Core planar cell polarity genes VANGL1 and VANGL2 in predisposition to congenital vertebral malformations.

Congenital scoliosis (CS), affecting approximately 0.5 to 1 in 1,000 live births, is commonly caused by congenital vertebral malformations (CVMs) arising from aberrant somitogenesis or somite differentiation. While Wnt/ß-catenin signaling has been implicated in somite development, the function of Wnt/planar cell polarity (Wnt/PCP) signaling in this process remains unclear. Here, we investigated the role of Vangl1 and Vangl2 in vertebral development and found that their deletion causes vertebral anomalies resembling human CVMs. Analysis of exome sequencing data from multiethnic CS patients revealed a number of rare and deleterious variants in VANGL1 and VANGL2, many of which exhibited loss-of-function and dominant-negative effects. Zebrafish models confirmed the pathogenicity of these variants. Furthermore, we found that Vangl1 knock-in (p.R258H) mice exhibited vertebral malformations in a Vangl gene dose- and environment-dependent manner. Our findings highlight critical roles for PCP signaling in vertebral development and predisposition to CVMs in CS patients, providing insights into the molecular mechanisms underlying this disorder.

A novel CCDC91 isoform associated with ossification of the posterior longitudinal ligament of the spine works as a non-coding RNA to regulate osteogenic genes.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common intractable disease that causes spinal stenosis and myelopathy. We have previously conducted genome-wide association studies for OPLL and identified 14 significant loci, but their biological implications remain mostly unclear. Here, we examined the 12p11.22 locus and identified a variant in the 5' UTR of a novel isoform of CCDC91 that was associated with OPLL. Using machine learning prediction models, we determined that higher expression of the novel CCDC91 isoform was associated with the G allele of rs35098487. The risk allele of rs35098487 showed higher affinity in the binding of nuclear proteins and transcription activity. Knockdown and overexpression of the CCDC91 isoform in mesenchymal stem cells and MG-63 cells showed paralleled expression of osteogenic genes, including RUNX2, the master transcription factor of osteogenic differentiation. The CCDC91 isoform directly interacted with MIR890, which bound to RUNX2 and decreased RUNX2 expression. Our findings suggest that the CCDC91 isoform acts as a competitive endogenous RNA by sponging MIR890 to increase RUNX2 expression.

Null and missense mutations of ERI1 cause a recessive phenotypic dichotomy in humans.

ERI1 is a 3'-to-5' exoribonuclease involved in RNA metabolic pathways including 5.8S rRNA processing and turnover of histone mRNAs. Its biological and medical significance remain unclear. Here, we uncover a phenotypic dichotomy associated with bi-allelic ERI1 variants by reporting eight affected individuals from seven unrelated families. A severe spondyloepimetaphyseal dysplasia (SEMD) was identified in five affected individuals with missense variants but not in those with bi-allelic null variants, who showed mild intellectual disability and digital anomalies. The ERI1 missense variants cause a loss of the exoribonuclease activity, leading to defective trimming of the 5.8S rRNA 3' end and a decreased degradation of replication-dependent histone mRNAs. Affected-individual-derived induced pluripotent stem cells (iPSCs) showed impaired in vitro chondrogenesis with downregulation of genes regulating skeletal patterning. Our study establishes an entity previously unreported in OMIM and provides a model showing a more severe effect of missense alleles than null alleles within recessive genotypes, suggesting a key role of ERI1-mediated RNA metabolism in human skeletal patterning and chondrogenesis.

Recapitulating the human segmentation clock with pluripotent stem cells.

