et al., Cell Death Discovery, 2019, 5, 91) and its ligand Dickkopf-1 (DKK-1), a secreted glycoprotein, and R-Spondin1.

[0051] In some embodiments, the KREMEN1 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks in the recognition sequence(s) in the KREMEN1 genomic nucleic acid molecule or in a DNA binding protein that binds to the recognition sequence. The recognition sequence can be located in the coding region of the KREMEN1 gene or in a regulatory region that affects the expression of the gene. The recognition sequence of the DNA binding protein or nuclease agent can be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region. The recognition sequence can include or be adjacent to the start codon of the KREMEN1 gene. For example, the recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon. As another example, two or more nuclease agents can be used, each of which targets a nuclease recognition sequence that includes or is adjacent to the start codon. As another example, two nuclease agents can be used, one targeting a nuclease recognition sequence containing or adjacent to a start codon, and the other targeting a nuclease recognition sequence containing or adjacent to a stop codon, and cleavage by these nuclease agents can result in the deletion of the coding region between the two nuclease recognition sequences.Any nuclease agent that induces a nick or double-strand break at the desired recognition sequence can be used in the methods and compositions disclosed herein.Any DNA binding protein that binds to the desired recognition sequence can be used in the methods and compositions disclosed herein.

[0052] Suitable nuclease agents and DNA binding proteins for use herein include, but are not limited to, zinc finger proteins or zinc finger nuclease (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The length of the recognition sequence can vary, but includes, for example, recognition sequences that are about 30-36 bp for zinc finger proteins or ZFN pairs, about 15-18 bp for each ZFN, about 36 bp for TALE proteins or TALEN, and about 20 bp for CRISPR / Cas guide RNA.

[0053] In some embodiments, the CRISPR / Cas system can be used to modify the KREMEN1 genomic nucleic acid molecule in a cell. The methods and compositions disclosed herein can employ the CRISPR-Cas system by utilizing a CRISPR complex (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of the KREMEN1 nucleic acid molecule.

[0054] Cas proteins generally contain at least one RNA recognition domain or RNA binding domain that can interact with gRNA. Cas proteins can also contain nuclease domains (e.g., DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 proteins and wild-type Cpf1 proteins (e.g., FnCpf1). Cas proteins can have full cleavage activity to create double-stranded breaks in KREMEN1 genomic nucleic acid molecules, or can be nickases that create single-stranded breaks in KREMEN1 genomic nucleic acid molecules. Additional examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cas proteins include, but are not limited to, Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, as well as homologs or variants thereof. Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, Cas proteins can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Cas proteins can be provided in any form. For example, the Cas protein can be provided in the form of a protein, e.g., a Cas protein complexed with a gRNA.Alternatively, the Cas protein can be provided in the form of a nucleic acid molecule, e.g., RNA or DNA, encoding the Cas protein.

[0055] In some embodiments, targeted genetic modification of KREMEN1 genomic nucleic acid molecule can be generated by contacting a cell with Cas protein and one or more gRNAs that hybridize with one or more gRNA recognition sequences in a target genomic locus in KREMEN1 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located within the region of SEQ ID NO: 1. The gRNA recognition sequence can include or be adjacent to the start codon of KREMEN1 genomic nucleic acid molecule or the stop codon of KREMEN1 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from the start codon or the stop codon.

[0056] The gRNA recognition sequence in the target genomic locus in the KREMEN1 genomic nucleic acid molecule is located near a protospacer adjacent motif (PAM) sequence, which is a 2-6 base pair DNA sequence that immediately follows the DNA sequence targeted by the Cas9 nuclease. A canonical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleobases. The gRNA can transport Cas9 anywhere in the genome for gene editing, but cannot edit at sites other than the site where Cas9 recognizes the PAM. In addition, 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells. Generally, the PAM is about 2-6 nucleotides downstream of the DNA sequence targeted by the gRNA. The PAM can be adjacent to the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM at the 3' end. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM at the 5' end. For example, the cleavage site of the Cas protein can be about 1 to about 10, about 2 to about 5, or 3 base pairs upstream or downstream of the PAM sequence. In some embodiments (e.g., when using Cas9 from S. pyogenes or a closely related Cas9), the PAM sequence of the non-complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and is immediately 3' to the gRNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand is 5'-CCN-3', where N is any DNA nucleotide and is immediately 5' to the gRNA recognition sequence of the complementary strand of the target DNA.

[0057] gRNA is an RNA molecule that binds to Cas protein and targets Cas protein to a specific location in KREMEN1 genomic nucleic acid molecule.Exemplary gRNA is an effective gRNA that induces Cas enzyme to bind to or cleave KREMEN1 genomic nucleic acid molecule, and wherein gRNA comprises a DNA targeting segment that hybridizes with gRNA recognition sequence in KREMEN1 genomic nucleic acid molecule.Exemplary gRNA comprises a DNA targeting segment that hybridizes with gRNA recognition sequence present in KREMEN1 genomic nucleic acid molecule that includes or is adjacent to start codon or stop codon. For example, a gRNA can be selected to hybridize with a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from a start codon, or with a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from a stop codon. Suitable gRNAs can include about 17 to about 25 nucleotides, about 17 to about 23 nucleotides, about 18 to about 22 nucleotides, or about 19 to about 21 nucleotides. In some embodiments, the gRNA can include 20 nucleotides.

[0058] Examples of suitable gRNA recognition sequences located within the human KREMEN1 reference gene are set forth in Table 1 as SEQ ID NOs: 17-36.

[0059] [Table 1]

[0060] The Cas protein and gRNA form a complex, and the Cas protein cuts the target KREMEN1 genomic nucleic acid molecule. The Cas protein can cut the nucleic acid molecule at a site inside or outside the nucleic acid sequence present in the target KREMEN1 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds. For example, the formation of a CRISPR complex (including the gRNA hybridized with the gRNA recognition sequence and complexed with the Cas protein) can cause one or both strands to be cut within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 50 or more base pairs) the nucleic acid sequence present in the KREMEN1 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds.

[0061] Such a method can produce a KREMEN1 genomic nucleic acid molecule in which, for example, a region of SEQ ID NO:1 is destroyed, the start codon is destroyed, the stop codon is destroyed, or the coding sequence is destroyed or deleted. Optionally, the cell can be further contacted with one or more additional gRNAs that hybridize with additional gRNA recognition sequences in the target genomic locus of the KREMEN1 genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (e.g., a second gRNA that hybridizes with a second gRNA recognition sequence), the cleavage by Cas protein can create two or more double-strand breaks or two or more single-strand breaks.

[0062] In some embodiments, the method of treatment further comprises detecting the presence or absence of a KREMEN1 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 in a biological sample from the subject. As used throughout this disclosure, a "KREMEN1 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1" is any KREMEN1 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes a KREMEN1 polypeptide having partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function.

[0063] The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or prevents bone mineral density loss, where the subject has or is at risk of developing bone mineral density loss. In some embodiments, the subject has bone mineral density loss. In some embodiments, the subject is at risk of developing bone mineral density loss. In some embodiments, the method comprises determining whether the subject has a KREMEN1 variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of KREMEN1 by obtaining or obtaining a biological sample from the subject and performing or performing a sequence analysis on the biological sample to determine whether the subject has a genotype that includes a KREMEN1 variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of KREMEN1. In some embodiments, the method further comprises administering or continuing to administer a therapeutic agent that treats or prevents bone mineral density loss at a standard dose to the subject who is a KREMEN1 norm, and / or administering a KREMEN1 inhibitor to the subject. In some embodiments, the method further comprises administering or continuing to administer a therapeutic agent for treating or preventing bone mineral density loss to a subject who is heterozygous for the KREMEN1 variant nucleic acid molecule at a dose equal to or less than the standard dose, and / or administering a KREMEN1 inhibitor to the subject. In some embodiments, the method further comprises administering or continuing to administer a therapeutic agent for treating or preventing bone mineral density loss to a subject who is homozygous for the KREMEN1 variant nucleic acid molecule at a dose equal to or less than the standard dose. The presence of a genotype having a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 indicates that the subject has a low risk of developing bone mineral density loss. In some embodiments, the subject is KREMEN1 standard. In some embodiments, the subject is heterozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1.

[0064] For subjects who have been genotyped or determined to be heterozygous for a KREMEN1 variant nucleic acid molecule that is a KREMEN1 standard or encodes a predicted loss-of-function polypeptide of KREMEN1, such subjects can be treated with a KREMEN1 inhibitor as described herein.

[0065] Detecting the presence or absence of a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 in a biological sample from a subject and / or determining whether a subject has a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from the subject.

[0066] In some embodiments, if the subject is KREMEN1 normative, the subject is administered a standard dose of a therapeutic agent for treating or preventing bone mineral density loss. In some embodiments, if the subject is heterozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1, the subject is administered a standard dose or a lower dose of a therapeutic agent for treating or preventing bone mineral density loss.

[0067] In some embodiments, the method of treatment further comprises detecting the presence or absence of a predicted loss-of-function polypeptide of KREMEN1 in a biological sample from the subject. In some embodiments, if the subject does not have a predicted loss-of-function polypeptide of KREMEN1, the subject is administered a standard dose of a therapeutic agent for treating or preventing bone mineral density loss. In some embodiments, if the subject has a predicted loss-of-function polypeptide of KREMEN1, the subject is administered a standard dose of a therapeutic agent for treating or preventing bone mineral density loss.

