Treating bone mineral loss with cluster of differentiation 109 (CD109) 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 an XML file of 355 kilobytes in size under the title 18923807402SEQ, created on July 28, 2022. This Sequence Listing is incorporated herein by reference.

[0002] The present disclosure relates generally to the treatment of subjects with bone mineral density loss with cluster of differentiation 109 (CD109) 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 less severe bone condition than osteoporosis, characterized by a loss of bone mass due to bone loss at a faster rate than new bone growth. Osteopenia manifests in bones with mineral density lower than normal peak bone mineral density, but not as low as 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 disease characterized by the gradual weakening of bones due to bone demineralization and / or problems with their structure. Osteoporosis manifests in bones by making them more susceptible to fracture. Hormonal deficiencies associated with menopause in women, and aging in both sexes, contribute to degenerative conditions of bone. In addition, insufficient dietary intake of minerals essential for bone growth and maintenance is a potential cause of bone loss. Genetic influences on osteoporosis have also been reported (Non-Patent Document 1).

[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.In addition, it is uncertain whether many of the proposed mineral and hormone supplements can actually reduce the risk of bone fracture.Moreover, many synthetic hormones or non-mammalian hormone therapy have additional undesirable side effects, such as increased risk of cardiovascular disorders, neurological disorders, or exacerbation of pre-existing conditions.

[0005] Cluster of differentiation 109 (CD109) is a member of the alpha2-macroglobulin / complement (AMCOM) family of thioester-containing proteins. This glycosylphosphatidylinositol (GPI)-linked glycoprotein is localized on the surface of platelets, activated T cells, and endothelial cells. In addition, CD109 is expressed on hematopoietic cell lineages and some epithelial cells. CD109 protein binds transforming growth factor beta (TGF-β) in keratinocytes, thereby negatively regulating signal transduction, and also binds TGF-βRI and TGF-βRII. In addition, CD109 may function as a protease inhibitor, like most members of the AMCOM family. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Morris et al.,Nature Genet.,2019,51,258-266 Summary of the Invention [Means for solving the problem]

[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 CD109 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 CD109 inhibitor. The 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 CD109 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 CD109. The 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 CD109 inhibitor.

[0010] The disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits 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 CD109 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109 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 CD109 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109; and i) administering or continuing to administer the therapeutic agent that treats or inhibits bone mineral density loss at a standard dosage to a subject that is CD109 normative, and and / or administering a CD109 inhibitor to the subject; ii) administering or continuing to administer to the subject who is heterozygous for the CD109 missense variant nucleic acid molecule the therapeutic agent that treats or inhibits bone mineral density loss at the same or a lower standard dosage, and / or administering a CD109 inhibitor to the subject; or iii) administering or continuing to administer to the subject who is homozygous for the CD109 missense variant nucleic acid molecule the therapeutic agent that treats or inhibits bone mineral density loss at the same or a lower standard dosage; the presence of a genotype having the CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 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 CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 in a biological sample obtained from the subject; if the subject is CD109 normative, the subject has an increased risk of developing bone mineral density loss; and if the subject is heterozygous or homozygous for the CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109, the subject has a decreased risk of developing bone mineral density loss.

[0012] The present disclosure also provides a therapeutic agent for treating or inhibiting bone mineral density loss, the therapeutic agent for use in treating bone mineral density loss in a subject having a CD109 missense variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109, a CD109 missense variant mRNA molecule encoding a predicted loss-of-function polypeptide of CD109, or a CD109 missense variant cDNA molecule encoding a predicted loss-of-function polypeptide of CD109.

[0013] The present disclosure also provides a CD109 inhibitor for use in treating bone mineral density loss in a subject that a) is reference to a CD109 genomic nucleic acid molecule, a CD109 mRNA molecule, or a CD109 cDNA molecule, or b) is heterozygous for i) a CD109 missense variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109; ii) a CD109 missense variant mRNA molecule encoding a predicted loss-of-function polypeptide of CD109; or iii) a CD109 missense variant cDNA molecule encoding a predicted loss-of-function polypeptide of CD109. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] 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.

[0015] 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.

[0016] 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.

[0017] As used herein, the term "comprising" may in certain embodiments be replaced with "consisting" or "consisting essentially of," as desired.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In accordance with the present disclosure, it has been observed that CD109 missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CD109 (whether these variants are homozygous or heterozygous in a particular subject) are associated with a reduced risk of developing bone mineral density loss. The details or direction of CD109's involvement in bone mineral density are unknown. The data presented herein are the first data showing that rare, nonsynonymous / loss-of-function variants in CD109 are associated with a reduced risk of developing bone mineral density loss. Thus, subjects who are CD109 norm or heterozygous for CD109 missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CD109 can be treated with CD109 inhibitors so that bone mineral density loss is inhibited, symptoms thereof are alleviated, and / or the onset of symptoms is suppressed. It is also believed that such subjects with bone mineral density loss can be further treated with therapeutic agents that treat or inhibit bone mineral density loss.

[0022] For the purpose of this disclosure, any particular subject, such as a human, can be classified as having one of three CD109 genotypes: i) CD109 standard; ii) heterozygous for CD109 missense variant nucleic acid molecule encoding predicted loss-of-function polypeptide of CD109; or iii) homozygous for CD109 missense variant nucleic acid molecule encoding predicted loss-of-function polypeptide of CD109.If the subject does not have a copy of CD109 missense variant nucleic acid molecule encoding predicted loss-of-function polypeptide of CD109, the subject is CD109 standard.If the subject has a single copy of CD109 missense variant nucleic acid molecule encoding predicted loss-of-function polypeptide of CD109, the subject is heterozygous for CD109 missense variant nucleic acid molecule encoding predicted loss-of-function polypeptide of CD109. A CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 is any nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) encoding a variant CD109 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 CD109 polypeptide with partial loss-of-function (or predicted partial loss-of-function) is hypomorphic for CD109. If a subject has two copies (same or different) of a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109, the subject is homozygous for a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109.