Pluripotent stem cells are increasingly used to model different aspects of embryogenesis and organ formation1. Despite recent advances in in vitro induction of major mesodermal lineages and cell types2,3, experimental model systems that can recapitulate more complex features of human mesoderm development and patterning are largely missing. Here we used induced pluripotent stem cells for the stepwise in vitro induction of presomitic mesoderm and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modelling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We observed oscillatory expression of core segmentation clock genes, including HES7 and DKK1, determined the period of the human segmentation clock to be around five hours, and demonstrated the presence of dynamic travelling-wave-like gene expression in in vitro-induced human presomitic mesoderm. Furthermore, we identified and compared oscillatory genes in human and mouse presomitic mesoderm derived from pluripotent stem cells, which revealed species-specific and shared molecular components and pathways associated with the putative mouse and human segmentation clocks. Using CRISPR-Cas9-based genome editing technology, we then targeted genes for which mutations in patients with segmentation defects of the vertebrae, such as spondylocostal dysostosis, have been reported (HES7, LFNG, DLL3 and MESP2). Subsequent analysis of patient-like and patient-derived induced pluripotent stem cells revealed gene-specific alterations in oscillation, synchronization or differentiation properties. Our findings provide insights into the human segmentation clock as well as diseases associated with human axial skeletogenesis.

Dyssegmental dysplasia Rolland-Desbuquois type is caused by pathogenic variants in HSPG2 - a founder haplotype shared in five patients.

Dyssegmental dysplasia (DD) is a severe skeletal dysplasia comprised of two subtypes: lethal Silverman-Handmaker type (DDSH) and nonlethal Rolland-Desbuquois type (DDRD). DDSH is caused by biallelic pathogenic variants in HSPG2 encoding perlecan, whereas the genetic cause of DDRD remains undetermined. Schwartz-Jampel syndrome (SJS) is also caused by biallelic pathogenic variants in HSPG2 and is an allelic disorder of DDSH. In SJS and DDSH, 44 and 8 pathogenic variants have been reported in HSPG2, respectively. Here, we report that five patients with DDRD carried four pathogenic variants in HSPG2: c.9970 G > A (p.G3324R), c.559 C > T (p.R187X), c7006 + 1 G > A, and c.11562 + 2 T > G. Two patients were homozygous for p.G3324R, and three patients were heterozygous for p.G3324R. Haplotype analysis revealed a founder haplotype spanning 85,973 bp shared in the five patients. SJS, DDRD, and DDSH are allelic disorders with pathogenic variants in HSPG2.

Heterozygous mutations in the straitjacket region of the latency-associated peptide domain of TGFB2 cause Camurati-Engelmann disease type II.

Camurati-Engelmann disease (CED) is an autosomal dominant bone dysplasia characterized by progressive hyperostosis of the skull base and diaphyses of the long bones. CED is further divided into two subtypes, CED1 and CED2, according to the presence or absence of TGFB1 mutations, respectively. In this study, we used exome sequencing to investigate the genetic cause of CED2 in three pedigrees and identified two de novo heterozygous mutations in TGFB2 among the three patients. Both mutations were located in the region of the gene encoding the straitjacket subdomain of the latency-associated peptide (LAP) of pro-TGF-ß2. Structural simulations of the mutant LAPs suggested that the mutations could cause significant conformational changes and lead to a reduction in TGF-ß2 inactivation. An activity assay confirmed a significant increase in TGF-ß2/SMAD signaling. In vitro osteogenic differentiation experiment using iPS cells from one of the CED2 patients showed significantly enhanced ossification, suggesting that the pathogenic mechanism of CED2 is increased activation of TGF-ß2 by loss-of-function of the LAP. These results, in combination with the difference in hyperostosis patterns between CED1 and CED2, suggest distinct functions between TGFB1 and TGFB2 in human skeletal development and homeostasis.

Compound heterozygous variants in RAB34 in a rare skeletal ciliopathy syndrome.