[0068] The disclosure also provides a method of treating a subject with a therapeutic agent that treats or prevents bone mineral density loss, where the subject has or is at risk of developing bone mineral density loss. In some embodiments, the subject has bone mineral density loss. In some embodiments, the subject is at risk of developing bone mineral density loss. In some embodiments, the method includes determining whether the subject has a predicted loss-of-function polypeptide of KREMEN1 by obtaining or obtaining a biological sample from the subject and performing or performing an assay on the biological sample to determine whether the subject has a predicted loss-of-function polypeptide of KREMEN1. If the subject does not have a predicted loss-of-function polypeptide of KREMEN1, a therapeutic agent that treats or prevents bone mineral density loss is administered or continues to be administered to the subject at a standard dose, and / or a KREMEN1 inhibitor is administered to the subject. If the subject has a predicted loss-of-function polypeptide of KREMEN1, a therapeutic agent for treating or preventing bone mineral density loss is administered or continues to be administered to the subject at the same or lower dose as the standard dose, and / or a KREMEN1 inhibitor is administered to the subject. The presence of a predicted loss-of-function polypeptide of KREMEN1 indicates that the subject has a low risk of developing bone mineral density loss. In some embodiments, the subject has a predicted loss-of-function polypeptide of KREMEN1. In some embodiments, the subject does not have a predicted loss-of-function polypeptide of KREMEN1.

[0069] Detecting the presence or absence of a predicted loss-of-function KREMEN1 polypeptide in a biological sample from a subject and / or determining whether a subject has a predicted loss-of-function KREMEN1 polypeptide can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the polypeptide can be present in a cell obtained from the subject.

[0070] Examples of therapeutic agents for treating or preventing bone mineral density loss include, but are not limited to, calcium and vitamin D supplementation (vitamin D2, vitamin D3, and cholecalciferol), bisphosphonates such as FOSAMAX® (alendronate), BONIVA® (ibandronate), RECLAST® (zoledronate), ACTONEL® (risedronate), MIACALCIN®, FORTICAL®, and CALCIMAR® (calcitonin), FORTEO® (teriparatide), PROLIA® (denosumab), hormone replacement therapy with estrogen and progesterone, and EVISTA® (raloxifene). In some embodiments, the therapeutic agent for treating or preventing bone mineral density loss is vitamin D2, vitamin D3, cholecalciferol, alendronate, ibandronate, zoledronate, risedronate, calcitonin, teriparatide, denosumab, EVENITY® (romosozumab), or raloxifene. In some embodiments, the therapeutic agent for treating or preventing bone mineral density loss is vitamin D2. In some embodiments, the therapeutic agent for treating or preventing bone mineral density loss is vitamin D3. In some embodiments, the therapeutic agent for treating or preventing bone mineral density loss is cholecalciferol. In some embodiments, the therapeutic agent for treating or preventing bone mineral density loss is alendronate. In some embodiments, the therapeutic agent for treating or preventing bone mineral density loss is ibandronate. In some embodiments, the therapeutic agent for treating or preventing bone mineral density loss is zoledronate. In some embodiments, the therapeutic agent for treating or preventing bone mineral density loss is risedronate. In some embodiments, the therapeutic agent for treating or preventing bone mineral density loss is calcitonin. In some embodiments, the therapeutic agent for treating or preventing bone mineral density loss is teriparatide. In some embodiments, the therapeutic agent for treating or preventing bone mineral density loss is denosumab.In some embodiments, the therapeutic agent for treating or preventing bone mineral density loss is raloxifene.

[0071] In some embodiments, the dose of a therapeutic agent for treating or preventing bone mineral density loss can be reduced (i.e., less than the standard dose) for subjects who are heterozygous for KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1 compared to subjects who are KREMEN1 norms (who may receive standard doses). In some embodiments, the dose of a therapeutic agent for treating or preventing bone mineral density loss can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, subjects who are heterozygous for KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1 can be administered less frequently compared to subjects who are KREMEN1 norms.

[0072] In some embodiments, the dose of a therapeutic agent for treating or preventing bone mineral density loss can be reduced by about 10%, about 20%, about 30%, about 40%, about 50% for a subject homozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1, compared to a subject heterozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1. In some embodiments, the dose of a therapeutic agent for treating or preventing bone mineral density loss can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, the dose of a therapeutic agent for treating or preventing bone mineral density loss in a subject homozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 can be administered less frequently than a subject heterozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1.

[0073] Administration of the therapeutic agent and / or KREMEN1 inhibitor for treating or preventing bone mineral density loss can be repeated, for example, after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, or 3 months. Repeated administration can be the same dose or different doses. Administration can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. For example, according to a particular dosing regimen, a subject can be treated for an extended period of time, for example, 6 months, 1 year, or more.

[0074] Administration of the therapeutic agent and / or KREMEN1 inhibitor for treating or preventing bone mineral density loss can occur by any suitable route, including, but not limited to, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are desirably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a single dose for administration). Pharmaceutical compositions can be formulated using one or more physiologically and pharmacologic acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration selected. The term "pharmaceutical acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially harmful to the recipient thereof.

[0075] As used herein, the terms "treat", "treating", and "treatment" and "prevent", "preventing", and "prevention" refer to eliciting a desired biological response, such as a therapeutic effect and a prophylactic effect, respectively. In some embodiments, a therapeutic effect includes one or more of the following following administration of an agent or a composition comprising an agent: reduction / alleviation of bone mineral density loss, reduction / alleviation of the severity of bone mineral density loss (e.g., reduction or inhibition of onset of bone mineral density loss), reduction / alleviation of symptoms and bone mineral density loss related effects, delaying the onset of symptoms and bone mineral density loss related effects, reducing the severity of symptoms of bone mineral density loss related effects, reducing the number of symptoms and bone mineral density loss related effects, reducing the latency of symptoms and bone mineral density loss related effects, ameliorating symptoms and bone mineral density loss related effects, reducing secondary symptoms, reducing secondary infections, preventing recurrence of bone mineral density loss, reducing the number or frequency of recurrent episodes, increasing the latency period between symptomatic episodes, increasing the time to sustained progression, accelerating recovery, or increasing the effectiveness or reducing resistance to alternative therapeutic agents, and / or increasing the survival time of an affected host animal. A prophylactic effect may include complete or partial avoidance / inhibition or delay (e.g., complete or partial avoidance / inhibition or delay) of the onset / progression of bone mineral density loss following administration of a treatment protocol, and increasing the survival time of an affected host animal. Treatment of bone mineral density loss includes treatment of subjects already diagnosed with some form of bone mineral density loss, either at a clinical stage or clinical symptoms, delaying the onset or progression or progression or worsening of symptoms or signs of bone mineral density loss, and / or preventing and / or reducing the severity of bone mineral density loss.

[0076] The present disclosure also provides a method for identifying a subject at high risk of developing bone mineral density loss. In some embodiments, the method includes determining or having determined the presence or absence of a KREMEN1 variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule and / or a cDNA molecule) encoding a predicted loss-of-function polypeptide of KREMEN1 in a biological sample obtained from the subject. If the subject lacks a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 (i.e., the subject is classified as KREMEN1 standard by genotyping), the subject has a high risk of developing bone mineral density loss. If the subject has a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 (i.e., the subject is heterozygous or homozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1), the subject has a low risk of developing bone mineral density loss.

[0077] Having a single copy of a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 further protects a subject from developing bone mineral density loss than not having a copy of a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1. Without intending to be limited to a particular theory or mechanism of action, it is believed that a single copy of a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 (i.e., heterozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1) protects a subject from developing bone mineral density loss, and it is also believed that having two copies of a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 (i.e., homozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1) may further protect a subject from developing bone mineral density loss compared to a subject having a single copy. Thus, in some embodiments, a single copy of a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 may not completely protect a subject from developing bone mineral density loss, but may instead provide partial or incomplete protection. Without wishing to be bound by any particular theory, there may be additional factors or molecules involved in the development of bone mineral density loss that are still present in a subject having a single copy of a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1, which may result in less than complete protection from the development of bone mineral density loss.

[0078] Determining whether a subject has a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 in a biological sample from a subject and / or determining whether a subject has a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from the subject.

[0079] In some embodiments, once a subject is identified as having a high risk of developing bone mineral density loss, the subject is treated with a therapeutic agent for treating or preventing bone mineral density loss, as described herein, and / or a KREMEN1 inhibitor. For example, if a subject is KREMEN1 criterion and therefore has a high risk of developing bone mineral density loss, the subject is administered a KREMEN1 inhibitor. In some embodiments, such a subject is also administered a therapeutic agent for treating or preventing bone mineral density loss. In some embodiments, if a subject is heterozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1, the subject is administered a therapeutic agent for treating or preventing bone mineral density loss at a dose equal to or less than the standard dose, and is also administered a KREMEN1 inhibitor. In some embodiments, such a subject is also administered a therapeutic agent for treating or preventing bone mineral density loss. In some embodiments, if the subject is homozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1, the subject is administered a therapeutic agent for treating or preventing bone mineral density loss at a dose equal to or less than the standard dose. In some embodiments, the subject is a KREMEN1 standard. In some embodiments, the subject is heterozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1. In some embodiments, the subject is homozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1.

