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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.272AG:p.(Lys91Arg), one with c.584CA:p.(Thr195Lys), and the other with c.701GT: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.

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.

Colony-stimulating factor 1 receptor (CSF1R) plays key roles in the development and function of the cells in the monocyte/macrophage lineage, including microglia and osteoclasts. It is well known that mono-allelic mutations of CSF1R cause hereditary diffuse leukoencephalopathy with spheroids (HDLS, OMIM # 221820), an adult-onset progressive neurodegenerative disorder. Recently, a more severe phenotypic spectrum has been identified in individuals with bi-allelic mutations of CSF1R. In addition to leukoencephalopathy of earlier onset than HDLS, the new disease shows brain malformations and skeletal dysplasia compatible with dysosteosclerosis (DOS), thus named "brain abnormalities, neurodegeneration, and dysosteosclerosis" (BANDDOS, OMIM # 618476). In addition, some individuals with bi-allelic missense mutations of CSF1R have been found to present with incomplete BANDDOS where skeletal dysplasia is absent. In this review, we summarize the monogenic disorders caused by mutations in CSF1R and their mutational spectra, and propose a dose-dependent model to explain the complex genotype-phenotype association.

Background Hypophosphatemic rickets (HR) is a rare genetic disorder associated with renal phosphate wasting and characterized by bone defects. Inactivating mutations in the phosphate regulating endopeptidase homolog X‑linked gene (PHEX) account for most cases of HR. The aim of this study was to identify causative variants in nine unrelated Chinese families associated with HR, and to determine potential pathogenicity of the identified variants. Methods Genomic DNA was isolated from the peripheral blood of HR patients and their healthy relatives, followed by next-generation sequencing and/or Sanger sequencing. In silico prediction combined with conservation analysis was performed to assess the effects of the variants, and 3D protein modeling was conducted to predict the functional effects on the encoded protein. Results All HR patients recruited in this study displayed bone deformities and tooth agenesis, as well as reduced serum phosphate levels and elevated urine phosphate levels. Nine PHEX variants were identified in eight families, including four novel variants (c.1661_1726del, c.980A > G, c.1078A > T, and c.1017_1051dup). Of the nine identified PHEX variants, five caused a truncated protein, two caused an altered amino acid, and the other two were the canonical splicing variants. Novel variants c.1336G > A and c.1364 T > C in SLC34A3 were also found in one family. Conservation analysis showed that all the amino acids corresponding to the missense variants were highly conserved. In silico analysis and 3D protein structure modeling confirmed the pathogenicity of these variants. Conclusions This study identified four novel variants in PHEX and two novel variants in SLC34A3 in a Chinese cohort with HR. Our findings highlight the dominant role of PHEX in HR, and expand the genotypic and phenotypic spectra of this disorder.

Dysosteosclerosis (DOS) is a rare sclerosing bone dysplasia characterized by unique osteosclerosis of the long tubular bones and platyspondyly. DOS is inherited in an autosomal recessive manner and is genetically and clinically heterogeneous. To date, four individuals with DOS who have five different TNFRSF11A mutations have been reported. Based on their data, it is hypothesized that mutations producing aberrant mutant RANK proteins (missense or truncated or elongated) cause DOS, while null mutations lead to osteopetrosis, autosomal recessive 7 (OPTB7). Herein, we present the fifth case of TNFRSF11A-associated DOS with a novel homozygous frame-shift mutation (c.19_31del; p.[Arg7CysfsTer172]). The mutation is predicted to cause nonsense mutation-mediated mRNA decay (NMD) in all RANK isoform transcripts, resulting in totally null allele. Our findings suggest genotype-phenotype relationship in TNFRSF11A-associated OPTB7 and DOS remains unclear, and that the deficiency of TNFRSF11A functions might cause DOS, rather than osteopetrosis. More data are necessary to understand the phenotypic spectrum caused by TNFRSF11A mutations.

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. (C) 2022 The Authors. Published by Elsevier Inc. on behalf of American College of Medical Genetics and Genomics.

