Exome-wide benchmark of difficult-to-sequence regions using short-read next-generation DNA sequencing.

Next-generation DNA sequencing (NGS) in short-read mode has recently been used for genetic testing in various clinical settings. NGS data accuracy is crucial in clinical settings, and several reports regarding quality control of NGS data, primarily focusing on establishing NGS sequence read accuracy, have been published thus far. Variant calling is another critical source of NGS errors that remains unexplored at the single-nucleotide level despite its established significance. In this study, we used a machine-learning-based method to establish an exome-wide benchmark of difficult-to-sequence regions at the nucleotide-residue resolution using 10 genome sequence features based on real-world NGS data accumulated in The Genome Aggregation Database (gnomAD) of the human reference genome sequence (GRCh38/hg38). The newly acquired metric, designated the 'UNMET score,' along with additional lines of structural information from the human genome, allowed us to assess the sequencing challenges within the exonic region of interest using conventional short-read NGS. Thus, the UNMET score could provide a basis for addressing potential sequential errors in protein-coding exons of the human reference genome sequence GRCh38/hg38 in clinical sequencing.

Chromosomal microdeletion leading to pituitary gigantism through hormone-gene overexpression.

Pituitary gigantism is a rare endocrinopathy characterized by tall stature due to growth hormone (GH) hypersecretion. This condition is generally linked to a genetic predisposition to tumors that produce GH or GH-releasing hormone (GHRH). Here, we report a Japanese woman who exhibited prominent body growth from infancy to reach an adult height of 197.4 cm (+7.4 standard deviation). Her blood GH levels were markedly elevated. She carried no pathogenic variants in known growth-controlling genes but had a hitherto unreported 752 kb heterozygous deletion at 20q11.23. The microdeletion was located 8.9 kb upstream of GHRH and encompassed exons 2-9 of a ubiquitously expressed gene TTI1 together with 12 other genes, pseudogenes and non-coding RNAs. Transcript analyses of the patient's leukocytes showed that the microdeletion produced chimeric mRNAs consisting of exon 1 of TTI1 and all coding exons of GHRH. In silico analysis detected promoter-associated genomic features around TTI1 exon 1. Genome-edited mice carrying the same microdeletion recapitulated accelerated body growth from a few weeks after birth. The mutant mice developed pituitary hyperplasia and exhibited ectopic Ghrh expression in all tissues examined. Thus, the extreme phenotype of pituitary gigantism in the patient likely reflects GHRH overexpression driven by an acquired promoter. The results of this study indicate that germline submicroscopic deletions have the potential to cause conspicuous developmental abnormalities due to gene overexpression. Furthermore, this study provides evidence that constitutive expression of a hormone-encoding gene can result in congenital disease.

Integrator complex subunit 15 controls mRNA splicing and is critical for eye development.

The eye and brain are composed of elaborately organized tissues, development of which is supported by spatiotemporally precise expression of a number of transcription factors and developmental regulators. Here we report the molecular and genetic characterization of Integrator complex subunit 15 (INTS15). INTS15 was identified in search for the causative gene(s) for an autosomal-dominant eye disease with variable individual manifestation found in a large pedigree. While homozygous Ints15 knockout mice are embryonic lethal, mutant mice lacking a small C-terminal region of Ints15 show ocular malformations similar to the human patients. INTS15 is highly expressed in the eye and brain during embryogenesis and stably interacts with the Integrator complex to support small nuclear RNA 3' end processing. Its knockdown resulted in missplicing of a large number of genes, probably as a secondary consequence, and substantially affected genes associated with eye and brain development. Moreover, studies using human iPS cells-derived neural progenitor cells showed that INTS15 is critical for axonal outgrowth in retinal ganglion cells. This study suggests a new link between general transcription machinery and a highly specific hereditary disease.

Two ovarian candidate enhancers, identified by time series enhancer RNA analyses, harbor rare genetic variations identified in ovarian insufficiency.