[0080] In some embodiments, any of the methods described herein may further comprise determining the total burden of the subject having a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 associated with a reduced risk of developing bone mineral density loss and / or a predicted loss-of-function variant polypeptide of KREMEN1. The gene burden is the sum of all variants of the KREMEN1 gene, which may be performed in an association analysis with bone mineral density loss. In some embodiments, the subject is homozygous for one or more KREMEN1 variant nucleic acid molecules encoding a predicted loss-of-function polypeptide of KREMEN1 associated with a reduced risk of developing bone mineral density loss. In some embodiments, the subject is heterozygous for one or more KREMEN1 variant nucleic acid molecules encoding a predicted loss-of-function polypeptide of KREMEN1 associated with a reduced risk of developing bone mineral density loss. The results of the association analysis suggest that the KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 is associated with a reduced risk of developing bone mineral density loss. If the subject has a lower total load, the subject is at higher risk of developing bone mineral density loss, and the subject is administered or continues to be administered a standard dose of a therapeutic agent for treating or preventing bone mineral density loss.If the subject has a higher total load, the subject is at lower risk of developing bone mineral density loss, and the subject is administered or continues to be administered a standard dose of a therapeutic agent for treating or preventing bone mineral density loss.The higher the total load, the lower the risk of developing bone mineral density loss.

[0081] KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1 and / or predicted loss-of-function mutant polypeptides of KREMEN1 used to determine a subject's total load include, but are not limited to, the variants listed in FIG. 3, Table 2, or listed elsewhere herein.

[0082] In some embodiments, the total load of subjects having any one or more KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1 represents a weighted sum of any multiple of the KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1. In some embodiments, the total burden is calculated using at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 10,000, at least about 100,000, or at least about 1,000,000 or more genetic variants present in or surrounding the KREMEN1 gene (up to 10 Mb), where the genetic burden is the number of alleles multiplied by the estimated association for each allele with bone mineral density loss or related outcome (e.g., a weighted polygenic burden score). This can include any genetic variants that are close to the KREMEN1 gene (up to 10Mb around the gene) that show non-zero association with bone mineral density loss related traits in gene association analysis, regardless of genome annotation.In some embodiments, if a subject has a total load higher than a desired threshold score, the subject has a low risk of developing bone mineral density loss.In some embodiments, if a subject has a total load lower than a desired threshold score, the subject has a high risk of developing bone mineral density loss.

[0083] In some embodiments, the total burden can be divided into quintiles, e.g., top quintile, middle quintile, and bottom quintile, with the top quintile of total burden corresponding to the lowest risk group and the bottom quintile of total burden corresponding to the highest risk group. In some embodiments, subjects with a larger total burden include the highest weighted total burden, including but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% total burden from the subject population. In some embodiments, the genetic variants include genetic variants that have an association with bone mineral density loss in the top 10%, top 20%, top 30%, top 40%, or top 50% of the p-value range for the association. In some embodiments, each of the identified genetic variants is within about 10 -2 , about 10 -3 , about 10 -4 , about 10 -5 , about 10 -6 , about 10 -7 , about 10 -8 , about 10 -9 , about 10 -10 , about 10 -11 , about 10 -12 , about 10 -13 , about 10 -14 , or about 10 -15 In some embodiments, the identified genetic variants are those with an association with reduced bone mineral density at a p-value of 5×10 -8In some embodiments, the identified genetic variants include genetic variants that are associated with bone mineral density loss in subjects at high risk compared to the remainder of a reference population with an odds ratio (OR) of about 1.5 or more, about 1.75 or more, about 2.0 or more, or about 2.25 or more for the top 20% of the distribution; or about 1.5 or more, about 1.75 or more, about 2.0 or more, about 2.25 or more, about 2.5 or more, or about 2.75 or more. In some embodiments, the odds ratio (OR) may range from about 1.0 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, or may be greater than 7.0. In some embodiments, high-risk subjects include subjects with a total burden in the bottom decile, quintile, or tertile in the reference population. The total burden threshold is determined based on the nature of the intended practical application and the risk difference that is considered meaningful for that practical application.

[0084] In some embodiments, once a subject is identified as having a high risk of developing bone mineral density loss, the subject is treated with a therapeutic agent for treating or preventing bone mineral density loss, as described herein, and / or a KREMEN1 inhibitor. For example, if a subject is KREMEN1-based and therefore has a high risk of developing bone mineral density loss, the subject is administered a KREMEN1 inhibitor. In some embodiments, such a subject is administered a therapeutic agent for treating or preventing bone mineral density loss. In some embodiments, if the subject is heterozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1, the subject is administered a therapeutic agent for treating or preventing bone mineral density loss at a dose equal to or less than the standard dose, and is also administered a KREMEN1 inhibitor. In some embodiments, the subject is KREMEN1-based. In some embodiments, the subject is heterozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1. Furthermore, if a subject has a lower overall burden of having KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1 and therefore has a higher risk of developing bone mineral density loss, the subject is administered a therapeutic agent for treating or preventing bone mineral density loss. In some embodiments, if a subject has a lower overall burden of having KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1, the subject is administered a therapeutic agent for treating or preventing bone mineral density loss at a dose equal to or higher than the standard dose administered to a subject with a higher overall burden of having KREMEN1 variant nucleic acid molecules encoding predicted loss-of-function polypeptides of KREMEN1.

[0085] The present disclosure also provides a method for detecting the presence or absence of KREMEN1 variant genomic nucleic acid molecules (i.e., genomic nucleic acid molecules, mRNA molecules, or cDNA molecules generated from mRNA molecules) that code for predicted loss-of-function polypeptides of KREMEN1 in a biological sample from a subject. It is understood that gene sequences within a population, and the mRNA molecules encoded by such genes, may differ due to polymorphisms, such as single nucleotide polymorphisms. The sequences provided herein for KREMEN1 variant genomic nucleic acid molecules, KREMEN1 variant mRNA molecules, and KREMEN1 variant cDNA molecules are merely exemplary sequences. Other sequences for KREMEN1 variant genomic nucleic acid molecules, variant mRNA molecules, and variant cDNA molecules are also possible.

[0086] The biological sample can be derived from any cell, tissue, or biological fluid of a subject. The biological sample may include any clinically relevant tissue, such as a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of a bodily fluid, such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine. In some cases, the sample includes a buccal swab. The biological sample used in the methods disclosed herein may vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. The biological sample may be treated differently depending on the assay employed. For example, when detecting KREMEN1 mutant nucleic acid molecules that encode predicted loss-of-function polypeptides of KREMEN1, a pretreatment designed to isolate or enrich the biological sample for genomic DNA may be employed. For this purpose, various techniques may be used. When detecting the level of KREMEN1 mutant mRNA molecules, various techniques may be used to enrich the biological sample with mRNA molecules. Various methods may be used to detect the presence or level of mRNA molecules, or the presence of a particular mutant genomic DNA locus.

[0087] In some embodiments, detecting a KREMEN1 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1 in a subject comprises performing sequence analysis on a biological sample obtained from the subject to determine whether a KREMEN1 genomic nucleic acid molecule in the biological sample, and / or a KREMEN1 mRNA molecule in the biological sample, and / or a KREMEN1 cDNA molecule generated from the mRNA molecule in the biological sample contains one or more mutations that cause or are predicted to cause loss-of-function (partial or complete).

[0088] In some embodiments, a method for detecting the presence or absence of a KREMEN1 variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule generated from an mRNA molecule) encoding a predicted loss-of-function polypeptide of KREMEN1 in a subject includes performing an assay on a biological sample obtained from the subject, the assay determining whether a nucleic acid molecule in the biological sample comprises a particular nucleotide sequence.

[0089] In some embodiments, the biological sample comprises cells or cell lysates. Such methods can further comprise, for example, obtaining a biological sample from a subject that comprises KREMEN1 genomic nucleic acid molecules or mRNA molecules, and, if mRNA, optionally reverse transcribing the mRNA into cDNA. Such assays can comprise, for example, determining the identity of these positions of a particular KREMEN1 nucleic acid molecule. In some embodiments, the methods are in vitro methods.

[0090] In some embodiments, the determining, detecting, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a KREMEN1 genomic nucleic acid molecule, a KREMEN1 mRNA molecule, or a KREMEN1 cDNA molecule in the biological sample, wherein the portion that is sequenced contains one or more mutations that cause or are predicted to cause a loss of function (partial or complete).

[0091] In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only the KREMEN1 genomic nucleic acid molecule is analyzed. In some embodiments, only the KREMEN1 mRNA is analyzed. In some embodiments, only the KREMEN1 cDNA obtained from the KREMEN1 mRNA is analyzed.

[0092] Mutation-specific polymerase chain reaction techniques can be used to detect mutations such as SNPs in nucleic acid sequences. Mutation-specific primers can be used because DNA polymerase will not extend if there is a mismatch with the template.

[0093] In some embodiments, the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the nucleic acid molecule is present in a cell obtained from the subject.

[0094] In some embodiments, the assay involves contacting the biological sample with a primer or probe, such as a mutation-specific primer or a mutation-specific probe, that specifically hybridizes to a KREMEN1 mutant genomic sequence, mutant mRNA sequence, or mutant cDNA sequence under stringent conditions but does not specifically hybridize to the corresponding KREMEN1 reference sequence, and determining whether hybridization occurs.

[0095] In some embodiments, the determining, detecting, or sequence analyzing step includes: a) amplifying at least a portion of a nucleic acid molecule encoding a KREMEN1 polypeptide; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support containing a mutation-specific probe; and d) detecting the detectable label.