Skeletal ciliopathies are a heterogenous group of congenital disorders characterized by multiple internal abnormalities, and distinct radiographic presentation. Pathogenic variants in at least 30 cilia genes are known to cause skeletal ciliopathies. Here we report a fetus with an atypical skeletal ciliopathy phenotype and compound heterozygous variants in the RAB34 gene. The affected fetus had multiple malformations, including posterior neck edema, micrognathia, low-set and small ears, auricular hypoplasia, cleft lip and palate, short extremities, and a combination of rarely occurring pre- and postaxial polydactyly. Genome sequencing identified compound heterozygous variants in the RAB34 gene: maternal c.254T>C, p.(Ile85Thr), and paternal c.691C>T, p.(Arg231*) variants. Only the paternal variant was present in the unaffected sibling. Evidence in the literature indicated that Rab34-/- mice displayed a ciliopathy phenotype with cleft palate and polydactyly. These features were consistent with malformations detected in our patient supporting the pathogenicity of the identified RAB34 variants. Overall, this case report further expands genetic landscape of human ciliopathy syndromes and suggests RAB34 as a candidate gene for skeletal ciliopathies.

Identification of a de novo PUF60 variant associated with craniofacial microsomia.

Craniofacial microsomia (CFM), also known as the oculo-auriculo-vertebral spectrum, is a congenital disorder characterized by hypoplasia of the mandible and external ear due to tissue malformations originating from the first and second branchial arches. However, distinguishing it from other syndromes of branchial arch abnormalities is difficult, and causal variants remain unidentified in many cases. In this report, we performed an exome sequencing analysis of a Brazilian family with CFM. The proband was a 12-month-old boy with clinical findings consistent with the diagnostic criteria for CFM, including unilateral mandibular hypoplasia, microtia, and external auditory canal abnormalities. A heterozygous de novo nonsense variant (c.713C>G, p.S238*) in PUF60 was identified, which was predicted to be pathogenic in silico. PUF60 has been reported as a causal gene in Verheij syndrome, but not in CFM. Although the boy showed craniofacial abnormalities and developmental delay that overlapped with Verheij syndrome, the facial asymmetry with unilateral hypoplasia of the mandible observed in this case did not match the previously reported phenotypes of PUF60 variants. Our findings expand the phenotypic range of PUF60 variants that cover CFM and Verheij syndrome.

A longitudinal population-based study identifies THBS2 as a susceptibility gene for intervertebral disc degeneration.

PURPOSE: Intervertebral disc degeneration (IDD) is a common degenerative disease associated with ageing. Additionally, IDD is recognized as one of the leading causes of low back pain and disability in the working-age population and is the first step in the process leading to degenerative spinal changes. However, the genetic factors and regulatory mechanisms of IDD remain unknown. Therefore, we selected eight single nucleotide polymorphisms of genes to reveal the progression of IDD in a 7-year longitudinal study of the general population in Japan. METHODS: IDD was evaluated in the Wakayama Spine Study (WSS), which is a population-based cohort study. Overall, 574 participants from the general population cohort who underwent whole spine magnetic resonance imaging and provided clinical information were included in this longitudinal survey. RESULTS: The progression of IDD was affected only by THBS2 at the lumbar region, T12-L1 (p = 0.0044) and L3-4 (p = 0.0045). The significant interaction between THBS2 and age with IDD negatively affected the thoracic spines and passively influenced both the thoracolumbar junction and thoracic spines. The higher progression per year of Pfirrmann's score was rapid in young people with age; however, this decelerated the IDD progression per year in different ages. CONCLUSION: Our longitudinal study found the genes associated with IDD progression and that genetic factors' impact on IDD differs depending on disc level and age.

Family History of Developmental Dysplasia of the Hip is a Risk Factor for the Progression of Hip Osteoarthritis.