[0023] For subjects who are genotyped or determined to be CD109-based, such subjects have an increased risk of developing bone mineral density loss, such as osteopenia, osteoporosis type I, osteoporosis type II, and / or secondary osteoporosis. For subjects who are genotyped or determined to be CD109-based or heterozygous for a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109, such subjects or subjects can be treated with a CD109 inhibitor.

[0024] In any of the embodiments described herein, the CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 can be any nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) encoding a CD109 variant polypeptide having partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. In some embodiments, the CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 is associated with a reduced in vitro response to a CD109 ligand compared to a reference CD109. In some embodiments, the CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 is a CD109 variant that results, or is predicted to result, in premature cleavage of the CD109 polypeptide compared to a human reference genome sequence. In some embodiments, the CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 is a variant predicted to be damaging by an in vitro prediction algorithm, such as Polyphen, SIFT, or a similar algorithm. In some embodiments, the CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 is a variant that causes, or is predicted to cause, a nonsynonymous amino acid substitution in CD109, the allele frequency of which is less than 1 / 100 alleles in the population from which the subject is selected. In some embodiments, the CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 is any rare missense 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 CD109 variant.

[0025] In any of the embodiments described herein, the predicted loss-of-function polypeptide of CD109 can be any CD109 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.

[0026] In any of the embodiments described herein, the CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 may comprise a mutation at position chromosome 6 (SEQ ID NO:1; ENSG00000156535.15 chr6:73,695,785-73,828,316 in the GRCh38 / hg38 human genome assembly) using the nucleotide sequence of the CD109 reference genomic nucleic acid molecule as the reference sequence.

[0027] There are a number of genetic variants in CD109 that result in subsequent changes in the CD109 polypeptide sequence, including but not limited to: 6:73730573:A:G, 6:73823473:GA:G (p.Ser1394fs, p.Ser1317fs, p.Ser1377fs), 6:73763607:C:A (p.Phe343Leu, p.Phe266Leu, p.Phe343Leu), 6:73803256:G:T (p.Gly972Val, p.Gly895Val, p.Gly972Val), 6:73818486:T:C (p .Val1337Ala, p.Val1260Ala, p.Val1320Ala), 6:73787379:G:A(p.Gly828Glu, p.Gly751Glu, p.Gly828Glu), 6:73771510:A:G(p.Ile586Val, p.Ile509Val, p .Ile586Val), 6:73806987:A:T(p.His1035Leu, p.His958Leu, p.His1035Leu), 6:73758991:A:G(p.Met241Val, p.Met164Val, p.Met241Val), 6:73823456:A:G 6:73762778:A:C(p.Glu298Ala, p.Glu221Ala, p.Glu298Ala), 6:73763660:A:G(p.Lys361Arg, p.Lys284Arg, p.Ly s361Arg), 6:73730573:A:G(p.Lys169Arg, p.Lys169Arg), 6:73806956:G:A(p.Gly1025Ser, p.Gly948Ser, p.Gly1 025Ser), 6:73792628:G:C(p.Asp902His, p.Asp825His, p.Asp902His), 6:73806926:A:T(p.Thr1015Ser, p.Thr93 8Ser, p.Thr1015Ser), 6:73771576:G:A(p.Glu608Lys, p.Glu531Lys, p.Glu608Lys), 6:73815026:C:T(p.Arg1272 * , p.Arg1195 * , p.Arg1255 *), 6:73765952:G:A(p.Gly377Asp, p.Gly300Asp, p.Gly377Asp).

[0028] Any one or more (i.e., any combination) of CD109 missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CD109 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 CD109 and an increased risk of developing bone mineral density loss.

[0029] 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.

[0030] 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.

[0031] 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 CD109 inhibitor.

[0032] 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 CD109 inhibitor. The 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 CD109 inhibitor.

[0033] 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 CD109. The 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 CD109 inhibitor.

[0034] In some embodiments, the CD109 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 CD109 nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes with a sequence in a CD109 genomic nucleic acid molecule or mRNA molecule and reduces the expression of a CD109 polypeptide in a cell of a subject. In some embodiments, the CD109 inhibitor comprises an antisense molecule that hybridizes with a CD109 genomic nucleic acid molecule or mRNA molecule and reduces the expression of a CD109 polypeptide in a cell of a subject. In some embodiments, the CD109 inhibitor comprises an siRNA that hybridizes with a CD109 genomic nucleic acid molecule or mRNA molecule and reduces the expression of a CD109 polypeptide in a cell of a subject. In some embodiments, CD109 inhibitors include shRNAs that hybridize with a CD109 genomic nucleic acid molecule or mRNA molecule and reduce expression of a CD109 polypeptide in cells of a subject.

[0035] 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.

[0036] 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.

[0037] The inhibitory nucleic acid molecule can 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 moieties. 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 different purine or pyrimidine bases (e.g., pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl, etc.). 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, (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo, etc.), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0038] Nucleotide analogs can also include modifications at the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural and synthetic modifications of the ribose and deoxyribose. 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 1-10 Alkyl or C 2-10 Alkenyl, and C 2-10 Exemplary 2' sugar modifications also 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 CH3)]2, where n and m are independently 1 to about 10. Other modifications at the 2' position include, but are not limited to, C 1-10These 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 can also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides, and the 5' position of a 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.

[0039] Nucleotide analogs can also be modified at the phosphate site. Modified phosphate sites 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. These phosphate linkages or modified phosphate linkages between two nucleotides can be through 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).

[0040] 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.

[0041] In some embodiments, the siRNA molecule has a 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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; * " are phosphorothioate backbone linkages.

[0046] 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.

[0047] 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.

[0048] In some embodiments, the CD109 inhibitor is or comprises LY294002, a PI3K inhibitor that suppresses expression of CD109. In some embodiments, the CD109 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks in the recognition sequence(s) in the CD109 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 CD109 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 CD109 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.

[0049] 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.

[0050] In some embodiments, the CRISPR / Cas system can be used to modify the CD109 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 CD109 nucleic acid molecule.

[0051] Cas proteins generally contain at least one RNA recognition or 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 CD109 genomic nucleic acid molecules, or can be nickases to create single-stranded breaks in CD109 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.