[0096] In some embodiments, the assay involves RNA sequencing (RNA-Seq). In some embodiments, the assay also involves reverse transcribing mRNA into cDNA, for example, by reverse transcription polymerase chain reaction (RT-PCR).

[0097] In some embodiments, the method utilizes probes and primers of sufficient nucleotide length to bind to the target nucleotide sequence and specifically detect and / or identify polynucleotides including mutant genomic nucleic acid molecules, mutant mRNA molecules, or mutant cDNA molecules of KREMEN1. Hybridization or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length that is sufficient for use in an optimal detection method, including any assay described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under highly stringent hybridization conditions. The probes and primers can have complete nucleotide sequence identity to consecutive nucleotides within the target nucleotide sequence, although probes that are different from the target nucleotide sequence and retain the ability to specifically detect and / or identify the target nucleotide sequence can be designed by conventional methods. The probes and primers can have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity or complementarity to the nucleotide sequence of the target nucleic acid molecule.

[0098] Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods include nucleic acid hybridization methods other than sequencing (fluorescence in situ hybridization (FISH)), including the use of labeled primers or labeled probes on purified DNA, amplified DNA, and fixed cell preparations. In some methods, the target nucleic acid molecule may be amplified prior to or simultaneously with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

[0099] Hybridization techniques can employ stringent conditions so that the probe or primer specifically hybridizes with its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other non-target sequences, for example, at least 2-fold, at least 3-fold, at least 4-fold or more above background, including more than 10-fold above background. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 2-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 3-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 4-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater extent than other nucleotide sequences, more than 10-fold above background. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0100] Suitable stringent conditions that promote DNA hybridization, such as 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by a 2x SSC wash at 50°C, are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection include conditions in which the salt concentration is less than about 1.5 M NaCl at pH 7.0-8.3. +ion, usually about 0.01 to 1.0 M Na + The conditions will be ionic concentration (or other salts) and temperature of at least about 30° C. for short probes (e.g., 10-50 nucleotides) and at least about 60° C. for longer probes (e.g., more than 50 nucleotides). Stringent conditions may be achieved by the addition of destabilizing agents such as formamide. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash will be at least long enough to reach equilibrium.

[0101] In some embodiments, such isolated nucleic acid molecules comprise at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55 , at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, or at least about 5000 nucleotides. In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 10 to about 35, about 10 to about 30, about 10 to about 25, about 12 to about 30, about 12 to about 28, about 12 to about 24, about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about 18 to about 22 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 18 to about 30 nucleotides.In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides to at least about 35 nucleotides.

[0102] In some embodiments, such isolated nucleic acid molecules hybridize under stringent conditions with KREMEN1 variant nucleic acid molecules (e.g., genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules). Such nucleic acid molecules can be used, for example, as probes, primers, mutation-specific probes, or mutation-specific primers as described or exemplified herein, including, but not limited to, primers, probes, antisense RNA, shRNA, and siRNA, each of which is described in more detail elsewhere herein and can be used in any of the methods described herein.

[0103] In some embodiments, the isolated nucleic acid molecule hybridizes to at least about 15 contiguous nucleotides of a nucleic acid molecule that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a KREMEN1 variant genomic nucleic acid molecule, a KREMEN1 variant mRNA molecule, and / or a KREMEN1 variant cDNA molecule. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 35 nucleotides.

[0104] In some embodiments, the mutation-specific probe and the mutation-specific primer comprise DNA. In some embodiments, the mutation-specific probe and the mutation-specific primer comprise RNA.

[0105] In some embodiments, the probes and primers described herein (including mutation-specific probes and mutation-specific primers) have nucleotide sequences that specifically hybridize to any of the nucleic acid molecules disclosed herein or their complements. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0106] In some embodiments, the primers can be used in second generation or high throughput sequencing, including mutation specific primers. Sometimes the primers can be modified, including mutation specific primers. In particular, the primers can include various modifications used in various steps of, for example, Massive Parallel Signature Sequencing (MPSS), Polony sequencing, and 454 pyrosequencing. Modified primers can be used in several steps of the process, including biotinylated primers in the cloning step, and fluorescently labeled primers used in the bead loading and detection steps. Polony sequencing is typically performed using paired-end tag libraries, where each molecule of DNA template is about 135 bp in length. Biotinylated primers are used in the bead loading and emulsion PCR steps. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. The adapters can contain 5'-biotin tags for immobilizing the DNA library on streptavidin-coated beads.

[0107] The probes and primers described herein can be used to detect nucleotide variations in any of the KREMEN1 mutant genomic nucleic acid molecules, KREMEN1 mutant mRNA molecules, and / or KREMEN1 mutant cDNA molecules disclosed herein. The primers described herein can be used to amplify KREMEN1 mutant genomic nucleic acid molecules, KREMEN1 mutant mRNA molecules, or KREMEN1 mutant cDNA molecules, or fragments thereof.

[0108] In the context of this disclosure, "specifically hybridizes" means that a probe or primer (e.g., a mutation-specific probe or a mutation-specific primer) does not hybridize to a nucleic acid sequence encoding a KREMEN1-based genomic nucleic acid molecule, a KREMEN1-based mRNA molecule, and / or a KREMEN1-based cDNA molecule.

[0109] In some embodiments, the probe (such as, for example, a mutation-specific probe) comprises a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin. The present disclosure also provides a support comprising a substrate to which any one or more of the probes disclosed herein are attached. A solid support is a solid-state substrate or support to which a molecule such as any of the probes disclosed herein can associate. A form of solid support is an array. Another form of solid support is an array detector. An array detector is a solid support to which a plurality of different probes are attached in an array, grid, or other organized pattern. A form of solid-state substrate is a microtiter dish such as a standard 96-well format. In some embodiments, a multi-well glass slide can be used, usually containing one array per well.

[0110] The nucleotide sequence of the KREMEN1 reference genomic nucleic acid molecule is set forth in SEQ ID NO:1 (ENSG00000183762.13; ENST00000327813.9; chr22:29073118-29168333; or chr22:29073035-29168333 or chr22:29073077-29168333 in the GRCh38 / hg38 human genome assembly).

[0111] The nucleotide sequence of a KREMEN1 reference mRNA molecule is set forth in SEQ ID NO:2. The nucleotide sequence of another KREMEN1 reference mRNA molecule is set forth in SEQ ID NO:3. The nucleotide sequence of another KREMEN1 reference mRNA molecule is set forth in SEQ ID NO:4. The nucleotide sequence of another KREMEN1 reference mRNA molecule is set forth in SEQ ID NO:5. The nucleotide sequence of another KREMEN1 reference mRNA molecule is set forth in SEQ ID NO:6. The nucleotide sequence of another KREMEN1 reference mRNA molecule is set forth in SEQ ID NO:7.

[0112] The nucleotide sequence of a KREMEN1 reference cDNA molecule is set forth in SEQ ID NO:8. The nucleotide sequence of another KREMEN1 reference cDNA molecule is set forth in SEQ ID NO:9. The nucleotide sequence of another KREMEN1 reference cDNA molecule is set forth in SEQ ID NO:10. The nucleotide sequence of another KREMEN1 reference cDNA molecule is set forth in SEQ ID NO:11. The nucleotide sequence of another KREMEN1 reference cDNA molecule is set forth in SEQ ID NO:12. The nucleotide sequence of another KREMEN1 reference cDNA molecule is set forth in SEQ ID NO:13.

[0113] The amino acid sequence of the KREMEN1 reference polypeptide is set forth in SEQ ID NO: 14 and is 492 amino acids in length. The nucleotide sequence of another KREMEN1 reference polypeptide is set forth in SEQ ID NO: 15 and is 458 amino acids in length. The nucleotide sequence of another KREMEN1 reference polypeptide is set forth in SEQ ID NO: 16 and is 473 amino acids in length.

[0114] The genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be derived from any organism. For example, the genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be an ortholog from human or another organism (e.g., non-human mammal, rodent, mouse, or rat). It is understood that gene sequences within a population can differ due to polymorphisms, such as single nucleotide polymorphisms. The examples provided herein are only exemplary sequences. Other sequences are also possible.

[0115] Also provided herein are functional polynucleotides that can interact with the disclosed nucleic acid molecules. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triplex-forming molecules, and external guide sequences. Functional polynucleotides can act as effectors, inhibitors, modulators, and stimulators of the specific activity of target molecules, or functional polynucleotides can have de novo activity independent of any other molecules.

[0116] The isolated nucleic acid molecules disclosed herein can include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules can also be linked or fused to heterologous nucleic acid sequences, for example in a vector, or heterologous labels. For example, the isolated nucleic acid molecules disclosed herein can be present as exogenous donor sequences in or containing a vector that includes the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules can also be linked or fused to heterologous labels. The labels can be directly detectable (e.g., fluorophores) or indirectly detectable (e.g., haptens, enzymes, or fluorophore quenchers). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The labels can also be, for example, chemiluminescent materials; metal-containing materials; or enzymes, where enzyme-dependent secondary generation of a signal occurs. The term "label" can also refer to a "tag" or hapten that can be selectively attached to a binding molecule such that the binding molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag together with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and probed using a colorimetric (e.g., tetramethylbenzidine (TMB)) or fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3xFLAG, 6xHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their colorimetric, fluorescent and chemiluminescent substrates, as well as other labels.