The genetic regulation of ovarian development remains largely unclear. Indeed, in most cases of impaired ovarian development-such as 46,XX disorders of sex development (DSD) without SRY, and premature ovarian insufficiency (POI)-the genetic causes have not been identified, and the vast majority of disease-associated sequence variants could lie within non-coding regulatory sequences. In this study, we aimed to identify enhancers of five ovarian genes known to play key roles in early ovarian development, basing our analysis on the expression of enhancer derived transcripts (eRNAs), which are considered to characterize active enhancers. Temporal expression profile changes in mouse WT1-positive ovarian cells were obtained from cap analysis of gene expression at E13.5, E16.5 and P0. We compared the chronological expression profiles of ovarian-specific eRNA with expression profiles for each of the ovarian-specific genes, yielding two candidate sequences for enhancers of Wnt4 and Rspo1. Both sequences are conserved between mouse and human, and we confirmed their enhancer activities using transient expression assays in murine granulosa cells. Furthermore, by sequencing the region in patients with impaired ovarian development in 24 patients, such as POI, gonadal dysgenesis and 46,XX DSD, we identified rare single nucleotide variants in both sequences. Our results demonstrate that combined analysis of the temporal expression profiles of eRNA and mRNA of target genes presents a powerful tool for locating cis-element enhancers, and a means of identifying disease-associated sequence variants that lie within non-coding regulatory sequences, thus advancing an important unmet need in forward human genetics.

Systematic molecular analyses for 115 karyotypically normal men with isolated non-obstructive azoospermia.

STUDY QUESTION: Do copy-number variations (CNVs) in the azoospermia factor (AZF) regions and monogenic mutations play a major role in the development of isolated (non-syndromic) non-obstructive azoospermia (NOA) in Japanese men with a normal 46, XY karyotype? SUMMARY ANSWER: Deleterious CNVs in the AZF regions and damaging sequence variants in eight genes likely constitute at least 8% and approximately 8% of the genetic causes, respectively, while variants in other genes play only a minor role. WHAT IS KNOWN ALREADY: Sex chromosomal abnormalities, AZF-linked microdeletions, and monogenic mutations have been implicated in isolated NOA. More than 160 genes have been reported as causative/susceptibility/candidate genes for NOA. STUDY DESIGN, SIZE, DURATION: Systematic molecular analyses were conducted for 115 patients with isolated NOA and a normal 46, XY karyotype, who visited our hospital between 2017 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied 115 unrelated Japanese patients. AZF-linked CNVs were examined using sequence-tagged PCR and multiplex ligation-dependent probe amplification, and nucleotide variants were screened using whole exome sequencing (WES). An optimized sequence kernel association test (SKAT-O), a gene-based association study using WES data, was performed to identify novel disease-associated genes in the genome. The results were compared to those of previous studies and our in-house control data. MAIN RESULTS AND THE ROLE OF CHANCE: Thirteen types of AZF-linked CNVs, including the hitherto unreported gr/gr triplication and partial AZFb deletion, were identified in 63 (54.8%) cases. When the gr/gr deletion, a common polymorphism in Japan, was excluded from data analyses, the total frequency of CNVs was 23/75 (30.7%). This frequency is higher than that of the reference data in Japan and China (11.1% and 14.7%, respectively). Known NOA-causative AZF-linked CNVs were found in nine (7.8%) cases. Rare damaging variants in known causative genes (DMRT1, PLK4, SYCP2, TEX11, and USP26) and hemizygous/multiple-heterozygous damaging variants in known spermatogenesis-associated genes (TAF7L, DNAH2, and DNAH17) were identified in nine cases (7.8% in total). Some patients carried rare damaging variants in multiple genes. SKAT-O detected no genes whose rare damaging variants were significantly accumulated in the patient group. LIMITATIONS, REASONS FOR CAUTION: The number of participants was relatively small, and the clinical information of each patient was fragmentary. Moreover, the pathogenicity of identified variants was assessed only by in silico analyses. WIDER IMPLICATIONS OF THE FINDINGS: This study showed that various AZF-linked CNVs are present in more than half of Japanese NOA patients. These results broadened the structural variations of AZF-linked CNVs, which should be considered for the molecular diagnosis of spermatogenic failure. Furthermore, the results of this study highlight the etiological heterogeneity and possible oligogenicity of isolated NOA. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Grants from the Japan Society for the Promotion of Science (21K19283 and 21H0246), the Japan Agency for Medical Research and Development (22ek0109464h0003), the National Center for Child Health and Development, the Canon Foundation, the Japan Endocrine Society, and the Takeda Science Foundation. The results of this study were based on samples and patient data obtained from the International Center for Reproductive Medicine, Dokkyo Medical University Saitama Medical Center, Koshigaya, Japan. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