[0052] In some embodiments, targeted genetic modification of a CD109 genomic nucleic acid molecule can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize with one or more gRNA recognition sequences in a target genomic locus in a CD109 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 a CD109 genomic nucleic acid molecule or the stop codon of a CD109 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.

[0053] The gRNA recognition sequence in the target genomic locus in the CD109 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.

[0054] gRNA is an RNA molecule that binds to Cas protein and targets Cas protein to a specific location in CD109 genomic nucleic acid molecule.Exemplary gRNA is an effective gRNA that induces Cas enzyme to bind to or cut CD109 genomic nucleic acid molecule, and gRNA comprises a DNA targeting segment that hybridizes with gRNA recognition sequence in CD109 genomic nucleic acid molecule.Exemplary gRNA comprises a DNA targeting segment that hybridizes with gRNA recognition sequence that exists in CD109 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.

[0055] Examples of suitable gRNA recognition sequences located within the human CD109 reference gene are set forth in Table 1 as SEQ ID NOs: 37-58.

[0056] [Table 1]

[0057] The Cas protein and gRNA form a complex, and the Cas protein cleaves the target CD109 genomic nucleic acid molecule. The Cas protein can cleave the nucleic acid molecule at a site inside or outside the nucleic acid sequence present in the target CD109 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 result in the cleavage of one or both strands 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 CD109 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds.

[0058] Such a method can result in a CD109 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 CD109 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 the Cas protein can create two or more double-strand breaks or two or more single-strand breaks.

[0059] In some embodiments, the method of treatment further comprises detecting the presence or absence of a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 in a biological sample from the subject. As used throughout this disclosure, a "CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109" is any CD109 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes a CD109 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.

[0060] The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits 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 CD109 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109 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 a CD109 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109. In some embodiments, the method further comprises administering or continuing to administer a therapeutic agent that treats or inhibits bone mineral density loss at a standard dose to the subject who is a CD109 norm, and / or administering a CD109 inhibitor to the subject. In some embodiments, the method further comprises administering or continuing to administer a therapeutic agent that treats or inhibits bone mineral density loss to a subject that is heterozygous for a CD109 missense variant nucleic acid molecule at or below a standard dose, and / or administering a CD109 inhibitor to the subject. In some embodiments, the method further comprises administering or continuing to administer a therapeutic agent that treats or inhibits bone mineral density loss to a subject that is homozygous for a CD109 missense variant nucleic acid molecule at or below a standard dose. The presence of a genotype with a CD109 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109 indicates that the subject has a low risk of developing bone mineral density loss. In some embodiments, the subject is CD109 normative. In some embodiments, the subject is heterozygous for a CD109 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109.

[0061] For subjects who have been genotyped or determined to be heterozygous for a CD109 missense variant nucleic acid molecule that is a CD109 standard or encodes a predicted loss-of-function polypeptide of CD109, such subjects can be treated with a CD109 inhibitor as described herein.

[0062] Detecting the presence or absence of a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 in a biological sample from a subject and / or determining whether a subject has a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 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.

[0063] In some embodiments, if the subject is a CD109 norm, the subject is administered a standard dose of a therapeutic agent that treats or inhibits bone mineral density loss. In some embodiments, if the subject is heterozygous for a CD109 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109, the subject is administered a standard dose or a lower dose of a therapeutic agent that treats or inhibits bone mineral density loss.

[0064] In some embodiments, the method of treatment further comprises detecting the presence or absence of a predicted loss-of-function polypeptide of CD109 in a biological sample from the subject. In some embodiments, if the subject does not have a predicted loss-of-function polypeptide of CD109, the subject is administered a standard dose of a therapeutic agent that treats or inhibits bone mineral density loss. In some embodiments, if the subject has a predicted loss-of-function polypeptide of CD109, the subject is administered a standard dose of a therapeutic agent that treats or inhibits bone mineral density loss.

[0065] The disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits 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 CD109 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 CD109. If the subject does not have a predicted loss-of-function polypeptide of CD109, a therapeutic agent that treats or inhibits bone mineral density loss is or continues to be administered to the subject at a standard dosage, and / or a CD109 inhibitor is administered to the subject. If the subject has a predicted loss-of-function polypeptide of CD109, a therapeutic agent that treats or inhibits bone mineral density loss is administered or continues to be administered to the subject at the same or lower standard dose, and / or a CD109 inhibitor is administered to the subject. The presence of a predicted loss-of-function polypeptide of CD109 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 CD109. In some embodiments, the subject does not have a predicted loss-of-function polypeptide of CD109.

[0066] Detecting the presence or absence of a predicted loss-of-function polypeptide of CD109 in a biological sample from a subject and / or determining whether a subject has a predicted loss-of-function polypeptide of CD109 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.

[0067] Examples of therapeutic agents that treat or inhibit 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), and EVENITY® (romosozumab). In some embodiments, the therapeutic agent treating or inhibiting bone mineral density loss is vitamin D2, vitamin D3, cholecalciferol, alendronate, ibandronate, zoledronate, risedronate, calcitonin, teriparatide, denosumab, or raloxifene. In some embodiments, the therapeutic agent treating or inhibiting bone mineral density loss is vitamin D2. In some embodiments, the therapeutic agent treating or inhibiting bone mineral density loss is vitamin D3. In some embodiments, the therapeutic agent treating or inhibiting bone mineral density loss is cholecalciferol. In some embodiments, the therapeutic agent treating or inhibiting bone mineral density loss is alendronate. In some embodiments, the therapeutic agent treating or inhibiting bone mineral density loss is ibandronate. In some embodiments, the therapeutic agent treating or inhibiting bone mineral density loss is zoledronate. In some embodiments, the therapeutic agent treating or inhibiting bone mineral density loss is risedronate. In some embodiments, the therapeutic agent treating or inhibiting bone mineral density loss is calcitonin. In some embodiments, the therapeutic agent treating or inhibiting bone mineral density loss is teriparatide. In some embodiments, the therapeutic agent treating or inhibiting bone mineral density loss is denosumab.In some embodiments, the therapeutic agent treating or inhibiting bone mineral density loss is raloxifene.