[0117] The isolated nucleic acid molecule or its complement can be present in a host cell. In some embodiments, the host cell can comprise a vector comprising any of the nucleic acid molecules described herein or their complements. In some embodiments, the nucleic acid molecule is operably linked to a promoter active in the host cell. In some embodiments, the promoter is an exogenous promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the host cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a yeast cell. In some embodiments, the host cell is an insect cell. In some embodiments, the host cell is a mammalian cell.

[0118] The disclosed nucleic acid molecules can include, for example, nucleotides, or non-natural or modified nucleotides, such as, for example, nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or nucleotides that incorporate non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides.

[0119] The nucleic acid molecules disclosed herein may also include one or more nucleotide analogs or nucleotide substitutes. A nucleotide analog is a nucleotide that contains modifications to either the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, as well as various purine or pyrimidine bases, such as, for example, pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I), and 2-aminoadenine-9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (cytosine), and thymine. Examples of uracils and cytosines include, but are not limited to, 4-isopropyl uracil, 4-isopropyl uracil, 8 ...

[0120] Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose, as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C-alkyl groups. 1~10 Alkyl or C 2~10 Alkenyl, and C 2~10Exemplary 2' sugar modifications include -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON [(CH2) n Other modifications at the 2' position include, but are not limited to, C 1~10 Examples of suitable substituents include, but are not limited to, alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications may be made at other positions on the sugar, particularly the 3' position of the sugar in the 3' terminal nucleotide or 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Modified sugars can also include those containing modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0121] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those in which the linkage between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. Such phosphate or modified phosphate linkages between two nucleotides can be via 3'-5' or 2'-5' linkages, and the linkages can contain reverse polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNAs).

[0122] The present disclosure also provides a vector comprising any one or more of the nucleic acid molecules disclosed herein. In some embodiments, the vector comprises any one or more of the nucleic acid molecules disclosed herein and a heterologous nucleic acid. The vector can be a viral vector or a non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or a cosmid (such as a circular double stranded DNA into which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast artificial chromosomes (YACs), Epstein-Barr (EBV) derived episomes, and other expression vectors known in the art.

[0123] Desirable regulatory sequences for mammalian host cell expression may include, for example, viral elements directing high levels of polypeptide expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), Simian Virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma derived promoters and / or enhancers, as well as strong mammalian promoters, such as native immunoglobulin promoters and actin promoters. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known. The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter).

[0124] Percent identity (%) (or percent complementarity) between specific stretches of nucleotide sequences in nucleic acid molecules or amino acid sequences in polypeptides can be routinely determined using the BLAST program (base local sequence comparison search tool) and PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings that use the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). When referring to percent sequence identity in this specification, a higher percent sequence identity is preferred over a lower one.

[0125] As used herein, the phrase "corresponding to" or grammatical variations thereof, when used in the context of numbering a particular nucleotide or sequence of nucleotides or position, refers to the numbering of a specified reference sequence when the particular nucleotide or sequence of nucleotides is compared to a reference sequence (e.g., SEQ ID NO:1). In other words, the residue (e.g., nucleotide or amino acid) number or residue (e.g., nucleotide or amino acid) position of a particular polymer is specified with reference to a reference sequence, not by the actual position number of the residue within the particular nucleotide or nucleotide sequence. For example, a particular nucleotide sequence can be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, although gaps exist, the numbering of the residues in the particular nucleotide or nucleotide sequence is done with reference to the reference sequence to which it is aligned.

[0126] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5'-end of the sequence and proceeding toward the 3'-end (i.e., from left to right in each sequence). Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by reference to the shown strand. The amino acid sequences follow the standard convention of beginning at the amino-terminus of the sequence and proceeding toward the carboxy-terminus (i.e., from left to right in each sequence).

[0127] The present disclosure also provides a therapeutic agent for treating or preventing bone mineral density loss, the therapeutic agent being used for treating or preventing bone mineral density loss in a subject having a KREMEN1 variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1, a KREMEN1 variant mRNA molecule encoding a predicted loss-of-function polypeptide of KREMEN1, or a KREMEN1 variant cDNA molecule encoding a predicted loss-of-function polypeptide of KREMEN1.Any of the therapeutic agents for treating or preventing bone mineral density loss described herein can be used in these methods.The subject may have or be at risk of developing bone mineral density loss, osteopenia, osteoporosis type I, osteoporosis type II, or secondary osteoporosis.

[0128] The present disclosure also provides the use of a therapeutic agent for treating or preventing bone mineral density loss, which is used to prepare a medicament for treating or preventing bone mineral density loss in a subject having a KREMEN1 variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1, a KREMEN1 variant mRNA molecule encoding a predicted loss-of-function polypeptide of KREMEN1, or a KREMEN1 variant cDNA molecule encoding a predicted loss-of-function polypeptide of KREMEN1.Any of the therapeutic agents for treating or preventing bone mineral density loss described herein can be used in these methods.The subject may have or be at risk of developing bone mineral density loss, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis.

[0129] The present disclosure also provides a KREMEN1 inhibitor for use in treating or preventing bone mineral density loss in a subject that is a) a reference for KREMEN1 genomic nucleic acid molecule, KREMEN1 mRNA molecule, or KREMEN1 cDNA molecule, or b) i) a KREMEN1 mutant genomic nucleic acid molecule that encodes a predicted loss-of-function polypeptide of KREMEN1; ii) a KREMEN1 mutant mRNA molecule that encodes a predicted loss-of-function polypeptide of KREMEN1; or iii) a KREMEN1 mutant cDNA molecule that encodes a predicted loss-of-function polypeptide of KREMEN1.Any of the KREMEN1 inhibitors described herein can be used in these methods.The subject may have or be at risk of developing bone mineral density loss, osteopenia, osteoporosis type I, osteoporosis type II, or secondary osteoporosis.

[0130] The present disclosure also provides the use of a KREMEN1 inhibitor in the preparation of a medicament for treating or preventing bone mineral density loss in a subject that is a) a reference for KREMEN1 genomic nucleic acid molecule, KREMEN1 mRNA molecule, or KREMEN1 cDNA molecule, or b) i) a KREMEN1 mutant genomic nucleic acid molecule that encodes a predicted loss-of-function polypeptide of KREMEN1; ii) a KREMEN1 mutant mRNA molecule that encodes a predicted loss-of-function polypeptide of KREMEN1; or iii) a KREMEN1 mutant cDNA molecule that encodes a predicted loss-of-function polypeptide of KREMEN1.Any of the KREMEN1 inhibitors described herein can be used in these methods.The subject may have or be at risk of developing bone mineral density loss, osteopenia, osteoporosis type I, osteoporosis type II, or secondary osteoporosis.

[0131] All patent documents, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual document was specifically and individually indicated to be so incorporated by reference. Where various versions of a sequence are associated with accession numbers at different times, the version associated with the accession number at the effective filing date of this application is meant. Effective filing date means the earlier of the actual filing date or the filing date of the priority application to which the accession number refers, if applicable. Similarly, where different versions of publications, websites, etc. have been published at different times, the version last published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present disclosure may be used in combination with any other feature, step, element, embodiment, or aspect, unless otherwise indicated. Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

[0132] The following examples are provided to further illustrate the embodiments. They are intended to illustrate, not limit, the claimed embodiments. The following examples provide those skilled in the art with a disclosure and explanation of how the compounds, compositions, articles, devices and / or methods described herein are made and evaluated, and are intended to be merely illustrative and are not intended to limit the scope of any claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some error and deviation can be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric pressure. EXAMPLES

[0133] Example 1: General Methodology Cohort description The United Kingdom (UK) Biobank (UKB) is a population-based cohort of subjects aged 40–69 years at baseline, recruited by 22 laboratories in the UK between 2006 and 2010 (Bycroft et al., Nature, 2018, 562, 203-209). We used genetic and phenotypic data from nearly 420,000 participants of European ancestry in the UKB. The study was approved by the relevant ethical committees, and participants provided informed consent for participation in the UKB.

[0134] Phenotype definition Data on quantitative ultrasound of the heel were extracted from the UKB. A combination of speed of sound (SOS) and bone ultrasound attenuation (BUA) was used to calculate eBMD trait values ​​(g / cm 2 Gender-specific quality control measures were derived for SOS (subjects were excluded if SOS ≤ 1,450 or ≥ 1,700 m / s for men and ≤ 1,455 or ≥ 1,700 m / s for women), BUA (subjects were excluded if BUA ≤ 27 or ≥ 138 dB / MHz for men and ≤ 22 or ≥ 138 dB / MHz for women), and eBMD (subjects ≤ 0.18 or ≥ 1.06 g / cm for men). 2 , ≤0.12 or ≥1.025 g / cm for women 2 eBMD phenotypic values ​​were first transformed using a rank-based inverse normal transformation applied separately to sexes within each pedigree group and adjusted for fine-mapped common (MAF ≥ 0.01) genetic variants associated with eBMD.