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

Dysosteosclerosis (DOS) is a distinct form of sclerosing bone disease characterized by platyspondyly and progressive osteosclerosis. DOS is genetically heterogeneous. Three causal genes, SLC29A3, CSF1R, and TNFRSF11A are reported. TNFRSF11A-associated DOS has been identified in two patients; however, TNFRSF11A is also a causal gene for osteopetrosis, autosomal recessive 7 (OP-AR7). Whole-exome sequencing in a patient with sclerosing bone disease identified novel compound heterozygous variants (c.414_427 + 7del, c.1664del) in TNFRSF11A. We examined the impact of the two variants on five splicing isoforms of TNFRSF11A by RT-PCR. We found that c.1664del resulted in elongated proteins (p.S555Cfs*121, etc.), while c.414_427 + 7del generated two aberrant splicing products (p.A139Wfs*19 and p.E132Dfs*19) that lead to nonsense mediated mRNA decay (NMD). In the previous two cases of TNFRSF11A-associated DOS, their mutations produced truncated TNFRSF11A protein isoforms. The mutations in all three cases thus contrast with TNFRSF11A mutations reported in OP-AR7, which does not generated truncated or elongated TNFRSF11A proteins. Thus, we identified the third case of TNFRSF11A-associated DOS and reinforced the genotype-phenotype correlation that aberrant protein-producing TNFRSF11A mutations cause DOS.

Osteonecrosis of the femoral head (ONFH) involves necrosis of bone and bone marrow of the femoral head caused by ischemia with unknown etiology. Previous genetic studies on ONFH failed to produce consistent results, presumably because ONFH has various causes with different genetic backgrounds and the underlying diseases confounded the associations. Steroid-associated ONFH (S-ONFH) accounts for one-half of all ONFH, and systemic lupus erythematosus (SLE) is a representative disease underlying S-ONFH. We performed a genome-wide association study (GWAS) to identify genetic risk factors for S-ONFH in patients with SLE. We conducted a two-staged GWAS on 636 SLE patients with S-ONFH and 95 588 non-SLE controls. Among the novel loci identified, we determined S-ONFH-specific loci by comparing allele frequencies between SLE patients without S-ONFH and non-SLE controls. We also used Korean datasets comprising 148 S-ONFH cases and 37 015 controls to assess overall significance. We evaluated the functional annotations of significant variants by in silico analyses. The Japanese GWAS identified 4 significant loci together with 12 known SLE susceptibility loci. The four significant variants showed comparable effect sizes on S-ONFH compared with SLE controls and non-SLE controls. Three of the four loci, MIR4293/MIR1265 [odds ratio (OR) = 1.99, P-value = 1.1 × 10−9)], TRIM49/NAALAD2 (OR = 1.65, P-value = 4.8 × 10−8) and MYO16 (OR = 3.91, P-value = 4.9 × 10−10), showed significant associations in the meta-analysis with Korean datasets. Bioinformatics analyses identified MIR4293, NAALAD2 and MYO16 as candidate causal genes. MIR4293 regulates a PPARG-related adipogenesis pathway relevant to S-ONFH. We identified three novel susceptibility loci for S-ONFH in SLE.

Congenital scoliosis (CS) is a form of scoliosis caused by congenital vertebral malformations. Genetic predisposition has been demonstrated in CS. We previously reported that TBX6 loss-of-function causes CS in a compound heterozygous model; however, this model can explain only 10% of CS. Many monogenic and polygenic CS genes remain to be elucidated. In this study, we analyzed exome sequencing (ES) data of 615 Chinese CS from the Deciphering Disorders Involving Scoliosis and COmorbidities (DISCO) project. Cosegregation studies for 103 familial CS identified a novel heterozygous nonsense variant, c.2649G>A (p.Trp883Ter) in FBN1. The association between FBN1 and CS was then analyzed by extracting FBN1 variants from ES data of 574 sporadic CS and 828 controls; 30 novel variants were identified and prioritized for further analyses. A mutational burden test showed that the deleterious FBN1 variants were significantly enriched in CS subjects (OR = 3.9, P = 0.03 by Fisher’s exact test). One missense variant, c.2613A>C (p.Leu871Phe) was recurrent in two unrelated CS subjects, and in vitro functional experiments for the variant suggest that FBN1 may contribute to CS by upregulating the transforming growth factor beta (TGF-β) signaling. Our study expanded the phenotypic spectrum of FBN1, and provided nove insights into the genetic etiology of CS.