[0068] In some embodiments, the dose of a therapeutic agent for treating or inhibiting bone mineral density loss can be reduced (i.e., less than the standard dose) for subjects who are heterozygous for a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 compared to subjects who are CD109 normative (who may receive the standard dose). In some embodiments, the dose of a therapeutic agent for treating or inhibiting 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 a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 can be administered less frequently compared to subjects who are CD109 normative.

[0069] In some embodiments, the dose of a therapeutic agent for treating or inhibiting bone mineral density loss can be reduced by about 10%, about 20%, about 30%, about 40%, about 50% in a subject who is homozygous for a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 compared to a subject who is heterozygous for a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109. In some embodiments, the dose of a therapeutic agent for treating or inhibiting 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 inhibiting bone mineral density loss in a subject who is homozygous for a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 can be administered less frequently compared to a subject who is heterozygous for a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109.

[0070] Administration of the therapeutic agent for treating or inhibiting bone mineral density loss and / or CD109 inhibitor 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.

[0071] Administration of the therapeutic agent for treating or inhibiting bone mineral density loss and / or CD109 inhibitor 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 deleterious to the recipient thereof.

[0072] 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.

[0073] 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 CD109 missense 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 CD109 in a biological sample obtained from the subject. If the subject lacks a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 (i.e., the subject is classified as CD109 norm by genotyping), the subject has a high risk of developing bone mineral density loss. If the subject has a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 (i.e., the subject is heterozygous or homozygous for a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109), the subject has a low risk of developing bone mineral density loss.

[0074] Having a single copy of a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 further protects a subject from developing bone mineral density loss than having no copies of a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109. Without intending to be limited to a particular theory or mechanism of action, it is believed that a single copy of a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 (i.e., heterozygous for a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109) protects a subject from developing bone mineral density loss, and it is also believed that having two copies of a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 (i.e., homozygous for a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109) 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 CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 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 CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109, resulting in less than complete protection from the development of bone mineral density loss.

[0075] Determining whether a subject has a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 in a biological sample from a subject and / or determining whether a subject has a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 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.

[0076] 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 that treats or inhibits bone mineral density loss, as described herein, and / or a CD109 inhibitor. For example, if a subject has a CD109 criteria and therefore has a high risk of developing bone mineral density loss, the subject is administered a CD109 inhibitor. In some embodiments, such a subject is also administered a therapeutic agent that treats or inhibits bone mineral density loss. In some embodiments, if a subject is heterozygous for a CD109 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109, the subject is administered a therapeutic agent that treats or inhibits bone mineral density loss at a dose equal to or less than the standard dose, and is also administered a CD109 inhibitor. In some embodiments, such a subject is also administered a therapeutic agent that treats or inhibits bone mineral density loss. In some embodiments, if the subject is homozygous for a CD109 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109, the subject is administered a therapeutic agent that treats or inhibits bone mineral density loss at a dose equal to or less than the standard dose. In some embodiments, the subject is CD109 norm. In some embodiments, the subject is heterozygous for a CD109 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109. In some embodiments, the subject is homozygous for a CD109 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109.

[0077] In some embodiments, any of the methods described herein may further comprise determining the gene burden of the subject having a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 associated with a reduced risk of developing bone mineral density loss and / or a predicted loss-of-function variant polypeptide of CD109. The gene burden is the sum of all variants of the CD109 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 CD109 missense variant nucleic acid molecules encoding a predicted loss-of-function polypeptide of CD109 associated with a reduced risk of developing bone mineral density loss. In some embodiments, the subject is heterozygous for one or more CD109 missense variant nucleic acid molecules encoding a predicted loss-of-function polypeptide of CD109 associated with a reduced risk of developing bone mineral density loss. The results of the association analysis suggest that the CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 is associated with a reduced risk of developing bone mineral density loss. If the subject has a lower genetic burden, 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 that treats or inhibits bone mineral density loss.If the subject has a higher genetic burden, 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 that treats or inhibits bone mineral density loss.The higher the genetic burden, the lower the risk of developing bone mineral density loss.

[0078] CD109 missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CD109 and / or predicted loss-of-function variant polypeptides of CD109 that can be used to determine the gene burden of a subject include, but are not limited to, 6:73730573:A:G, 6:73823473:GA:G (p.Ser1394fs, p.Ser1317fs, p.Ser1377fs), 6:73763607:C:A (p.Phe343Leu, p.Phe266Leu, p.Phe343Leu), 6:73803256:G:T (p.Gly972Val, p.Gly895Val, p.Gly972Val ...972Val, p.Gly972Val), 6:73803256:G:T (p.Gly972Val, p.Gly972Val, p.Gly972Val), 6:73803256:G:T (p.Gly972Val, p.Gly972Val, p.Gly972Val), 6:73803256:G:T (p :73818486:T:C(p.Val1337Ala, p.Val1260Ala, p.Val1320Ala), 6:73787379:G:A( p.Gly828Glu, p.Gly751Glu, p.Gly828Glu), 6:73771510:A:G(p.Ile586Val, p.Ile5 09Val, p.Ile586Val), 6:73806987:A:T(p.His1035Leu, p.His958Leu, p.His1035L eu), 6:73758991:A:G(p.Met241Val, p.Met164Val, p.Met241Val), 6:73823456:A:G 6:73762778:A:C(p.Glu298Ala, p.Glu221Ala, p.Glu298Ala), 6:73763660:A:G(p.Lys361Arg, p.Lys284Arg, p.Ly s361Arg), 6:73730573:A:G(p.Lys169Arg, p.Lys169Arg), 6:73806956:G:A(p.Gly1025Ser, p.Gly948Ser, p.Gly1 025Ser), 6:73792628:G:C(p.Asp902His, p.Asp825His, p.Asp902His), 6:73806926:A:T(p.Thr1015Ser, p.Thr93 8Ser, p.Thr1015Ser), 6:73771576:G:A(p.Glu608Lys, p.Glu531Lys, p.Glu608Lys), 6:73815026:C:T(p.Arg1272 * , p.Arg1195 * , p.Arg1255 *), 6:73765952:G:A (p.Gly377Asp, p.Gly300Asp, p.Gly377Asp).