[0135] Genotype data High-coverage whole-exome sequencing was performed as previously reported (Dewey et al., Science, 2016, 354, 6319:aaf6814, and Van Hout et al., Nature, 2020, 586, 749-756) and summarized below. A modified version of the xGen design available from Integrated DNA Technologies (IDT) was used for targeted sequence capture of the exome. During library preparation, a unique 10 bp barcode (IDT) was added to each DNA fragment to facilitate multiplexed exome capture and sequencing. Equal amounts of samples were pooled prior to exome capture. Sequencing was performed on an Illumina NovaSeq instrument using 75 bp paired-end reads. Sequencing had sufficient coverage depth (i.e., the number of sequence reads covering each nucleotide within the targeted region of the genome) to provide greater than 20-fold coverage for 90% of the targeted bases in 99% of the IDT samples. Data processing steps included demultiplexing samples using Illumina software, alignment to the GRCh38 human genome reference sequence including generation of binary alignment and mapping files (BAM), and processing of the BAM files (e.g., marking of duplicate reads and other read mapping assessments). Variant calling was performed using the GLNexus system (Lin et al., bioRxiv, 2018, 343970). Mapping and annotation of variants was based on the GRCh38 human genome reference sequence and Ensembl v85 gene definitions using snpEff software. snpEff predictions related to protein-coding transcripts with annotated start and stop points were then combined into a single functional impact prediction by selecting the most deleterious functional effect class for each gene. The hierarchy of these annotations (from most to least deleterious) was: frameshift, stopgain, stoploss, splice acceptor, splice donor, stoploss, in-frame indel, missense, and other annotations.Predicted LoF gene variants included a) insertions or deletions resulting in frameshifts, b) insertions, deletions or single nucleotide variants resulting in the introduction of premature stop codons or loss of transcription start or stop sites, and c) donor or acceptor splice site variants.For possible functional impact, missense variants were classified according to the number of in silico prediction algorithms that predicted deleteriousness using SIFT (Vaser et al., Nature Protocols, 2016, 11, 1-9), Polyphen2_HDIV and Polyphen2_HVAR (Adzhubei et al., Nat. Methods, 2010, 7, 248-249), LRT (Chun et al., Genome Res., 2009, 19, 1553-1561), and MutationTaster (Schwarz et al., Nat. Methods, 2010, 7, 575-576). For each gene, the alternative allele frequency (AAF) and functional annotation of each variant determined inclusion into seven gene burden exposures: 1) pLoF variants with AAF<1%, 2) pLoF or missense variants predicted as deleterious by 5 of 5 algorithms with AAF<1%, 3) pLoF or missense variants predicted as deleterious by 5 of 5 algorithms with AAF<0.1%, 4) pLoF or missense variants predicted as deleterious by at least 1 of 5 algorithms with AAF<1%, 5) pLoF or missense variants predicted as deleterious by at least 1 of 5 algorithms with AAF<0.1%, 6) pLoF or any missense with AAF<1%, and 7) pLoF or any missense variant with AAF<0.1%. Results described elsewhere in this document as relating to "pLoF or predicted deleterious missense variants" refer to analyses performed using the total burden of pLoF variants or missense variants predicted to be deleterious by 5 out of 5 algorithms.

[0136] Association analysis of the gene burden of rare pLoF and missense mutations in KREMEN1 We used REGENIE v1.0 (Mbatchou et al., Nature Genetics, 2021) to examine associations between rare pLoF or missense variant burden in a given gene and eBMD by fitting linear regression models adjusted for polygenic scores that approximate the genomic relationship matrix. Analyses were performed with age, age, and genomic relationship matrix. 2 , gender, age by gender and age by gender 2 We adjusted for interaction terms, experimental batch-related covariates, principal components from 10 common variants, and principal components from 20 rare variants. Association analyses were performed using single variants and using gene burden testing, in which all subjects are labeled as heterozygous if they carry one or more eligible rare variants (based on frequency and functional annotation as described above) and as homozygous if they carry any eligible variant in the homozygous state. This "composite genotype" is then used to test for association.

[0137] Example 2: Loss of function of KREMEN1 is associated with higher estimated bone mineral density Whole-exome sequencing of 419,737 subjects of European ancestry in the UK Biobank (UKB) was performed to identify protein-coding variants in each gene in the genome. We examined the association of each sequenced gene and gene variant with estimated bone mineral density (eBMD, measured using heel ultrasound). eBMD is a commonly used biomarker of bone density and strength that is highly correlated with bone mineral density measured using dual-energy X-ray absorptiometry (DXA) technology. Reduced levels of bone mineral density are strongly associated with increased risk of osteoporotic fractures.

[0138] Exome-wide analysis in UKB revealed that the burden of rare (alternative allele frequency [AAF] <1%) predicted loss-of-function (pLoF) or predicted deleterious missense variants (predicted deleteriousness of missense variants was based on concordance between five in silico prediction algorithms) in the KREMEN1 gene was 0.13 standard deviation units (or 0.015 g / cm 2 units) was found to be associated with higher eBMD (P value = 2.1 × 10 -7 , Bonferroni-corrected exome-wide statistical significance threshold of P < 3.6 × 10 -7 (corrected for an aggregation model of 20,000 genes and 7 variants with an alpha of 0.05) (see Figure 1).

[0139] A nominally significant association was observed between the total burden of KREMEN1 pLoF variants only (excluding missense variants) and higher eBMD (see Figure 2). The effect estimate for pLoF variant burden (as shown in Figure 2) was 0.18 SD or 0.022 g / cm per KREMEN1 allele copy. 2 High eBMD) was associated with the effect of pLoF or predicted deleterious missense variant burden (0.13 SD or 0.015 g / cm per KREMEN1 allele copy, as shown in Figure 1 ). 2 This suggests that most of the missense variants included in our analysis result in loss of KREMEN1 function and that the association with higher eBMD can be attributed to KREMEN1 loss of function.

[0140] FIG. 3 shows all pLoFs and predicted deleterious missense variants included in the KREMEN1 gene burden analysis of eBMD. Example 3: Integrated evidence from exome sequencing and common variants implicates KREMEN1 in osteoporosis UKB cohort A total of 291,932 participants (278,807 of European ancestry and 13,125 of African, East Asian, or South Asian ancestry) from within the UKB with whole-exome sequencing and eBMD data available were included in the analysis.

[0141] Whole-exome sequencing in UKB Sample preparation and sequencing of UKB samples was performed and are briefly summarized below. A modified version of the xGen exome design available from Integrated DNA Technologies was used for targeted DNA capture. Sequencing was performed on an Illumina NovaSeq instrument using 75 bp paired-end reads. Sequencing had sufficient coverage depth to provide >20-fold coverage for 90% of the targeted bases in 99% of the samples. Variant calling and annotation was based on the GRCh38 human genome reference sequence and Ensembl v85 gene definitions using snpEff software. Variants were annotated according to their most deleterious functional effect in the following order (in descending order of deleteriousness): frameshift, stop gain, stop loss, splice acceptor, splice donor, in-frame indel, missense, other annotation. Predicted LOF variants included a) insertions or deletions resulting in a frameshift, b) insertions, deletions, or single-nucleotide variants resulting in the introduction of a premature stop codon or loss of a transcription start or stop site, and c) donor or acceptor splice site variants.Missense variants were classified for their predicted functional impact using multiple in silico prediction algorithms that predicted deleteriousness (SIFT, PolyPhen2(HDIV), PolyPhen2(HVAR), LRT, and MutationTaster).For each gene, the alternative allele frequency (AAF) and functional annotation of each variant determined inclusion in seven gene burden exposures as previously reported (Akbari et al., 2021, Science 373, eabf8683): ​​1) pLOF variants with AAF<1%, 2) pLOF or missense variants predicted to be deleterious by 5 of 5 algorithms with AAF<1%, 3) pLOF or missense variants predicted to be deleterious by 5 of 5 algorithms with AAF<0.1%, 4) pLOF or missense variants predicted to be deleterious by at least 1 of 5 algorithms with AAF<1%, 5) pLOF or missense variants predicted to be deleterious by at least 1 of 5 algorithms with AAF<0.1%, 6) pLOF or any missense with AAF<1%, and 7) pLOF or any missense variants with AAF<0.1%. Genotyping and imputation of SNP arrays was performed at UKB as previously reported.

[0142] UKB Phenotype Definition We derived eBMD at the heel from quantitative ultrasound SOS and broadband ultrasound attenuation using a previously published model (Morris et al., Nat. Genet., 2018, 51, 258-66). A meticulous data curation pipeline resulted in high quality eBMD data while maximizing the number of participants compared to using direct BMD measurements at the heel reported in the UKB as reported in a previous study. eBMD is used as a surrogate for BMD because it is highly correlated with BMD derived by dual energy X-ray absorptiometry (DXA) (Pearson correlation r = 0.69) and eBMD has a strong association with osteoporotic fracture risk. Prior to analysis, we performed a rank inverse normal transformation of the eBMD phenotype by sex and within each lineage.

[0143] Exome-wide association analysis in UKB Associations of genetic variants or their gene burdens with eBMD were estimated by fitting mixed-effects regression models using REGENIE v1.0.6.8. REGENIE accounts for association, polygenicity, and population structure by approximating a genomic association matrix using genotype-based predictions of individual trait values ​​from the entire genome. Associations of genetic variants or their gene burdens are then estimated conditional on their polygenic predictors along with other covariates. Covariates in the association model included age, age 2 , sex, interaction term of age by sex, age by sex 2 The covariates included interaction terms for , experimental batch-related covariates, principal components derived from 10 common variants, and principal components derived from 20 rare variants. To ensure that associations of rare coding variants or gene burden were statistically independent from common genetic variants associated with eBMD, exome association analysis for index common variants (MAF ≥ 1%) identified by fine-mapping genome-wide associations of common alleles with eBMD was further adjusted as previously reported (Akbari et al., 2021, Science 373, eabf8683). Meta-analysis between subgroup results was performed using a fixed-effects inverse variance weighted model. The exome-wide level of statistical significance for gene burden analysis was Bonferroni-corrected at a type I error rate of 0.05 assuming 20,000 genes and considering seven variant selection models used per gene, p < 3.6 × 10 -7 (Akbari et al., 2021, Science 373, eabf8683). Secondary analyses estimated associations between individual nonsynonymous and / or pLOF variants (minor allele frequency <1% and minor allele count ≥25) identified by exome sequencing and eBMD. p < 5 × 10, Bonferroni-corrected based on 1 million effective numbers of independent tests at a type I error rate of 0.05. -8 A threshold of was used to identify exome-wide significant single variants as previously reported (Akbari et al., 2021, Science 373, eabf8683).