SHOX and sex difference in height: a hypothesis.

The mean height is taller in males than in females, except for early teens. In this regard, previous studies have revealed that (1) distribution of the mean adult heights in subjects with disorders accompanied by discordance between sex chromosome complement and bioactive sex steroids and in control subjects (the British height standards) indicates that, of the ~12.5 cm of sex difference in the mean adult height, ~9 cm is accounted for by the difference in the sex chromosome complement and the remaining ~3.5 cm is explained by the dimorphism in sex steroids (primarily due to the growth-promoting effect of gonadal androgens); (2) according to the infancy-childhood-puberty growth model, the sex difference in the childhood growth function produces height differences of ~1 cm in childhood and 8-10 cm at 18-20 years of age, whereas the sex difference in the pubertal growth function yields height difference of ~4.5 cm at 18-20 years of age; and (3) SHOX expression and methylation analyses using knee cartilage tissues and cultured chondrocytes have shown lower SHOX expression levels in female samples than in male samples and methylation patterns consistent with partial spreading of X-inactivation affecting SHOX in female samples. These findings suggest that small but persistent sex difference in SHOX expression dosage leads to the variation in the sex steroid independent childhood growth function, thereby yielding the sex difference in height which remains small in childhood but becomes obvious in adulthood.

Y Chromosome Genomic Variations and Biological Significance in Human Diseases and Health.

The Y chromosome is a haploid genome unique to males with no genes essential for life. It is easily transmitted to the next generation without being repaired by recombination, even if a major genomic structural alteration occurs. On the other hand, the Y chromosome genome is basically a region transmitted only from father to son, reflecting a male-specific inheritance between generations. The Y chromosome exhibits genomic structural differences among different ethnic groups and individuals. The Y chromosome was previously thought to affect only male-specific phenotypes, but recent studies have revealed associations between the Y chromosomes and phenotypes common to both males and females, such as certain types of cancer and neuropsychiatric disorders. This evidence was discovered with the finding of the mosaic loss of the Y chromosome in somatic cells. This phenomenon is also affected by environmental factors, such as smoking and aging. In the past, functional analysis of the Y chromosome has been elucidated by assessing the function of Y chromosome-specific genes and the association between Y chromosome haplogroups and human phenotypes. These studies are currently being conducted intensively. Additionally, the recent advance of large-scale genome cohort studies has increased the amount of Y chromosome genomic information available for analysis, making it possible to conduct more precise studies of the relationship between genome structures and phenotypes. In this review, we will introduce recent analyses using large-scale genome cohort data and previously reported association studies between Y chromosome haplogroups and human phenotypes, such as male infertility, cancer, cardiovascular system traits, and neuropsychiatric disorders. The function and biological role of the Y chromosome in human phenotypes will also be discussed.

Rare sequence variants associated with the risk of non-syndromic biliary atresia.