To characterize clinically measurable endophenotypes, implicating the TBX6 compound inheritance model. Patients with congenital scoliosis (CS) from China(N = 345, cohort 1), Japan (N = 142, cohort 2), and the United States (N = 10, cohort 3) were studied. Clinically measurable endophenotypes were compared according to the TBX6 genotypes. A mouse model for Tbx6 compound inheritance (N = 52) was investigated by micro computed tomography (micro-CT). A clinical diagnostic algorithm (TACScore) was developed to assist in clinical recognition of TBX6-associated CS (TACS). In cohort 1, TACS patients (N = 33) were significantly younger at onset than the remaining CS patients (P = 0.02), presented with one or more hemivertebrae/butterfly vertebrae (P = 4.9 × 10‒8), and exhibited vertebral malformations involving the lower part of the spine (T8–S5, P = 4.4 × 10‒3); observations were confirmed in two replication cohorts. Simple rib anomalies were prevalent in TACS patients (P = 3.1 × 10‒7), while intraspinal anomalies were uncommon (P = 7.0 × 10‒7). A clinically usable TACScore was developed with an area under the curve (AUC) of 0.9 (P = 1.6 × 10‒15). A Tbx6-/mh (mild-hypomorphic) mouse model supported that a gene dosage effect underlies the TACS phenotype. TACS is a clinically distinguishable entity with consistent clinically measurable endophenotypes. The type and distribution of vertebral column abnormalities in TBX6/Tbx6 compound inheritance implicate subtle perturbations in gene dosage as a cause of spine developmental birth defects responsible for about 10% of CS.

BackgroundCongenital scoliosis (CS) is a common vertebral malformation. Spondylocostal dysostosis (SCD) is a rare skeletal dysplasia characterised by multiple vertebral malformations and rib anomalies. In a previous study, a compound heterozygosity for a null mutation and a risk haplotype composed by three single-nucleotide polymorphisms inTBX6have been reported as a disease-causing model of CS. Another study identified bi-allelic missense variants in a SCD patient. The purpose of our study is to identifyTBX6variants in CS and SCD and examine their pathogenicity.MethodsWe recruited 200 patients with CS or SCD and investigatedTBX6variants. We evaluated the pathogenicity of the variants by in silico prediction and in vitro experiments.ResultsWe identified five 16p11.2 deletions, one splice-site variant and five missense variants in 10 patients. In vitro functional assays for missense variants identified in the previous and present studies demonstrated that most of the variants caused abnormal localisation of TBX6 proteins. We confirmed mislocalisation of TBX6 proteins in presomitic mesoderm cells induced from SCD patient-derived iPS cells. In induced cells, we found decreased mRNA expressions ofTBX6and its downstream genes were involved in somite formation. All CS patients with missense variants had the risk haplotype in the opposite allele, while a SCD patient with bi-allelic missense variants did not have the haplotype.ConclusionsOur study suggests that bi-allelic loss of function variants ofTBX6cause a spectrum of phenotypes including CS and SCD, depending on the severity of the loss ofTBX6function.

Dysosteosclerosis (DOS) is a rare sclerosing bone dysplasia characterized by osteosclerosis and platyspondyly. DOS is genetically heterogeneous and causally associated with mutations in three genes, SLC29A3, CSF1R, and TNFRSF11A. TNFRSF11A has been known as the causal gene for osteopetrosis, autosomal recessive 7, and is recently reported to cause DOS in three cases, which show a complex genotype-phenotype relationship. The phenotypic spectrum of TNFRSF11A-associated sclerosing bone dysplasia remains unclear and needs to be characterized further in more cases with molecular genetic diagnosis. Here, we report another TNFRSF11A-associated DOS case with a homozygous missense mutation (p.R129C). The mutation effect is different from the previous three cases, in which truncated or elongated RANK proteins were generated in isoform specific manner, thus enriching our understanding of the genotype-phenotype association in TNFRSF11A-associated sclerosing bone dysplasia. Besides DOS, our case presented with intracranial extramedullary hematopoiesis, which is an extremely rare condition and has not been identified in any other sclerosing bone dysplasias with molecular genetic diagnosis. Our findings provide the fourth case of TNFRSF11A-associated DOS and further expand its phenotypic spectrum.