[0079] In some embodiments, the genetic burden of a subject having any one or more CD109 missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CD109 represents a weighted sum of any multiple of the CD109 missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CD109. In some embodiments, the genetic 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 (up to 10 Mb) the CD109 gene, 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 burden score). This can include any genetic variants that are close to the CD109 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 genetic burden 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 genetic burden lower than a desired threshold score, the subject has a high risk of developing bone mineral density loss.

[0080] In some embodiments, the genetic burden can be divided into quintiles, for example, top quintile, middle quintile, and bottom quintile, with the top quintile of genetic burden corresponding to the lowest risk group and the bottom quintile of genetic burden corresponding to the highest risk group. In some embodiments, subjects with greater genetic burden include the highest weighted genetic burden, including but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% genetic 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 genetic burden in the bottom decile, quintile, or tertile in the reference population. The genetic 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.

[0081] 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 that treats or inhibits bone mineral density loss, as described herein, and / or a CD109 inhibitor. For example, if a subject is CD109-based and therefore has a high risk of developing bone mineral density loss, the subject is administered a CD109 inhibitor. In some embodiments, such a subject is administered a therapeutic agent that treats or inhibits bone mineral density loss. In some embodiments, if the subject is heterozygous for a CD109 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109, the subject is administered a therapeutic agent that treats or inhibits bone mineral density loss at a dose equal to or less than the standard dose, and is also administered a CD109 inhibitor. In some embodiments, the subject is CD109-based. In some embodiments, the subject is heterozygous for a CD109 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109. Furthermore, if a subject has a lower genetic burden for carrying a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 and therefore has a higher risk of developing bone mineral density loss, the subject is administered a therapeutic agent that treats or inhibits bone mineral density loss. In some embodiments, if a subject has a lower genetic burden for carrying a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109, the subject is administered a therapeutic agent that treats or inhibits bone mineral density loss at a dose equal to or higher than the standard dose administered to a subject with a higher genetic burden for carrying a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109.

[0082] CD109 variants available for gene burden analysis include any one or more, or any combination of the following (the variant column indicates the chromosome, 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 variations are provided according to the Human Genome Variation Society nomenclature and refer to the three annotated CD109 transcripts in the Ensembl database (URL: worldwide@useast.ensembl.org / index.html), annotations for these three transcripts are reported in the table in the following order: ENST00000287097: ENST00000422508: ENST00000437994).

[0083] [Table 2-1]

[0084] [Table 2-2]

[0085] [Table 2-3]

[0086] [Table 2-4]

[0087] [Table 2-5]

[0088] [Table 2-6]

[0089]

Table 2-7

[0090]

Table 2-8

[0091]

Table 2-9

[0092]

Table 2-10

[0093]

Table 2-11

[0094]

Table 2-12

[0095]

Table 2-13

[0096]

Table 2-14

[0097] The present disclosure also provides a method for detecting the presence or absence of CD109 missense variant genomic nucleic acid molecules (i.e., genomic nucleic acid molecules, mRNA molecules, or cDNA molecules generated from mRNA molecules) encoding predicted loss-of-function polypeptides of CD109 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 CD109 variant genomic nucleic acid molecules, CD109 variant mRNA molecules, and CD109 variant cDNA molecules are only exemplary sequences. Other sequences for CD109 variant genomic nucleic acid molecules, variant mRNA molecules, and variant cDNA molecules are also possible.

[0098] 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 CD109 missense variant nucleic acid molecules that encode predicted loss-of-function polypeptides of CD109, 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 CD109 variant mRNA molecules, various techniques may be used to enrich the biological sample for mRNA molecules. Various methods may be used to detect the presence or level of mRNA molecules, or the presence of a particular variant genomic DNA locus.

[0099] In some embodiments, detecting a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109 in a subject comprises performing sequence analysis on a biological sample obtained from the subject to determine whether a CD109 genomic nucleic acid molecule in the biological sample, and / or a CD109 mRNA molecule in the biological sample, and / or a CD109 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).

[0100] In some embodiments, a method for detecting the presence or absence of a CD109 missense 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 CD109 in a subject comprises 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.

[0101] 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 CD109 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 CD109 nucleic acid molecule. In some embodiments, the methods are in vitro methods.

[0102] In some embodiments, the determining, detecting, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a CD109 genomic nucleic acid molecule, a CD109 mRNA molecule, or a CD109 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).

[0103] In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only the CD109 genomic nucleic acid molecule is analyzed. In some embodiments, only the CD109 mRNA is analyzed. In some embodiments, only the CD109 cDNA obtained from the CD109 mRNA is analyzed.

[0104] 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.

[0105] 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.

[0106] 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 CD109 variant genomic sequence, variant mRNA sequence, or variant cDNA sequence under stringent conditions but not to the corresponding CD109 reference sequence, and determining whether hybridization occurs.

[0107] In some embodiments, the determining, detecting, or sequence analyzing step comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a CD109 polypeptide; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe; and d) detecting the detectable label.

[0108] 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).

[0109] 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 comprising CD109 variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules. 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 the detection method of choice, 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 distinct from the target nucleotide sequence and that 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.

[0110] 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).

[0111] 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.

[0112] 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.

[0113] 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.

[0114] In some embodiments, such isolated nucleic acid molecules hybridize under stringent conditions to CD109 missense 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.

[0115] 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 CD109 missense variant genomic nucleic acid molecule, a CD109 missense variant mRNA molecule, and / or a CD109 missense 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] The probes and primers described herein can be used to detect nucleotide variations in any of the CD109 variant missense genomic nucleic acid molecules, CD109 missense variant mRNA molecules, and / or CD109 missense variant cDNA molecules disclosed herein. The primers described herein can be used to amplify CD109 variant missense variant genomic nucleic acid molecules, CD109 missense variant mRNA molecules, or CD109 missense variant cDNA molecules, or fragments thereof.

[0120] In the context of this disclosure, "specifically hybridizes" means that a probe or primer (e.g., a mutation-specific probe or mutation-specific primer) does not hybridize to a nucleic acid sequence encoding a CD109-based genomic nucleic acid molecule, a CD109-based mRNA molecule, and / or a CD109-based cDNA molecule.