BACKGROUND: Developmental dysplasia of the hip (DDH) is considered to have genetic predisposition and presents many intrafamilial occurrences. However, there is no report that evaluates the effect of DDH family history on the progression after the onset of hip osteoarthritis (OA). METHODS: Medical interviews about detailed clinical information including family history were conducted on 298 consecutive patients who had undergone surgery for OA due to DDH. Clinical or radiographic items that are associated with the severity of DDH (total hip arthroplasty [THA], involvement of bilateral DDH, onset age of hip pain, and three radiological indices of DDH: center-edge angle, sharp angle, and acetabular roof obliquity) were collected and evaluated in multivariate analyses for their associations with DDH family history in a qualitative or quantitative manner. Survival time analyses for THA as the endpoint was also performed to evaluate the effects of DDH family history on the progression of OA. RESULTS: The DDH family history showed significant associations with bilateral involvement of DDH (odds ratio = 2.09 [95% confidence interval {CI} 1.05 to 4.16]; P = .037), early onset of hip pain (P = .0065), and radiological severity of DDH (P = .016). The DDH family history showed a significant association with undergoing THA (odds ratio = 2.25 [95% CI 1.09 to 4.66]; P = .029), further supported by the Cox regression analyses (hazards ratio = 1.56 [95% CI 1.15 to 2.11]; P = .0044). CONCLUSION: A DDH family history is a risk factor for the progression of hip OA. Stronger genetic predisposition to DDH leads to faster onset and progression of hip OA.

Genetic factors affecting survival in Japanese patients with sporadic amyotrophic lateral sclerosis: a genome-wide association study and verification in iPSC-derived motor neurons from patients.

BACKGROUND: Several genetic factors are associated with the pathogenesis of sporadic amyotrophic lateral sclerosis (ALS) and its phenotypes, such as disease progression. Here, in this study, we aimed to identify the genes that affect the survival of patients with sporadic ALS. METHODS: We enrolled 1076 Japanese patients with sporadic ALS with imputed genotype data of 7 908 526 variants. We used Cox proportional hazards regression analysis with an additive model adjusted for sex, age at onset and the first two principal components calculated from genotyped data to conduct a genome-wide association study. We further analysed messenger RNA (mRNA) and phenotype expression in motor neurons derived from induced pluripotent stem cells (iPSC-MNs) of patients with ALS. RESULTS: Three novel loci were significantly associated with the survival of patients with sporadic ALS-FGF1 at 5q31.3 (rs11738209, HR=2.36 (95% CI, 1.77 to 3.15), p=4.85×10-9), THSD7A at 7p21.3 (rs2354952, 1.38 (95% CI, 1.24 to 1.55), p=1.61×10-8) and LRP1 at 12q13.3 (rs60565245, 2.18 (95% CI, 1.66 to 2.86), p=2.35×10-8). FGF1 and THSD7A variants were associated with decreased mRNA expression of each gene in iPSC-MNs and reduced in vitro survival of iPSC-MNs obtained from patients with ALS. The iPSC-MN in vitro survival was reduced when the expression of FGF1 and THSD7A was partially disrupted. The rs60565245 was not associated with LRP1 mRNA expression. CONCLUSIONS: We identified three loci associated with the survival of patients with sporadic ALS, decreased mRNA expression of FGF1 and THSD7A and the viability of iPSC-MNs from patients. The iPSC-MN model reflects the association between patient prognosis and genotype and can contribute to target screening and validation for therapeutic intervention.