Background Congenital scoliosis (CS) is defined as a lateral curvature of the spine due to the vertebral malformations and has an incidence of 0.5–1/1,000 births. We previously examined TBX6 in Japanese CS patients and revealed that approximately 10% of CS was caused by TBX6 mutations. However, the genetic cause of remaining CS is unknown. Methods We recruited 78 CS patients without TBX6 mutations and major comorbidities, and investigated the genes previously reported to be associated with CS and congenital vertebral malformations by whole‐exome sequencing. Results We identified the compound heterozygous missense variants in LFNG in one patient. No likely disease‐causing variants were identified in other patients, however. LFNG encodes a GlcNAc‐transferase. The LFNG variants showed loss of their enzyme function. Conclusions A LFNG mutation is reported in a case of spondylocostal dysostosis (SCD), a skeletal dysplasia with severe malformations of vertebra and rib. The CS patient with LFNG mutations had multiple vertebral malformations including hemivertebrae, butterfly vertebrae, and block vertebrae, and rib malformations. LFNG mutations may cause a spectrum of phenotypes including CS and SCD. The current list of known disease genes could explain only a small fraction of genetic cause of CS.

Scoliosis represents the most common musculoskeletal disorder in children and affects approximately 3% of the world population. Scoliosis is separated into two major phenotypic classifications: congenital and idiopathic. Idiopathic scoliosis is defined as a curvature of the spine of 10° or greater visualized on plane radiograph and does not have associated vertebral malformations (VM). “Congenital” scoliosis (CS) due to malformations in vertebrae is frequently associated with other birth defects. Recently, significant advances have been made in understanding the genetic basis of both conditions. There is evidence that both conditions are etiologically related. A 2-day conference entitled “Genomic Approaches to Understanding and Treating Scoliosis” was held at Scottish Rite Hospital for Children in Dallas, Texas, to synergize research in this field. This first combined, multidisciplinary conference featured international scoliosis researchers in basic and clinical sciences. A major outcome of the conference advancing scoliosis research was the proposal and subsequent vote in favor of merging the International Consortium for Vertebral Anomalies and Scoliosis (ICVAS) and International Consortium for Scoliosis Genetics (ICSG) into a single entity called International Consortium for Spinal Genetics, Development, and Disease (ICSGDD). The ICSGDD is proposed to meet annually as a forum to synergize multidisciplinary spine deformity research.

Adolescent idiopathic scoliosis (AIS) is a common spinal deformity affecting millions of children. Since treatment and prognosis of AIS depend on curve progression, identifying factors related to AIS curve progression is important in its management. Although several genetic loci for AIS occurrence are reported, no locus for curve progression has been identified. To identify genes associated with AIS progression, we conducted a genome-wide association study followed by a replication study using a total of 2,543 AIS subjects who were evaluated for the curve progression. We identified a significantly associated locus on chromosome 11q14.1 (P = 1.98 × 10-9, odds ratio = 1.56). In silico and in vitro analyses identified a functional variant, rs35333564 in MIR4300HG, the host gene of a microRNA, MIR4300. The genomic region containing rs35333564 had enhancer activity, which was decreased in its risk allele. Our data suggest that decrease of MIR4300 is related to AIS progression.

We present two unrelated Japanese pedigrees with achondrogenesis type 1b (ACG1B), characterized by prenatally lethal fetal hydrops and severe micromelia. The affected members in these pedigrees carried a common homozygous missense point mutation in solute carrier family 26 member 2 (SLC26A2), a gene associated with ACG1B (NM_000112:c.1987G>A). This loss-of-function point mutation causes substitution of glycine 663 with arginine in a highly conserved loop domain of SLC26A2. Interestingly, only a few cases of this mutation have been registered in Japanese genomic databases, and there are no reports of this mutation in any major genomic databases outside Japan. Furthermore, we confirmed the presence of a homozygous stretch of approximately 75 kb surrounding the pathogenic variant. Our findings suggest that this missense point mutation in SLC26A2, which is likely the cause of the ACG1B phenotypes in these unrelated fetuses, is distributed exclusively in Japan.