[0121] 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.

[0122] The nucleotide sequence of the CD109 reference genomic nucleic acid molecule is set forth in SEQ ID NO:1 (ENSG00000156535.15 chr6:73,695,785-73,828,316 in the GRCh38 / hg38 human genome assembly).

[0123] The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO:2. The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO:3. The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO:4. The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO:5. The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO:6. The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO:7. The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO:8. The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO:9. The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO:10. The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO:11. The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO:12. The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO:13. The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO: 14. The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO: 15. The nucleotide sequence of another CD109 reference mRNA molecule is set forth in SEQ ID NO: 16.

[0124] The nucleotide sequence of another CD109 reference cDNA molecule is set forth in SEQ ID NO: 17. The nucleotide sequence of another CD109 reference cDNA molecule is set forth in SEQ ID NO: 18. The nucleotide sequence of another CD109 reference cDNA molecule is set forth in SEQ ID NO: 19. The nucleotide sequence of another CD109 reference cDNA molecule is set forth in SEQ ID NO: 20. The nucleotide sequence of another CD109 reference cDNA molecule is set forth in SEQ ID NO: 21. The nucleotide sequence of another CD109 reference cDNA molecule is set forth in SEQ ID NO: 22. The nucleotide sequence of another CD109 reference cDNA molecule is set forth in SEQ ID NO: 23. The nucleotide sequence of another CD109 reference cDNA molecule is set forth in SEQ ID NO: 24. The nucleotide sequence of another CD109 reference cDNA molecule is set forth in SEQ ID NO: 25. The nucleotide sequence of another CD109 reference cDNA molecule is set forth in SEQ ID NO: 26. The nucleotide sequence of another CD109 reference cDNA molecule is set forth in SEQ ID NO: 27. The nucleotide sequence of another CD109 reference cDNA molecule is set forth in SEQ ID NO: 28. The nucleotide sequence of another CD109 reference cDNA molecule is set forth in SEQ ID NO:29.

[0125] The amino acid sequence of the CD109 reference polypeptide is set forth in SEQ ID NO:30 and is 1,428 amino acids in length. The nucleotide sequence of another CD109 reference polypeptide is set forth in SEQ ID NO:31 and is 1,368 amino acids in length. The nucleotide sequence of another CD109 reference polypeptide is set forth in SEQ ID NO:32 and is 1,445 amino acids in length. The nucleotide sequence of another CD109 reference polypeptide is set forth in SEQ ID NO:33 and is 665 amino acids in length. The nucleotide sequence of another CD109 reference polypeptide is set forth in SEQ ID NO:34 and is 1,374 amino acids in length. The nucleotide sequence of another CD109 reference polypeptide is set forth in SEQ ID NO:35 and is 854 amino acids in length. The nucleotide sequence of another CD109 reference polypeptide is set forth in SEQ ID NO:36 and is 847 amino acids in length.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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 (basic local alignment 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.

[0130] 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.

[0131] 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).

[0132] The present disclosure also provides a therapeutic agent for treating or inhibiting bone mineral density loss, the therapeutic agent being for use in treating bone mineral density loss in a subject having a CD109 missense variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109, a CD109 missense variant mRNA molecule encoding a predicted loss-of-function polypeptide of CD109, or a CD109 missense variant cDNA molecule encoding a predicted loss-of-function polypeptide of CD109.Any of the therapeutic agents for treating or inhibiting 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.

[0133] The present disclosure also provides the use of a therapeutic agent for treating or inhibiting bone mineral density loss, for use in preparing a medicament for treating bone mineral density loss in a subject having a CD109 missense variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109, a CD109 missense variant mRNA molecule encoding a predicted loss-of-function polypeptide of CD109, or a CD109 missense variant cDNA molecule encoding a predicted loss-of-function polypeptide of CD109.Any of the therapeutic agents for treating or inhibiting 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.

[0134] The present disclosure also provides a CD109 inhibitor for use in treating bone mineral density loss in a subject that is a) a reference for CD109 genomic nucleic acid molecule, CD109 mRNA molecule, or CD109 cDNA molecule, or b) heterozygous for i) CD109 missense variant genomic nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109; ii) CD109 missense variant mRNA molecule that encodes a predicted loss-of-function polypeptide of CD109; or iii) CD109 missense variant cDNA molecule that encodes a predicted loss-of-function polypeptide of CD109.Any of the CD109 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.

[0135] The present disclosure also provides the use of CD109 inhibitor in preparing a drug for treating bone mineral density loss in a subject that is a) a reference for CD109 genomic nucleic acid molecule, CD109 mRNA molecule, or CD109 cDNA molecule, or b) i) a CD109 missense variant genomic nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CD109; ii) a CD109 missense variant mRNA molecule that encodes a predicted loss-of-function polypeptide of CD109; or iii) a CD109 missense variant cDNA molecule that encodes a predicted loss-of-function polypeptide of CD109.Any of the CD109 inhibitors described herein can be used in these methods.The subject can have or be at risk of developing bone mineral density loss, osteopenia, osteoporosis type I, osteoporosis type II, or secondary osteoporosis.

[0136] 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.

[0137] 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

[0138] General Methodology UK Biobank cohort description Genetic associations were investigated in the United Kingdom (UK) Biobank (UKB), a population-based cohort of subjects aged 40-69 years recruited through 22 testing centers in the UK between 2006 and 2010. We used genetic and phenotypic information from nearly 300,000 participants of European ancestry in the UKB.

[0139] 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 2Phenotypic values ​​of eBMD were first transformed using a rank-based inverse normal transformation applied separately to sexes within each pedigree group and adjusted for fine-mapped common genetic variants associated with eBMD.

[0140] Genotype data High-coverage whole-exome sequencing was performed as previously reported (Dewey et al., Science, 2016, 354,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 of samples using Illumina software, alignment to the GRCh38 human genome reference sequence including generation of binary alignment and mapping files (BAM), 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., 2018, bioRxiv: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 relating to protein-coding transcripts with annotated start and end 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 deleterious to least deleterious) was frameshift, stop gain, stop loss, splice acceptor, splice donor, stop lost, 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 in seven gene burden exposures: 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 variant with AAF<0.1%.