CDC5L promotes early chondrocyte differentiation and proliferation by modulating pre-mRNA splicing of SOX9, COL2A1, and WEE1.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is a common pathological condition that causes intractable myelopathy and radiculopathy, mainly the result of an endochondral ossification-like process. Our previous genome-wide association study identified six susceptibility loci for OPLL, including the cell division cycle 5-like (CDC5L) gene region. Here, we found CDC5L to be expressed in type II collagen-producing chondrocyte-like fibroblasts in human OPLL specimens, as well as in differentiating ATDC5 chondrocytes. Cdc5l siRNA transfection in murine chondrocytes decreased the expression of the early chondrogenic genes Sox9 and Col2a1, diminished the cartilage matrix production, and enhanced the expression of parathyroid-hormone-related protein (a resting chondrocyte marker). We also showed that Cdc5l shRNA suppressed the growth of cultured murine embryonal metatarsal cartilage rudiments and that Cdc5l knockdown suppressed the growth of ATDC5 cells. Fluorescence-activated cell sorting analysis revealed that the G2/M cell cycle transition was blocked; our data showed that Cdc5l siRNA transfection enhanced expression of Wee1, an inhibitor of the G2/M transition. Cdc5l siRNA also decreased the pre-mRNA splicing efficiency of Sox9 and Col2a1 genes in both ATDC5 cells and primary chondrocytes; conversely, loss of Cdc5l resulted in enhanced splicing of Wee1 pre-mRNA. Finally, an RNA-binding protein immunoprecipitation assay revealed that Cdc5l bound directly to these target gene transcripts. Overall, we conclude that Cdc5l promotes both early chondrogenesis and cartilage growth and may play a role in the etiology of OPLL, at least in part by fine-tuning the pre-mRNA splicing of chondrogenic genes and Wee1, thus initiating the endochondral ossification process.

Hypomorphic Mutations in TONSL Cause SPONASTRIME Dysplasia.

SPONASTRIME dysplasia is a rare, recessive skeletal dysplasia characterized by short stature, facial dysmorphism, and aberrant radiographic findings of the spine and long bone metaphysis. No causative genetic alterations for SPONASTRIME dysplasia have yet been determined. Using whole-exome sequencing (WES), we identified bi-allelic TONSL mutations in 10 of 13 individuals with SPONASTRIME dysplasia. TONSL is a multi-domain scaffold protein that interacts with DNA replication and repair factors and which plays critical roles in resistance to replication stress and the maintenance of genome integrity. We show here that cellular defects in dermal fibroblasts from affected individuals are complemented by the expression of wild-type TONSL. In addition, in vitro cell-based assays and in silico analyses of TONSL structure support the pathogenicity of those TONSL variants. Intriguingly, a knock-in (KI) Tonsl mouse model leads to embryonic lethality, implying the physiological importance of TONSL. Overall, these findings indicate that genetic variants resulting in reduced function of TONSL cause SPONASTRIME dysplasia and highlight the importance of TONSL in embryonic development and postnatal growth.

Bi-allelic CSF1R Mutations Cause Skeletal Dysplasia of Dysosteosclerosis-Pyle Disease Spectrum and Degenerative Encephalopathy with Brain Malformation.

Colony stimulating factor 1 receptor (CSF1R) plays key roles in regulating development and function of the monocyte/macrophage lineage, including microglia and osteoclasts. Mono-allelic mutations of CSF1R are known to cause hereditary diffuse leukoencephalopathy with spheroids (HDLS), an adult-onset progressive neurodegenerative disorder. Here, we report seven affected individuals from three unrelated families who had bi-allelic CSF1R mutations. In addition to early-onset HDLS-like neurological disorders, they had brain malformations and skeletal dysplasia compatible to dysosteosclerosis (DOS) or Pyle disease. We identified five CSF1R mutations that were homozygous or compound heterozygous in these affected individuals. Two of them were deep intronic mutations resulting in abnormal inclusion of intron sequences in the mRNA. Compared with Csf1r-null mice, the skeletal and neural phenotypes of the affected individuals appeared milder and variable, suggesting that at least one of the mutations in each affected individual is hypomorphic. Our results characterized a unique human skeletal phenotype caused by CSF1R deficiency and implied that bi-allelic CSF1R mutations cause a spectrum of neurological and skeletal disorders, probably depending on the residual CSF1R function.

SOX11 variants cause a neurodevelopmental disorder with infrequent ocular malformations and hypogonadotropic hypogonadism and with distinct DNA methylation profile.