Congenital scoliosis (CS) occurs as a result of vertebral malformations and has an incidence of 0.5-1/1,000 births. Recently, TBX6 on chromosome 16p11.2 was reported as a disease gene for CS; about 10% of Chinese CS patients were compound heterozygotes for rare null mutations and a common haplotype defined by three SNPs in TBX6. All patients had hemivertebrae. We recruited 94 Japanese CS patients, investigated the TBX6 locus for both mutations and the risk haplotype, examined transcriptional activities of mutant TBX6 in vitro, and evaluated clinical and radiographic features. We identified TBX6 null mutations in nine patients, including a missense mutation that had a loss of function in vitro. All had the risk haplotype in the opposite allele. One of the mutations showed dominant negative effect. Although all Chinese patients had one or more hemivertebrae, two Japanese patients did not have hemivertebra. The compound heterozygosity of null mutations and the common risk haplotype in TBX6 also causes CS in Japanese patients with similar incidence. Hemivertebra was not a specific type of spinal malformation in TBX6-associated CS (TACS). A heterozygous TBX6 loss-of-function mutation has been reported in a family with autosomal-dominant spondylocostal dysostosis, but it may represent a spectrum of the same disease with TACS.

Mutations in genes encoding enzymes responsible for the biosynthesis and structural diversity of glycosaminoglycans (GAGs) cause a variety of disorders affecting bone and connective tissues, including Desbuquois dysplasia (DD). In an infant with prenatal-onset disproportionate short stature, joint laxity, and radiographic findings typical for DD compound-heterozygosity for a large intragenic deletion, and a p.Pro384Arg missense mutation in CSGALNACT1 was found. CSGALNACT1 encodes chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1, ChGn-1), which initiates chondroitin sulfate (CS) chain biosynthesis on the so-called GAG-protein linker region tetrasaccharide. Biochemical studies revealed a reduced GalNAc-transferase activity of the Arg-384 mutant protein, whereas no differences in proteoglycan synthesis in fibroblasts and the GAG content in the urine were found between patient and controls. This is the first description of bi-allelic loss-of-function mutations in CSGALNACT1 that produce a skeletal dysplasia reminiscent of the skeletal dysplasia of Csgalnact1-/- mice, and adds to the genetic heterogeneity of DD.

Adolescent idiopathic scoliosis (AIS) is the most common type of scoliosis. Controlling its curve progression is the most important clinical task. Although recent genome-wide association studies (GWASs) identified several susceptibility loci associated with the development of AIS, the etiology of curve progression has been still unknown. Our previous GWAS has identified that rs12946942 showed significant association with severe AIS. To confirm the association, we conducted an international meta-analysis using four cohorts with different ethnicity. We analyzed 2272 severe AIS cases and 13,859 controls in total, and found the replication of significant association of rs12946942 (combined P = 7.23×10−13; odds ratio = 1.36, 95% confidence interval = 1.25−1.49). In silico analyses suggested that SOX9 is the most likely susceptibility gene for AIS curve progression in the locus.

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.

Adolescent idiopathic scoliosis (AIS) is the most common musculoskeletal disorder of childhood development. The genetic architecture of AIS is complex, and the great majority of risk factors are undiscovered. To identify new AIS susceptibility loci, we conducted the first genome-wide meta-analysis of AIS genome-wide association studies, including 7956 cases and 88459 controls from 3 ancestral groups. Three novel loci that surpassed genome-wide significance were uncovered in intragenic regions of the CDH13 (P-value_rs4513093=1.7E-15), ABO (P-value_ rs687621=7.3E-10) and SOX6 (P-value_rs1455114=2.98E-08) genes. Restricting the analysis to females improved the associations at multiple loci, most notably with variants within CDH13 despite the reduction in sample size. Genome-wide gene-functional enrichment analysis identified significant perturbation of pathways involving cartilage and connective tissue development. Expression of both SOX6 and CDH13 was detected in cartilage chondrocytes and chromatin immunoprecipitation sequencing experiments in that tissue revealed multiple HeK27ac-positive peaks overlapping associated loci. Our results further define the genetic architecture of AIS and highlight the importance of vertebral cartilage development in its pathogenesis.