[0144] For all secondary analyses involving false discovery rate (FDR)-corrected results, FDR-adjusted p-values ​​were obtained by first preselecting each gene and each gene burden exposure with the strongest association (lowest p-value), and then correcting for multiple testing using the Benjamini-Hochberg approach for all genes in this subset. Thus, the reported 1% FDR threshold (unadjusted p-value threshold 1.49 × 10 -5 (corresponding to ) is applied to the 18,866 genes after selection of the best gene burden exposure per gene. This corresponds to an FDR threshold of 2.05% if the FDR correction is applied to the entire analysis rather than to a preselected subset.

[0145] Fine mapping of GWAS common variants A genome-wide association study based on imputed genetic variants was performed to identify common variants associated with eBMD. Imputation was based on the HRC reference panel supplemented with UK10K. Genome-wide association analysis was performed in UKB by fitting a mixed-effects linear regression model using REGENIE v1.0.6.8. In each lineage, p<5×10 -8 Fine mapping was performed using FINEMAP software on genomic regions harboring genetic variants associated with eBMD, with a genome-wide significance threshold of 0. Linkage disequilibrium was estimated using genetic data from the exact same set of subjects included in each lineage-specific genome-wide association analysis.

[0146] Study of association with fractures and osteoporosis Genes that met exome-wide statistical significance levels in the eBMD gene burden analysis were tested for association with fracture and osteoporosis in the UK Biobank. Fracture cases were defined as subjects with a history of fracture recorded in the electronic medical record or self-reported (not including fractures of the skull, facial bones, hands, or toes, if possible), and subjects with a history of any type of fracture were excluded from the control group. Osteoporosis cases were defined as subjects with a history of osteoporosis recorded in the electronic medical record or self-reported. Subjects with a self-reported history of osteopenia were further excluded from the control group.

[0147] Enrichment study of positive control genes for osteoporosis To evaluate the ability of WES to detect effector genes for osteoporosis, a set of positive control genes for the disease was identified. 56 protein-coding genes that are either known drug targets for osteoporosis or whose perturbation leads to Mendelian osteoporosis or bone mass disorders, resulting in altered bone mineralization or bone mass were included as positive control genes (Morris et al., Nat. Genet., 2018, 51, 258-66). Fisher's test was used to estimate the enrichment of positive control genes among exome-wide significant genes in the gene burden analysis.

[0148] Effector index of eBMD effector genes The development of the effector index (Ei) was recently reported (Forgetta et al., Hum. Genet., 2022, (World Wide Web "doi.org / 10.1007 / s00439-022-02434-z"). The goal of Ei is to generate a causal probability for each protein-coding gene in a genome-wide association study (GWAS) locus, assigning a score between 0 and 1. GWAS loci were defined by 500 kb surrounding the lead GWAS SNP after linkage disequilibrium (LD) clumping (Forgetta et al. al., Hum. Genet., 2022, World Wide Web "doi.org / 10.1007 / s00439-022-02434-z"). Protein-coding genes with at least 50% of their gene body located within a GWAS locus were included, and overlapping GWAS loci were merged. Briefly, to generate the Ei score for eBMD, 12 diseases and traits (type 2 diabetes, low-density lipoprotein cholesterol level, adult height, calcium level, hypothyroidism, triglyceride level, eBMD, glutamate, and thyroid function) were included. Positive control genes for the following disease-specific variables (course level, red blood cell count, systolic blood pressure, diastolic blood pressure, and direct bilirubin level) were selected. Fine mapping was performed for each disease after GWAS to predict positive control genes using genomic annotations at the GWAS loci as features. This was achieved by first training a gradient boosted tree algorithm (XGBoost) to generate causal probabilities of genes at the GWAS loci for 11 diseases and traits (excluding eBMD), and then applying this trained algorithm to derive Ei scores from the eBMD GWAS data. A generalized linear model implemented in R was used to evaluate the association between Ei scores and odds of being a gene exome-wide significant.An additional complementary gene prioritization method, called Polygenic Priority Score (PoPS), was used to identify effector genes for eBMD from GWAS data (Weeks et al., medRxiv, 2020, World Wide Web "doi:10.1101 / 2020.09.08.20190561").

[0149] Enrichment study of Ei-prioritized genes at loci identified using exome-wide gene burden results for osteoporosis A 2x2 contingency table was created comparing genes prioritized by Ei with genes identified from the exome-wide analysis per locus. Data were then pooled across all of these loci and tested for enrichment using a stratified Fisher's exact test approach. Estimation of odds ratios and their confidence intervals were then based on conditional maximum likelihood estimates and exact confidence limit estimates using a tailed approach for discrete distributions, respectively.

[0150] Two-sample Mendelian randomization A two-sample Mendelian randomization (MR) analysis was performed to identify circulating proteins that affect eBMD. Two-sample MR uses genetic variants (pQTLs) that are strongly and specifically associated with circulating protein levels as instrumental variables to estimate the causal relationship between a given protein and an outcome (in this case, eBMD). This approach is less susceptible to confounding and reverse causality than observational epidemiological biomarker studies. The MR framework is based on three main assumptions: first, SNPs are robustly associated with the exposure. Second, SNPs are not associated with factors that confound the relationship between the exposure and the outcome. Third, SNPs have no effect on the outcome that is independent of the exposure (i.e., there is no horizontal pleiotropy). Of these, the third assumption is the most difficult to assess because the biological mechanistic effect of SNPs on outcomes such as eBMD is unknown in most cases. However, for circulating proteins, SNPs that are associated with protein levels and close to genes that code for the protein are more likely to have effects mediated through the protein level by affecting the transcription or translation of the gene into protein. Such SNPs, called cis-SNPs, can help reduce potential bias from horizontal pleiotropy.

[0151] To select genetic measures for circulating proteins, we used summary-level data from two proteomic GWAS studies that measured serum protein levels on the SOMAlogic platform. The primary analysis used the INTERVAL study as the source of pQTL data, which included measurements of 1,478 serum proteins in 3,301 subjects. The replication analysis used the AGES study, which included measurements of 4,137 serum proteins in 3,200 subjects. Proteins were selected for inclusion in the analysis if they had cis-acting associated SNPs ("cis-SNPs") because such measures may be less susceptible to horizontal pleiotropy (Swerdlow et al., Int. J. Epidemiol. 2016, 45, 1600-16). cis-SNPs from INTERVAL were independent, genome-wide significant SNPs (P<1.5×10) located within 1 Mb of the transcription start site (TSS) of a protein-coding gene. -11 , genome-wide significance threshold after multiple testing correction previously employed in INTERVAL). To select these cis-SNPs, independent SNP clumping and selection was performed for each protein using PLINK and the 1000 Genomes Project European Reference Panel (1KG EUR) (R 2 <0.001, distance 1000 kb). cis-SNPs from AGES were compared with index cis-SNPs located within 300 kb of the corresponding protein-coding genes (P < 5 × 10 -8The genome-wide significant SNPs with the lowest P-value for each protein were selected from the cis-SNPs (Milsson et al., Science, 2018, 1327, 1-12). Associations between each cis-SNP and eBMD (i.e., the outcome in the MR analysis) were obtained from a recent eBMD GWAS including 426,824 British Caucasian subjects (Surakka et al., Nat. Commun., 2020, 11, 4093). Palindromic cis-SNPs with minor allele frequency (MAF) >0.42 (as recommended by the TwoSampleMR R package) were removed prior to MR to prevent allelic mismatches. For cis-SNPs that were not present in the eBMD GWAS, LD R 2 SNPs with MAF >0.8 and MAF <0.42 were selected as proxies. For sequence comparison of SNP proxies, MAF >0.3 was used as the threshold for removing palindromic SNPs.

[0152] After matching protein cis-SNPs with eBMD GWAS and removing palindromic SNPs, 550 SOMAmer reagents (517 proteins) from INTERVAL (containing 515 matching cis-SNPs and 59 LD proxy cis-SNPs) and 749 circulating proteins from AGES (containing 706 unique matching cis-SNPs, 41 LD proxy cis-SNPs, and two cis-SNPs each for two proteins) were included in the MR analysis. Independent cis-pQTL data were selected from the INTERVAL data (p<1.5×10 -11 ).

[0153] MR analysis was performed using the TwoSampleMR package in R, and Wald ratios (β eBMD / β protein For proteins with multiple independent cis-SNPs, the inverse variance weighting (IVW) method was used to estimate their combined effect. 64A meta-analysis was performed using Bonferroni correction to control for the number of proteins independently tested in INTERVAL and AGES.