AIM: The etiology of non-syndromic biliary atresia (BA) remains largely unknown. In this study, we performed genome-wide screening of genes associated with the risk of non-syndromic BA. METHODS: We analyzed exome data of 15 Japanese patients with non-syndromic BA and 509 control individuals using an optimal sequence kernel association test (SKAT-O), a gene-based association study optimized for small-number subjects. Furthermore, we examined the frequencies of known BA-related single-nucleotide polymorphisms in the BA and control groups. RESULTS: SKAT-O showed that rare damaging variants of MFHAS1, a ubiquitously expressed gene encoding a Toll-like receptor-associated protein, were more common in the BA group than in the control group (Bonferroni corrected p-value = 0.0097). Specifically, p.Val106Gly and p.Arg556Cys significantly accumulated in the patient group. These variants resided within functionally important domains. SKAT-O excluded the presence of other genes significantly associated with the disease risk. Of 60 known BA-associated single-nucleotide polymorphisms, only eight were identified in the BA group. In particular, p.Ile3421Met of MYO15A and p.Ala421Thr of THOC2 were more common in the BA group than in the control group. However, the significance of these two variants is questionable, because MYO15A has been linked to deafness, but not to BA, and the p.Ala421Thr of THOC2 represents a relatively common single-nucleotide polymorphism in Asia. CONCLUSIONS: The results of this study indicate that rare damaging variants in MFHAS1 may constitute a risk factor for non-syndromic BA, whereas the contribution of other monogenic variants to the disease predisposition is limited.

CDKN1C hyperexpression in two patients with severe growth failure and microdeletions affecting the paternally inherited KCNQ1OT1:TSS-DMR.

BACKGROUND: Two imprinting control centres, H19/IGF2:IG-differentialy methylated region (DMR) and KCNQ1OT1:TSS-DMR, reside on chromosome 11p15.5. Paternal deletions involving the KCNQ1OT1:TSS-DMR result in variable phenotypes, namely, normal phenotype, Silver-Russel syndrome (SRS) and fetal demise. However, expression analyses for CDKN1C in these patients are very limited. CASES: Patient 1 (adult woman) and patient 2 (boy in early childhood) showed prenatal and postnatal growth failure and clinical suspicion of SRS. MOLECULAR ANALYSES: Both patients showed hypermethylation of the KCNQ1OT1:TSS-DMR caused by the paternal heterozygous de novo deletions involving the KCNQ1OT1:TSS-DMR, but not including CDKN1C enhancers. The deletion sizes were 5 kb and 12 kb for patients 1 and 2, respectively. CDKN1C gene expressions in immortalised leucocytes of both patients were increased compared with those of controls. CONCLUSION: Paternal deletions involving the KCNQ1OT1:TSS-DMR, but not including CDKN1C enhancers, disrupt KCNQ1OT1 expression, strongly activate CDKN1C expression and consequently cause severe growth failure.

The Structural Abnormalities Are Deeply Involved in the Cause of RPGRIP1-Related Retinal Dystrophy in Japanese Patients.

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophy. RPGRIP1-related LCA accounts for 5-6% of LCA. We performed whole-exome sequencing and whole-genome sequencing (WGS) on 29 patients with clinically suspected LCA and examined ophthalmic findings in patients with biallelic pathogenic variants of RPGRIP1. In addition to five previously reported cases, we identified five cases from four families with compound heterozygous RPGRIP1 variants using WGS. Five patients had null variants comprising frameshift variants, an Alu insertion, and microdeletions. A previously reported 1339 bp deletion involving exon 18 was found in four cases, and the deletion was relatively prevalent in the Japanese population (allele frequency: 0.002). Microdeletions involving exon 1 were detected in four cases. In patients with RPGRIP1 variants, visual acuity remained low, ranging from light perception to 0.2, and showed no correlation with age. In optical coherence tomography images, the ellipsoid zone (EZ) length decreased with age in all but one case of unimpaired EZ. The retinal structure was relatively preserved in all cases; however, there were cases with great differences in visual function compared to their siblings and a 56-year-old patient who still had a faint EZ line. Structural abnormalities may be important genetic causes of RPGRIP1-related retinal dystrophy in Japanese patients, and WGS was useful for detecting them.