Developmental dysplasia of the hip (DDH) is one of the most common congenital malformations and covers a spectrum of hip disorders from mild dysplasia to irreducible dislocation. The pathological mechanisms of DDH are poorly understood, which hampers the development of diagnostic tools and treatments. To gain insight into its disease mechanism, we explored the potential biological processes that underlie DDH by integrating pathway analysis tools and performing a genome-wide association study (GWAS). A total of 406 DDH-associated genes (P < 0.001) were identified by our GWAS using a Chinese Han cohort consisting of 386 DDH cases and 500 healthy controls (Set A). We verified the significant loci (P < 10-5 ) in another Chinese Han cohort consisting of 574 DDH patients and 569 healthy controls (Set B). An intronic Single Nucleotide Polymorphism (SNP) (rs61930502) showed significant association in Set A and Set B (P = 2.65 × 10-7 and 2.0 × 10-4 , respectively). The minor allele, rs61930502-A, which tended to prevent DDH showed a dominant effect. Heat shock 70 kDa protein 8 (HSPA8) showed the most direct interactions with other proteins which were coded by DDH-associated genes in the protein-protein interaction analysis. Interestingly, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested a relation between DDH and the genes involved in type II diabetes mellitus pathway (P = 0.0067). Our genetic and protein interaction evidence could open avenues for future studies of DDH.

Spondylocostal dysostosis (SCDO) is a heterogeneous group of skeletal disorders characterized by multiple segmentation defects involving vertebrae and ribs. Seven disease genes have been reported as causal genes for SCDO: DLL3, MESP2, TBX6, HES7, RIPPLY2, DMRT2, and LFNG. Here we report a Japanese SCDO case with multiple severe vertebral anomalies from cervical to sacral spine. The patient was a compound heterozygote for c.372delG (p.K124Nfs*) and c.601G>A (p.D201N) variants of LFNG, which encodes a glycosyltransferase (O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase). The missense variant was in the DxD motif, an active-site motif of the glycosyltransferase, and its loss of the enzyme function was confirmed by an in vitro enzyme assay. This is the second report of LFNG mutations in SCDO.

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 as-says 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.

Lumbar disc degeneration (LDD) is age-related break-down in the fibrocartilaginous joints between lumbar vertebrae. It is a major cause of low back pain and is conventionally assessed by magnetic resonance imaging (MRI). Like most other complex traits, LDD is likely polygenic and influenced by both genetic and environmental factors. However, genome-wide association studies (GWASs) of LDD have uncovered few susceptibility loci due to the limited sample size. Previous epidemiology studies of LDD also reported multiple heritable risk factors, including height, body mass index (BMI), bone mineral density (BMD), lipid levels, etc. Genetics can help elucidate causality between traits and suggest loci with pleiotropic effects. One such approach is polygenic score (PGS) which summarizes the effect of multiple variants by the summation of alleles weighted by estimated effects from GWAS. To investigate genetic overlaps of LDD and related heritable risk factors, we calculated the PGS of height, BMI, BMD and lipid levels in a Chinese population-based cohort with spine MRI examination and a Japanese case-control cohort of lumbar disc herniation (LDH) requiring surgery. Because most large-scale GWASs were done in European populations, PGS of corresponding traits were created using weights from European GWASs. We calibrated their prediction performance in independent Chinese samples, then tested associations with MRI-derived LDD scores and LDH affection status. The PGS of height, BMI, BMD and lipid levels were strongly associated with respective phenotypes in Chinese, although phenotype variances explained were lower than in Europeans which would reduce the power to detect genetic overlaps. Despite of this, the PGS of BMI and lumbar spine BMD were significantly associated with LDD scores; and the PGS of height was associated with the increased the liability of LDH. Furthermore, linkage disequilibrium score regression suggested that, osteoarthritis, another degenerative disorder that shares common features with LDD, also showed genetic correlations with height, BMI and BMD. The findings suggest a common key contribution of biomechanical stress to the pathogenesis of LDD and will direct the future search for pleiotropic genes.