[0154] result Whole-exome sequencing was performed on approximately 300,000 individuals from the UK Biobank cohort (UKB) to estimate the association of rare nonsynonymous and / or pLOF variant burden with eBMD for each gene in the genome. In a larger European ancestry subset of the UKB (N=278,807), KREMEN1 was identified (p<3.6×10 -7 ). These WES analyses were designed to be independent of eBMD-associated fine-mapped common alleles, so this association did not arise from a common genetic variant. Exome-wide multi-lineage meta-analysis identified two additional genes (WNT5B and KREMEN1) with exome-wide significance (Figures 4 and 5), yielding exome-wide significant genes. Table 2 shows all variants observed in only one lineage in the KREMEN1 gene burden study.

[0155] [Table 2-1]

[0156] [Table 2-2]

[0157] [Table 2-3]

[0158] [Table 2-4]

[0159] [Table 2-5]

[0160]

Table 2-6

[0161]

Table 2-7

[0162]

Table 2-8

[0163]

Table 2-9

[0164]

Table 2-10

[0165]

Table 2-11

[0166]

Table 2-12

[0167]

Table 2-13

[0168]

Table 2-14

[0169]

Table 2-15

[0170] [Table 2-16]

[0171] [Table 2-17]

[0172] A different GWAS effector gene prioritization method, gene-level polygenic prioritization score (PoPS), yielded similar results as Ei. KREMEN1 as a gene associated with eBMD in exome-wide significance and their evidence from common variant GWAS predicted by PoPS. "Positive control" indicates whether the gene is included in a subset of 56 expert-curated genes involved in bone mineral density by Mendelian genetics or pharmacological validation. The eBMD PoP score was calculated for all genes in the genome, whereas the PoPS rank was derived only for genes in GWAS loci (a total of 857 eBMD GWAS loci were included).

[0173] Table 3 shows that KREMEN1 was found exclusively in a multi-pedigree meta-analysis of eBMD (gene exposure, variant type; frequency cutoff (%) = pLOF + deleterious missense (5 / 5); AAF < 1%). Abbreviations: European ancestry, EUR; African ancestry, AFR; South Asian ancestry, SAS; East Asian ancestry, EAS; predicted loss of function, pLOF; alternative allele frequency, AAF; confidence interval, CI; standard deviation, SD; estimated bone mineral density, eBMD; P value, p; reference-reference genotype, RR; reference-alternate genotype, RA; alternative-alternate genotype, AA; gram per square centimeter, g / cm 2 ; the ratio of true heterogeneity to total observed mutations, I2.

[0174] [Table 3]

[0175] [Table 4]

[0176] Mendelian randomization of circulating protein abundance by eBMD Further evidence for the involvement of KREMEN1 in bone mineral density was obtained by utilizing large-scale proteomic data. Two-sample Mendelian randomization (MR) to identify circulating proteins genetically associated with eBMD. First, cis-SNPs associated with the levels of 863 circulating proteins were identified from two proteomic GWAS studies, the INTERVAL study and the AGES study. Both studies measured circulating proteins using the SomaScan platform and included 3,301 and 3,200 subjects of European ancestry, respectively. MR analysis showed that KREMEN1 (P<9.2×10) from INTERVAL was associated with circulating proteins genetically associated with eBMD. -5 , corresponding to the Bonferroni-corrected value for KREMEN1 tested in INTERVAL) and KREMEN1 from AGES ( P < 6.5 × 10 -5 , corresponding to the Bonferroni-corrected value for KREMEN1 tested in AGES) was associated with eBMD. In addition, KREMEN1 as a protein was associated with eBMD at INTERVAL (MR pval < 9.2 × 10 -5 ) and AGES (MR pval < 6.45 × 10 -5 ) was significantly associated with eBMD from beta: effect estimate of eBMD in SD per SD increase in protein level.

[0177] In addition to those described herein, various modifications of the described subject matter will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, etc.) is incorporated herein by reference in its entirety and for all purposes.

Claims

1. An in vitro method for identifying the susceptibility of a subject to the onset of reduced bone mineral density, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis, said method comprising: determining, or having determined, the presence or absence of a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of kringle-containing transmembrane protein 1 (KREMEN1) in a biological sample obtained from said subject; wherein said subject being a KREMEN1 reference indicates that the subject has a high risk of developing reduced bone mineral density, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis, and wherein said subject being heterozygous or homozygous for said KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of said KREMEN1 indicates that the subject has a low risk of developing reduced bone mineral density, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis.

2. The method of claim 1, wherein said KREMEN1 variant nucleic acid molecule is a splice site variant, a stop gain variant, a start loss variant, a stop loss variant, a frameshift variant, or an in-frame indel variant, or a variant encoding a predicted loss-of-function polypeptide of truncated KREMEN1.

3. The method of claim 1 or claim 2, wherein said predicted loss-of-function variant nucleic acid molecule of KREMEN1 is an mRNA molecule listed in FIG. 3 or generated therefrom, or a cDNA molecule generated from said mRNA molecule.

4. The method of claim 1 or claim 2, wherein said KREMEN1 variant nucleic acid molecule encodes a predicted loss-of-function polypeptide of truncated KREMEN1. Use of a therapeutic agent for treating or preventing decreased bone mineral density in the preparation of a medicament for treating or preventing decreased bone mineral density, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis in a subject, wherein the subject is a KREMEN1 variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of kringle-containing transmembrane protein 1 (KREMEN1), or a KREMEN1 variant mRNA molecule encoding a predicted loss-of-function polypeptide of KREMEN1 and is heterozygous with respect to it.

6. The use according to claim 5, wherein the KREMEN1 variant genomic nucleic acid molecule or the KREMEN1 variant mRNA molecule is a splice site variant, a stop gain variant, a start loss variant, a stop loss variant, a frameshift variant, or an in-frame indel variant, or a variant encoding a predicted loss-of-function polypeptide of truncated KREMEN1.

7. The use according to claim 5, wherein the KREMEN1 variant genomic nucleic acid molecule is included in FIG.

3.

8. The use according to claim 5, wherein the KREMEN1 variant genomic nucleic acid molecule or the KREMEN1 variant mRNA molecule encodes a predicted loss-of-function polypeptide of truncated KREMEN1.

9. The use according to any one of claims 5 to 8, wherein the therapeutic agent comprises calcium and vitamin D supplementation, a bisphosphonate formulation, or hormone replacement therapy.

10. The vitamin D supplementation comprises vitamin D2, vitamin D3, or colecalciferol, the bisphosphonate formulation comprises alendronate, ibandronate, zoledronate, risedronate, calcitonin, teriparatide, or denosumab, and the hormone replacement therapy comprises estrogen, progesterone, or raloxifene, The use according to claim 9. Use of a kringle-containing transmembrane protein 1 (KREMEN1) inhibitor in the preparation of a medicament for treating or preventing bone mineral density reduction, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis in a subject, wherein the subject is a) a reference for a KREMEN1 genomic nucleic acid molecule or a KREMEN1 mRNA molecule, or b) i) a KREMEN1 variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of KREMEN1; or ii) a KREMEN1 variant mRNA molecule encoding a predicted loss-of-function polypeptide of KREMEN1 and is heterozygous for. **Claim 12** The use according to claim 11, wherein the KREMEN1 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to a KREMEN1 nucleic acid molecule. **Claim 13** The use according to claim 12, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA). **Claim 14** The use according to claim 11, wherein the KREMEN1 inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within a KREMEN1 genomic nucleic acid molecule. **Claim 15** The use according to claim 14, wherein the Cas protein is Cas9 or Cpf1. **Claim 16** The use according to claim 14, wherein the gRNA recognition sequence is located within SEQ ID NO:

1. **Claim 17** The use according to claim 14, wherein the protospacer adjacent motif (PAM) sequence is about 2 to about 6 nucleotides downstream of the gRNA recognition sequence. **Claim 18** The use according to claim 14, wherein the gRNA comprises about 17 to about 23 nucleotides. **Claim 19** The use according to claim 14, wherein the gRNA recognition sequence comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 17 to 36. **Claim 20** The use according to claim 11, wherein the KREMEN1 mutant genomic nucleic acid molecule or the KREMEN1 mutant mRNA molecule is a splice site mutant, a stop gain mutant, a start loss mutant, a stop loss mutant, a frameshift mutant, or an in-frame indel mutant, or a mutant encoding a predicted loss-of-function polypeptide of truncated KREMEN1. **Claim 21** The use according to claim 11, wherein the KREMEN1 mutant genomic nucleic acid molecule is shown in Figure 3. **Claim 22** The use according to any one of claims 11 to 19, wherein the KREMEN1 mutant genomic nucleic acid molecule or the KREMEN1 mutant mRNA molecule encodes a predicted loss-of-function polypeptide of truncated KREMEN1. **Claim 23** A pharmaceutical composition for treating reduced bone mineral density, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis in a subject, wherein the subject is heterozygous for a KREMEN1 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of truncated kringle-containing transmembrane protein 1 (KREMEN1), and the pharmaceutical composition comprises, as an active ingredient, a therapeutic agent for treating or preventing reduced bone mineral density. **Claim 24** A pharmaceutical composition for treating reduced bone mineral density, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis in a subject, wherein the subject is a) a kringle-containing transmembrane protein 1 (KREMEN1) genomic nucleic acid molecule or a KREMEN1 mRNA molecule as a reference, or b) It is heterozygous for a KREMEN1 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of the cleavage-type KREMEN1, A pharmaceutical composition, wherein the pharmaceutical composition contains a KREMEN1 inhibitor as an active ingredient.

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