Genetic variants of G-protein coupled receptors associated with pubertal disorders.

Background: The human hypothalamic-pituitary-gonadal (HPG) axis is the regulatory center for pubertal development. This axis involves six G-protein coupled receptors (GPCRs) encoded by KISS1R, TACR3, PROKR2, GNRHR, LHCGR, and FSHR. Methods: Previous studies have identified several rare variants of the six GPCR genes in patients with pubertal disorders. In vitro assays and animal studies have provided information on the function of wild-type and variant GPCRs. Main Findings: Of the six GPCRs, those encoded by KISS1R and TACR3 are likely to reside at the top of the HPG axis. Several loss-of-function variants in the six genes were shown to cause late/absent puberty. In particular, variants in KISS1R, TACR3, PROKR2, and GNRHR lead to hypogonadotropic hypogonadism in autosomal dominant, recessive, and oligogenic manners. Furthermore, a few gain-of-function variants of KISS1R, PROKR2, and LHCGR have been implicated in precocious puberty. The human HPG axis may contain additional GPCRs. Conclusion: The six GPCRs in the HPG axis govern pubertal development through fine-tuning of hormone secretion. Rare sequence variants in these genes jointly account for a certain percentage of genetic causes of pubertal disorders. Still, much remains to be clarified about the molecular network involving the six GPCRs.

Exome-based genome-wide screening of rare variants associated with the risk of polycystic ovary syndrome.

Purpose: Genetic factors associated with the risk of polycystic ovary syndrome (PCOS) remain largely unknown. Here, we conducted an optimal sequence kernel association test (SKAT-O), an exome-based rare variant association study, to clarify whether rare variants in specific genes contribute to the development of PCOS. Methods: SKAT-O was performed using exome data of 44 Japanese patients with PCOS and 301 control women. We analyzed frequencies of rare probably damaging variants in the genome. Results: Rare variants of GSTO2 were more commonly identified in the patient group than in the control group (6/44 vs. 1/301; Bonferroni-corrected p-value, 0.028), while the frequencies of variants in other genes were comparable between the two groups. The identified GSTO2 variants were predicted to affect the function, structure, stability, hydrophobicity, and/or the formation of intrinsically disordered regions of the protein. GSTO2 encodes a glutathione transferase that mediates the oxidative stress response and arsenic metabolism. Previously, common variants in GSTO2 and its paralog GSTO1 were associated with the risk of PCOS. Conclusions: The results indicate that there are no genes whose rare variants account for a large fraction of the etiology of PCOS, although rare damaging variants in GSTO2 may constitute a risk factor in some cases.

Optical Genome Mapping for a Patient with a Congenital Disorder and Chromosomal Translocation.

We performed optical genome mapping (OGM), a newly developed cytogenetic technique, for a patient with a disorder of sex development (DSD) and a 46,XX,t(9;11)(p22;p13) karyotype. The results of OGM were validated using other methods. OGM detected a 9;11 reciprocal translocation and successfully mapped its breakpoints to small regions of 0.9-12.3 kb. OGM identified 46 additional small structural variants, only three of which were detected by array-based comparative genomic hybridization. OGM suggested the presence of complex rearrangements on chromosome 10; however, these variants appeared to be artifacts. The 9;11 translocation was unlikely to be associated with DSD, while the pathogenicity of the other structural variants remained unknown. These results indicate that OGM is a powerful tool for detecting and characterizing chromosomal structural variations, although the current methods of OGM data analyses need to be improved.

ACAN biallelic variants in a girl with severe idiopathic short stature.