With the recent advance in genome-wide association studies (GWAS), disease-associated single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) have been extensively reported. Accordingly, the issue of incorrect identification of recombination events that can induce the misannotation of multi-allelic or hemizygous variants has received more attention. However, the potential distorted calculation bias or significance of a detected association in a GWAS that may result due to the coexistence of CNVs and SNPs in the same genomic region may remain under-recognized. Here we performed the association study within a congenital scoliosis (CS) cohort whose genetic etiology was recently elucidated as a compound inheritance model including mostly one rare variant deletion CNV allele and one common variant noncoding hypomorphic haplotype of the TBX6 gene. We demonstrate that the existence of a deletion in TBX6 led to an overestimation of the contribution of the SNPs on the hypomorphic allele. Furthermore, we generalized a model to explain the calculation bias, or distorted significance calculation for an association study that can be ‘induced’ by CNVs at a locus. Meanwhile, overlapping between the disease-associated SNPs from published GWAS and common CNVs (overlap 10%) and pathogenic/likely pathogenic CNVs (overlap 99.69%) was significantly higher than the random distribution (p

Dysosteosclerosis (DOS) is a form of sclerosing bone disease characterized by irregular osteosclerosis and platyspondyly. Its mode of inheritance is autosomal recessive. SLC29A3 mutations have been reported as the causal gene in two DOS families, however, genetic heterogeneity has been suggested. By whole-exome sequencing in a Turkish patient with DOS, we found a novel splice-site mutation in TNFRSF11A. TNFRSF11A mutations have previously been reported in two autosomal dominant diseases (osteolysis, familial expansile and Paget disease of bone 2, early-onset) and an autosomal recessive disease (osteopetrosis, autosomal recessive 7). The biallelic mutation, c.616+3A>G, identified in our study was located in the splice donor site of intron 6 of TNFRSF11A. Exon trapping assay indicated the mutation caused skipping of exon 6, which was predicted to induce a frame-shift and an early termination codon in all known alternative transcript variants of TNFRSF11A. The predicted effect of the mutation for the isoforms was different from those of the previously reported mutations, which could explain the difference of their phenotypes. Thus, our study identified the second disease gene for DOS. TNFRSF11A isoforms may have the different roles in skeletal development and metabolism.

Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity. We previously conducted a genome-wide association study (GWAS) and detected two loci associated with AIS. To identify additional loci, we extended our GWAS by increasing the number of cohorts (2,109 affected subjects and 11,140 control subjects in total) and conducting a whole-genome imputation. Through the extended GWAS and replication studies using independent Japanese and Chinese populations, we identified a susceptibility locus on chromosome 9p22.2 (p = 2.46 × 10(-13); odds ratio = 1.21). The most significantly associated SNPs were in intron 3 of BNC2, which encodes a zinc finger transcription factor, basonuclin-2. Expression quantitative trait loci data suggested that the associated SNPs have the potential to regulate the BNC2 transcriptional activity and that the susceptibility alleles increase BNC2 expression. We identified a functional SNP, rs10738445 in BNC2, whose susceptibility allele showed both higher binding to a transcription factor, YY1 (yin and yang 1), and higher BNC2 enhancer activity than the non-susceptibility allele. BNC2 overexpression produced body curvature in developing zebrafish in a gene-dosage-dependent manner. Our results suggest that increased BNC2 expression is implicated in the etiology of AIS.

Desbuquois dysplasia (DBQD) is a rare skeletal dysplasia characterized by severe short stature, laxity, dislocation of multiple joints and developmental delay. DBQD is clinically heterogeneous. Distinct radiographic hand abnormalities such as the presence of extra-ossification distal to the second metacarpal or normal hand has led to its classification into types 1 and 2. Furthermore, the third type of DBQD, Kim type has been reported which is characterized by short metacarpals and elongated phalanges. However, DBQD Kim type has been exclusively reported in Japanese and Korean and its clinical characteristics remain to be delineated. Mutations in the calcium-activated nucleotidase 1 (CANT1) gene have been reported in all three types of DBQD. Previously reported patients with DBQD Kim type had a common mutation c.676G>A (p.Val226Met), which had a common founder between Japanese and Korean. Here, we report 3 Indian patients with DBQD, Kim type from 2 families which were unrelated to each other. We identified a novel mutation of CANT1, c.467C>T (p.Ser156Phe), in all the patients in the homozygous form. Our results show that DBQD Kim type is not exclusive to East Asians and also report a novel mutation from the Indian subcontinent.