PURPOSE: This study aimed to undertake a multidisciplinary characterization of the phenotype associated with SOX11 variants. METHODS: Individuals with protein altering variants in SOX11 were identified through exome and genome sequencing and international data sharing. Deep clinical phenotyping was undertaken by referring clinicians. Blood DNA methylation was assessed using Infinium MethylationEPIC array. The expression pattern of SOX11 in developing human brain was defined using RNAscope. RESULTS: We reported 38 new patients with SOX11 variants. Idiopathic hypogonadotropic hypogonadism was confirmed as a feature of SOX11 syndrome. A distinctive pattern of blood DNA methylation was identified in SOX11 syndrome, separating SOX11 syndrome from other BAFopathies. CONCLUSION: SOX11 syndrome is a distinct clinical entity with characteristic clinical features and episignature differentiating it from BAFopathies.

Genome-wide association study of colorectal polyps identified highly overlapping polygenic architecture with colorectal cancer.

No genome-wide association studies (GWAS) were reported for colorectal polyps and the overlap in polygenic backgrounds conferring risk of colorectal cancer and polyps remains unclear. We performed GWAS on subjects with colorectal polyps using the BioBank Japan data with 4447 cases and 157,226 controls. We evaluated genetic correlations between colorectal polyps and cancer, and effects on colorectal polyps of single nucleotide polymorphisms (SNPs) known to be associated with colorectal cancer. We identified CUX2, a known genetic locus to colorectal cancer, as a susceptibility locus to colorectal polyps (p value = 1.1 × 10-15). Subsequent fine-mapping analysis indicated that rs11065828 in CUX2 is the causal variant for colorectal polyps. We found that known colorectal cancer-susceptible SNPs were also associated with colorectal polyps. The genetic correlation between colorectal cancer and polyps is very high (r = 0.98 and p value = 0.0006). We additionally identified 14 significant loci of colorectal polyps and three significant loci of colorectal cancer by applying the multi-trait analysis of GWAS of colorectal cancer and colorectal polyps. We showed very similar germline polygenic features, which gives us the additional insight into potential cancers at polygenic levels for patients with polyps who are followed up at outpatients' clinic; thus, close observation and polypectomy is critical to prevent colorectal cancers.

De novo heterozygous variants in KIF5B cause kyphomelic dysplasia.

Kyphomelic dysplasia is a heterogeneous group of skeletal dysplasias characterized by severe bowing of the limbs associated with other variable findings, such as narrow thorax and abnormal facies. We searched for the genetic etiology of this disorder. Four individuals diagnosed with kyphomelic dysplasia were enrolled. We performed whole-exome sequencing and evaluated the pathogenicity of the identified variants. All individuals had de novo heterozygous variants in KIF5B encoding kinesin-1 heavy chain: two with c.272A>G:p.(Lys91Arg), one with c.584C>A:p.(Thr195Lys), and the other with c.701G>T:p.(Gly234Val). All variants involved conserved amino acids in or close to the ATPase activity-related motifs in the catalytic motor domain of the KIF5B protein. All individuals had sharp angulation of the femora and humeri, distinctive facial features, and neonatal respiratory distress. Short stature was observed in three individuals. Three developed postnatal osteoporosis with subsequent fractures, two showed brachycephaly, and two were diagnosed with optic atrophy. Our findings suggest that heterozygous KIF5B deleterious variants cause a specific form of kyphomelic dysplasia. Furthermore, alterations in kinesins cause various symptoms known as kinesinopathies, and our findings also extend the phenotypic spectrum of kinesinopathies.

Biallelic variants in CHST3 cause Spondyloepiphyseal dysplasia with joint dislocations in three Pakistani kindreds.