Although ACAN heterozygous loss-of-function variants often cause idiopathic short stature (ISS) phenotype, there is no report describing ISS phenotype caused by ACAN biallelic loss-of-function variants. We encountered a 4 1/12-year-old Japanese girl with a height of 80.4 cm (-5.2 SD), a weight of 11.4 kg (-1.9 SD), a head circumference of 48.7 cm (-0.6 SD), and an arm span/height ratio of 1.0 (+1.1 SD). Endocrine studies and bone survey showed no abnormal findings. Whole exome sequencing revealed biallelic rare variants in ACAN, i.e., NM_013227.4:c.4214delC:p.(Pro1405Leufs*3) derived from her father and paternal grandfather with short stature (-2.9 and -2.0 SD, respectively) and NM_013227.4:c.7124 A>G:p.(Gln2375Arg) inherited from her mother and maternal grandmother with short stature (-2.1 and -3.0 SD, respectively). The frameshift variant underwent nonsense mediated mRNA decay, and the missense variant was assessed to have high pathogenicity. The results imply for the first time that ACAN biallelic loss-of-function variants can cause severe ISS phenotype.

Frequency and clinical characteristics of distinct etiologies in patients with Silver-Russell syndrome diagnosed based on the Netchine-Harbison clinical scoring system.

Silver-Russel syndrome (SRS) is a representative imprinting disorder (ID) characterized by growth failure and diagnosed by clinical features. Recently, international consensus has recommended using the Netchine-Harbison clinical scoring system (NH-CSS) as clinical diagnostic criteria. Loss of methylation of H19/IGF2:intergenic differentially methylated region (H19LOM) and maternal uniparental disomy chromosome 7 (UPD(7)mat) are common etiologies of SRS; however, other IDs, pathogenic variants (PVs) of genes, and pathogenic copy number variants (PCNVs) have been reported in patients meeting NH-CSS. To clarify the frequency and clinical characteristics of each etiology, we conducted (epi)genetic analysis in 173 patients satisfying NH-CSS. H19LOM and UPD(7)mat were identified in 34.1%. PCNVs, other IDs, and PVs were in 15.0%. Patients with all six NH-CSS items were most frequently observed with H19LOM and UPD(7)mat. This study confirmed the suitability of NH-CSS as clinical diagnostic criteria, the (epi)genetic heterogeneity of SRS, and showed the necessity of further discussion regarding the "SRS spectrum".

Retrotransposition disrupting EBP in a girl and her mother with X-linked dominant chondrodysplasia punctata.

X-linked dominant chondrodysplasia punctata (CDPX2) is a rare congenital disorder caused by pathogenic variants in EBP on Xp11.23. We encountered a girl and her mother with CDPX2-compatible phenotypes including punctiform calcification in the neonatal period of the girl, and asymmetric limb shortening and ichthyosis following the Blaschko lines in both subjects. Although Sanger direct sequencing failed to reveal a disease-causing variant in EBP, whole genome sequencing (WGS) followed by Manta analysis identified a ~ 4.5 kb insertion at EBP exon 2 of both subjects. The insertion was associated with the hallmarks of retrotransposition such as an antisense poly(A) tail, a target site duplication, and a consensus endonuclease cleavage site, and the inserted sequence harbored full-length SVA_F1 element with 5'- and 3'-transductions containing the Alu sequence. The results imply the relevance of retrotransposition to the human genetic diseases and the usefulness of WGS in the identification of retrotransposition.

SHOX far-downstream deletion in a patient with nonsyndromic short stature.

Haploinsufficiency of SHOX represents one of the major genetic causes of nonsyndromic short stature. To date, eight DNA elements around SHOX exons have been proposed as putative enhancer regions. Although six copy-number variations (CNVs) downstream to the known enhancer regions have recently been identified in patients with short stature, the pathogenicity of these CNVs remains uncertain. Here, we identified a paternally derived SHOX far-downstream deletion in a boy. The deletion involved a ~100 kb genomic interval at a position >60 kb away from the known enhancer regions. The boy exhibited moderate short stature with nonspecific skeletal changes. The height of the father was within the normal range but lower than the mid-parental height. The deletion of the boy and the six previously reported CNVs mostly overlapped; however, all CNVs had unique breakpoints. The deletion of our case encompassed a ~30 kb genomic interval that has previously been associated with a 4C-seq peak, as well as several SHOX-regulatory SNPs/indels. These results indicate that the SHOX far-downstream region contains a novel cis-acting enhancer, whose deletion leads to nonsyndromic short stature of various degree. In addition, our data highlight genomic instability of SHOX-flanking regions that underlies diverse nonrecurrent CNVs.