[0141] Association analysis of the gene burden of rare pLOF and missense mutations in CD109 We investigated the association between the burden of rare predicted loss-of-function or missense variants in CD109 and eBMD by fitting linear regression models including adjustment for a polygenic score that approximates a genomic relationship matrix using REGENIE v1.0 (Mbatchou et al., Nature Genetics, 2021). Analyses were performed with age, age, and genomic relationship matrix (Grade 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 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.

[0142] Effector index for eBMD causative genes A novel machine learning algorithm, Effector Index, has been described elsewhere in the literature (Forgetta et al., bioRxiv:2021, 2020.2006.2028.171561). Training data was generated by performing GWAS analyses for 11 diseases and traits (type 2 diabetes, low-density lipoprotein cholesterol levels, adult height, calcium levels, hypothyroidism, triglyceride levels, glucose levels, red blood cell count systolic blood pressure, diastolic blood pressure, and direct bilirubin levels). Fine mapping was performed for each GWAS dataset, and genomic annotations were used as features to predict positive control genes at fine-mapped GWAS loci using a gradient boosting tree algorithm (XGBoost). This trained algorithm was then tested against fine-mapped and annotated eBMD-associated data at the CD109 locus to test the probability that the CD109 gene is a gene at this locus that affects eBMD.

[0143] Mendelian randomization analysis of circulating CD109 We used two-sample Mendelian randomization (MR) to examine the association between genetically predicted circulating CD109 and eBMD. This approach uses common genetic variants (termed protein quantitative trait loci, or pQTLs) associated with CD109 protein concentrations as instrumental variables. Lead CD109 cis-pQTLs were identified in two previously published studies conducted in the INTERVAL (N=3,301) and AGES (N=3,200) cohorts (Sun et al., Nature, 2018, 558, 73-79; and Emilsson et al., Science, 2018, eaaq1327). pQTL-outcome associations for this analysis were extracted from a previously published GWAS of eBMD in UKB, and MR analysis was performed using the Wald ratio method, using the TwoSampleMR R package. Colocalization analysis was performed to examine the effects of confounding due to linkage disequilibrium. This involved assessing whether gene association signals for CD109 protein concentration may share the same causal variants as eBMD gene association signals in CD109. These colocalization analyses were performed using two previously published algorithms, Coloc (Giambartolomei et al., PLOS Genetics, 2014, 10, e1004383) and eCAVIAR (Hormozdiari et al., Am. J. Hum. Genet., 2016, 99, 1245-1260).

[0144] Example 1: Loss of function of CD109 is associated with higher estimated bone mineral density Whole exome sequencing of 278,807 subjects of European ancestry in the UK Biobank (UKB) was performed to identify predicted loss-of-function (pLoF) and missense gene variants in each gene in the genome. We examined the association of each gene and gene variant sequenced in the UKB 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.

[0145] Exome-wide analysis in UKB found that rare (alternative allele frequency [AAF] < 1%) pLoF variant burden in the CD109 gene was associated with 0.18 standard deviation units higher eBMD (P value = 1.20 × 10 -09 , 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) (Table 2; association estimates were derived in UKB in association with CD109 pLoF variant burden of AAF<1%).

[0146] [Table 3]

[0147] Genotype counts indicate the number of subjects in each of the three genotype categories: RR indicates subjects who do not carry a rare pLoF variant in CD109; RA indicates subjects who carry a rare pLoF variant in a single CD109 allele; AA indicates subjects who carry a rare pLoF variant in both CD109 alleles. AAF indicates the alternative allele frequency of the pLoF variants included in this analysis. g / cm 2 is grams per square centimeter, SD is standard deviation, and CI is confidence interval.

[0148] When examining the genetic burden of rare pLoF or predicted damaging missense variants in CD109, the association of CD109 variants with higher eBMD was also significant (Table 3; association estimates were derived in UKB in association with CD109 pLoF or predicted damaging missense variant burden of AAF<1% or <0.1% (see genotype data below for description of the in silico algorithm used to identify predicted damaging missense variants)). These genetic data suggest that loss of function of CD109 results in higher eBMD in humans.

[0149] [Table 4]

[0150] Genotype counts indicate the number of subjects in each of the three genotype categories: RR indicates subjects carrying no rare pLoF variants in CD109; RA indicates subjects carrying rare pLoF or damaging missense variants in a single CD109 allele; AA indicates subjects carrying rare pLoF or damaging missense variants in both CD109 alleles. AAF is the alternative allele frequency of the variants included in this analysis. g / cm 2 is grams per square centimeter, SD is standard deviation, and CI is confidence interval.

[0151] Example 2: Variants associated with low CD109 protein concentrations in the blood are also associated with high eBMD. Using Mendelian randomization, we found that lower circulating CD109 protein (encoded by the CD109 gene) due to common genetic variants at the CD109 locus was associated with higher eBMD (Table 4; lead cis protein quantitative trait loci (pQTL) for CD109 were obtained in two independent cohorts: INTERVAL and AGES. Two-sample Mendelian randomization analysis was performed using the UKB eBMD GWAS data as outcome dataset). This association was further supported by colocalization analysis performed using two different algorithms (Coloc and eCAVIAR; Coloc posterior probability for H3 = 0.042, Coloc posterior probability for H4 = 0.958; eCAVIAR CLPP C1 = 0.024, CLPP C2 = 0.002). These results provided complementary evidence to the results reported in Table 2, which showed that loss of function of CD109 was associated with higher eBMD. Several individual rare pLoF and missense variants in CD109 showed nominal evidence of association with eBMD in UKB (P value for association < 0.05; Table 4).

[0152] [Table 5]

[0153] Example 3: Machine learning algorithms applied to common genetic variants in CD109 revealed further evidence implicating CD109 as a causative gene involved in the association with eBMD We applied a machine learning algorithm (Effector Index) to the eBMD genome-wide association data and identified strong evidence suggesting that CD109 is a causative gene involved in the eBMD GWAS associations in this genomic region (Effector Index = 0.96, indicating a high probability that CD109 is a causative gene at this locus).