BACKGROUND: Skeletal dysplasia is a heterogeneous group of disorders. Spondyloepiphyseal dysplasias comprise one subgroup. Deficiency of carbohydrate sulfotransferase 3 has been reported in a small number of patients with recessively inherited spondyloepiphyseal dysplasia with joint dislocation, short stature and scoliosis. We report here molecular and clinical findings of affected individuals in three consanguineous Pakistani families. Affected individuals in all three families had a uniform phenotype including severe short stature, multiple dislocated joints, progressive scoliosis and facial dysmorphism. METHODS: Clinical evaluation was done for three unrelated families. Radiological survey of bones was completed for patients from two of the families. Whole exome sequencing index patients from each family was performed followed by Sanger sequencing for validation of segregation of identified variants in respective families. In-silico analysis for determining pathogenicity of identified variants and conservation was done. RESULTS: Whole-exome sequencing revealed biallelic variants c.590 T > C;p.(Leu197Pro), c.603C > A;p.(Tyr201Ter) and c.661C > T;p.(Arg221Cys) in CHST3 (NM_004273.5) in the three families with eight, five and two affected individuals, respectively. Contrary to previous reports, affected individuals in none of the families exhibited a hearing loss. CONCLUSION: We describe genotypic and phenotypic findings of three unrelated families with spondyloepiphyseal dysplasia. Our study confirms phenotypic variability and adds to the genotypic spectrum of spondyloepiphyseal dysplasia.

Eight novel susceptibility loci and putative causal variants in atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is the most common allergic disease in the world. While genetic components play critical roles in its pathophysiology, a large proportion of its genetic background is still unexplored. OBJECTIVES: This study sought to illuminate the genetic associations with AD using genome-wide association study (GWAS) and its downstream analyses. METHODS: This study conducted a GWAS for AD comprising 2,639 cases and 115,648 controls in the Japanese population, followed by a trans-ethnic meta-analysis with UK Biobank data and downstream analyses including partitioning heritability analysis by linkage disequilibrium score regression. RESULTS: This study identified 17 significant susceptibility loci, among which 4 loci-AFF1, ITGB8, EHMT1, and EGR2-were novel in the Japanese GWAS. The trans-ethnic meta-analysis revealed 4 additional novel loci, namely-ZBTB38,LOC105755953/LOC101928272, TRAF3, andIQGAP1. This study found a missense variant (R243W) with a deleterious functional effect in NLRP10 and a variant altering expression of CCDC80 via enhancer expression as highly likely causal variants. These 2 regions were Asian-specific, and these population-specific associations could be explained by the frequency of causal variants. The gene-based test showed SMAD4 as an additional novel significant locus. Downstream analyses revealed substantial overlap of GWAS significant signals in enhancers of skin cells and immune cells, especially CD4 T cells. A highly shared polygenic architecture of AD between Europeans and Asians was also found. CONCLUSIONS: This study identified Japanese-specific loci and novel significant loci shared by different populations. Two putative causal variants were illuminated in Japanese-specific loci. Trans-ethnic analyses revealed strong heritability enrichment in immune-related pathways, and relevant cell types shared among populations.

Efficient detection of copy-number variations using exome data: Batch- and sex-based analyses.

Many algorithms to detect copy number variations (CNVs) using exome sequencing (ES) data have been reported and evaluated on their sensitivity and specificity, reproducibility, and precision. However, operational optimization of such algorithms for a better performance has not been fully addressed. ES of 1199 samples including 763 patients with different disease profiles was performed. ES data were analyzed to detect CNVs by both the eXome Hidden Markov Model (XHMM) and modified Nord's method. To efficiently detect rare CNVs, we aimed to decrease sequencing biases by analyzing, at the same time, the data of all unrelated samples sequenced in the same flow cell as a batch, and to eliminate sex effects of X-linked CNVs by analyzing female and male sequences separately. We also applied several filtering steps for more efficient CNV selection. The average number of CNVs detected in one sample was <5. This optimization together with targeted CNV analysis by Nord's method identified pathogenic/likely pathogenic CNVs in 34 patients (4.5%, 34/763). In particular, among 142 patients with epilepsy, the current protocol detected clinically relevant CNVs in 19 (13.4%) patients, whereas the previous protocol identified them in only 14 (9.9%) patients. Thus, this batch-based XHMM analysis efficiently selected rare pathogenic CNVs in genetic diseases.