Quantification of Maternal Microchimeric Cells in the Liver of Children With Biliary Atresia.

ABSTRACT: Biliary atresia (BA) is a rare disorder of unknown etiology. There is a debate as to whether maternal microchimerism plays a significant role in the development of BA or in graft tolerance after liver transplantation. Here, we performed quantitative-PCR-based assays for liver tissues of children with BA and other diseases. Maternal cells were detected in 4/13 and 1/3 of the BA and control groups, respectively. The estimated number of maternal cells ranged between 0 and 34.7 per 106 total cells. The frequency and severity of maternal microchimerism were similar between the BA and control groups, and between patients with and without acute rejection of maternal grafts. These results highlight the high frequency of maternal microchimerism in the liver. This study provides no evidence for roles of microchimerism in the etiology of BA or in graft tolerance. Thus, the biological consequences of maternal microchimerism need to be clarified in future studies.

Mosaic loss of the Y chromosome and men's health.

Background: Although Y chromosomal genes are involved in male sex development, spermatogenesis, and height growth, these genes play no role in the survival or mitosis of somatic cells. Therefore, somatic cells lacking the Y chromosome can stay and proliferate in the body. Methods: Several molecular technologies, including next-generation sequencing and multiplex PCR-based assays, are used to detect mosaic loss of the Y chromosome (mLOY) in the blood of men. Main findings: Accumulating evidence suggests that mLOY represents the most common acquired chromosomal alteration in humans, affecting >40% of men over 70 years of age. Advanced age, tobacco smoking, and some SNPs in cell cycle genes are known to increase the frequency of mLOY. The developmental process of mLOY in elderly men remains to be clarified, but it possibly reflects recurrent mitotic elimination of Y chromosomes or clonal expansion of 45,X cell lineages. In rare cases, mLOY also occurs in young men and fetuses. MLOY has been associated with early death, cancers, and other disorders in elderly men, infertility in reproductive-aged men, and developmental defects in children. Conclusion: Y chromosomes in men can be lost at every life stage and Y chromosomal loss is associated with various health problems.

MYRF haploinsufficiency causes 46,XY and 46,XX disorders of sex development: bioinformatics consideration.

Disorders of sex development (DSDs) are defined as congenital conditions in which chromosomal, gonadal or anatomical sex is atypical. In many DSD cases, genetic causes remain to be elucidated. Here, we performed a case-control exome sequencing study comparing gene-based burdens of rare damaging variants between 26 DSD cases and 2625 controls. We found exome-wide significant enrichment of rare heterozygous truncating variants in the MYRF gene encoding myelin regulatory factor, a transcription factor essential for oligodendrocyte development. All three variants occurred de novo. We identified an additional 46,XY DSD case of a de novo damaging missense variant in an independent cohort. The clinical symptoms included hypoplasia of Müllerian derivatives and ovaries in 46,XX DSD patients, defective development of Sertoli and Leydig cells in 46,XY DSD patients and congenital diaphragmatic hernia in one 46,XY DSD patient. As all of these cells and tissues are or partly consist of coelomic epithelium (CE)-derived cells (CEDC) and CEDC developed from CE via proliferaiton and migration, MYRF might be related to these processes. Consistent with this hypothesis, single-cell RNA sequencing of foetal gonads revealed high expression of MYRF in CE and CEDC. Reanalysis of public chromatin immunoprecipitation sequencing data for rat Myrf showed that genes regulating proliferation and migration were enriched among putative target genes of Myrf. These results suggested that MYRF is a novel causative gene of 46,XY and 46,XX DSD and MYRF is a transcription factor regulating CD and/or CEDC proliferation and migration, which is essential for development of multiple organs.