Claims

**Claim 1** An in vitro method for identifying a subject's susceptibility to the onset of reduced bone mineral density, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis, the method comprising: determining, or having determined, the presence or absence of a CD109 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of cluster of differentiation 109 (CD109) in a biological sample obtained from the subject; wherein a subject who is a CD109 criterion indicates a subject at high risk of developing reduced bone mineral density, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis; and a subject who is heterozygous or homozygous for the CD109 missense variant nucleic acid molecule encoding the predicted loss-of-function polypeptide of CD109 indicates a subject at low risk of developing reduced bone mineral density, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis. **Claim 2** The method according to claim 1, wherein the CD109 missense 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 CD109. **Claim 3** The method according to claim 1 or claim 2, wherein the predicted loss-of-function variant nucleic acid molecule of CD109 is 6:73730573:A:G, 6:73823473:GA:G, 6:73763607:C:A, 6:73803256:G:T, 6:73818486:T:C, 6:73787379:G:A, 6:73771510:A:G, 6:73806987:A:T, 6:73758991:A:G, 6:73823456:A:G, 6:73762778:A:C, 6:73763660:A:G, 6:73730573:A:G, 6:73806956:G:A, 6:73792628:G:C, 6:73806926:A:T, 6:73771576:G:A, 6:73815026:C:T, 6:73765952:G:A or an mRNA molecule produced therefrom, or a cDNA molecule produced from the mRNA molecule. **Claim 4** The method according to claim 1 or claim 2, wherein the CD109 missense mutant nucleic acid molecule encodes a predicted loss-of-function polypeptide of truncated CD109.

5. Use of a therapeutic agent for treating or inhibiting bone mineral density reduction in the preparation of a medicament for treating bone mineral density reduction, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis in a subject, wherein the subject has a CD109 missense mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of differentiation antigen group 109 (CD109).

6. The use according to claim 5, wherein the therapeutic agent is selected from alendronate, ibandronate, zoledronate, risedronate, calcitonin, teriparatide, denosumab, estrogen and progesterone, or raloxifene.

7. Use of a differentiation antigen group 109 (CD109) inhibitor in the preparation of a medicament for treating bone mineral density reduction, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis in a subject, wherein the subject a) is a reference for a CD109 genomic nucleic acid molecule or a CD109 mRNA molecule, or b) is heterozygous for a CD109 missense mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109.

8. The use according to claim 7, wherein the CD109 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to a CD109 nucleic acid molecule.

9. The use according to claim 8, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA).

10. The use according to claim 7, wherein the CD109 inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within a CD109 genomic nucleic acid molecule.

11. The use according to claim 10, wherein the Cas protein is Cas9 or Cpf1.

12. The use according to claim 10 or claim 11, wherein the gRNA recognition sequence is located within SEQ ID NO:

1.

13. The use according to claim 10 or claim 11, wherein the protospacer adjacent motif (PAM) sequence is about 2 to about 6 nucleotides downstream of the gRNA recognition sequence.

14. The use according to claim 10 or claim 11, wherein the gRNA comprises about 17 to about 23 nucleotides.

15. The use according to claim 10 or claim 11, wherein the gRNA recognition sequence comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 37 to 58.

16. The predicted loss-of-function mutant nucleic acid molecule of CD109 is 6:73730573:A:G, 6:73823473:GA:G, 6:73763607:C:A, 6:73803256:G:T, 6:73818486:T:C, 6:73787379:G:A, 6:73771510:A:G, 6:73806987:A:T, 6:73758991:A:G, 6:73823456:A:G, 6:73762778:A:C, 6:73763660:A:G, 6:73730573:A:G, 6:73806956:G:A, 6:73792628:G:C, 6:73806926:A:T, 6:73771576:G:A, 6:73815026:C:T, 6:73765952:G:A or an mRNA molecule produced therefrom, or a cDNA molecule produced from said mRNA molecule, the use according to claim 7 or claim 8.

17. The use according to claim 5 or claim 6, wherein the CD109 missense mutant nucleic acid 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 CD109. **Claim 18**: The use according to claim 5 or claim 6, wherein the predicted loss-of-function mutant nucleic acid molecule of CD109 is 6:73730573:A:G, 6:73823473:GA:G, 6:73763607:C:A, 6:73803256:G:T, 6:73818486:T:C, 6:73787379:G:A, 6:73771510:A:G, 6:73806987:A:T, 6:73758991:A:G, 6:73823456:A:G, 6:73762778:A:C, 6:73763660:A:G, 6:73730573:A:G, 6:73806956:G:A, 6:73792628:G:C, 6:73806926:A:T, 6:73771576:G:A, 6:73815026:C:T, 6:73765952:G:A or an mRNA molecule produced therefrom, or a cDNA molecule produced from said mRNA molecule. **Claim 19**: The use according to claim 5 or claim 6, wherein the CD109 missense mutant nucleic acid molecule encodes a predicted loss-of-function polypeptide of truncated CD109. **Claim 20**: The use according to claim 7 or claim 8, wherein the CD109 missense mutant nucleic acid 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 CD109. **Claim 21**: The use according to claim 20, wherein the CD109 missense mutant nucleic acid molecule encodes a predicted loss-of-function polypeptide of truncated CD109. **Claim 22**: In a pharmaceutical composition for treating decreased bone mineral density, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis in a patient, wherein the patient is heterozygous for a CD109 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of cluster of differentiation 109 (CD109), and the pharmaceutical composition comprises, as an active ingredient, a therapeutic agent for treating or preventing decreased bone mineral density. In a pharmaceutical composition for treating decreased bone mineral density, osteopenia, type I osteoporosis, type II osteoporosis, or secondary osteoporosis in a patient, the patient is a) a reference with respect to a cluster of differentiation 109 (CD109) genomic nucleic acid molecule or a CD109 mRNA molecule, or b) heterozygous with respect to a CD109 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CD109, and the pharmaceutical composition comprises a CD109 inhibitor as an active ingredient.