Spondylo-meta-epiphyseal dysplasia, short limb-abnormal calcification type, is a rare autosomal recessive disorder of the skeleton characterized by disproportionate short stature with narrow chest and dysmorphic facial features. The skeletal manifestations include platyspondyly, short flared ribs, short tubular bones with abnormal metaphyses and epiphyses, severe brachydactyly, and premature stippled calcifications in the cartilage. The abnormal calcifications are so distinctive as to point to the definitive diagnosis. However, they may be too subtle to attract diagnostic attention in infancy. Homozygous variants in DDR2 cause this disorder. We report on a 5-year-old girl with the classic phenotype of SMED, SL-AC in whom a novel homozygous nonsense mutation in DDR2 was detected using exome sequencing.

Guo, Long/0000-0002-9660-6941; Albayrak, Hatice Mutlu/0000-0001-5624-3878; Ikegawa, Shiro/0000-0003-0316-2147; Mizumoto, Shuji/0000-0002-4641-1505 WOS: 000406281300011 PubMed: 28331220 Spondylo-epi-metaphyseal dysplasia (SEMD) is a group of inherited skeletal diseases characterized by the anomalies in spine, epiphyses and metaphyses. SEMD is highly heterogeneous and 420 distinct entities have been identified. Here we describe a novel type of SEMD in two unrelated Turkish patients who presented with severe platyspondyly, kyphoscoliosis, pelvic distortion, constriction of the proximal femora and brachydactyly. Although these phenotypes overlap considerably with some known SEMDs, they had a novel causal gene, exostosin-like glycosyltransferase 3 (EXTL3), that encodes a glycosyltransferase involved in the synthesis of heparin and heparan sulfate. The EXTL3 mutation identified in the patients was a homozygous missense mutation (c. 953C>T) that caused a substitution in a highly conserved amino acid (p.P318L). The enzyme activity of the mutant EXTL3 protein was significantly decreased compared to the wild-type protein. Both patients had spinal cord compression at the cranio-vertebral junction and multiple liver cysts since early infancy. One of the patients showed severe immunodeficiency, which is considered non-fortuitous association. Our findings would help define a novel type of SEMD caused by EXTL3 mutations. Japan Agency For Medical Research and Development (AMED)Japan Agency for Medical Research and Development (AMED) [14525125]; Japan Society for the Promotion of Science, JapanMinistry of Education, Culture, Sports, Science and Technology, Japan (MEXT)Japan Society for the Promotion of Science [16K08251]; Nakatomi Foundation We thank the patients and their families for their help to the study. This study was supported in part by research grants from Japan Agency For Medical Research and Development (AMED; contract No 14525125), by a Grant-in-Aid for Scientific Research (C) 16K08251 (to SM) from the Japan Society for the Promotion of Science, Japan, and by the Nakatomi Foundation (to SM).

Osteogenesis imperfecta (OI) type V is a specific OI phenotype with interosseous membrane calcification of the forearm and hyperplastic callus formation as typical features. The causative gene mutation for OI type V has been recently discovered. The purpose of this report is to review the clinical and radiographic characteristics of mutation confirmed OI type V in detail. Sixteen (nine familial and seven sporadic) patients were enrolled in the study. Blue sclera and dentinogenesis imperfecta were not evident in any patient. However, hypodontia in the permanent teeth, ectopic eruption, and short roots in molars were additionally observed in 11 patients. Of the radiographic abnormalities, cortical thickening and bony excrescence of interosseous margin of the ulna was the most common finding, followed by overgrowth of the olecranon and/or coronoid process of the ulna. Slender ribs and sloping of the posterior ribs with or without fractures were also a consistent finding. Hyperplastic callus was detected in 75% of patients and was commonly encountered at the femur. Heterotopic ossification in the muscles and tendon insertion sites were noted in four patients, which resulted in bony ankylosis or contracture of joints. The current study confirms common clinical and radiographic findings of OI type V and reports additional phenotypic information. These observations provide clues to recognize OI type V more promptly and guide to direct targeted molecular study. © 2013 Wiley Periodicals, Inc.

Background Osteoporotic fracture (OF) as a clinical endpoint is a major complication of osteoporosis. To screen for OF susceptibility genes, we performed a genome-wide association study and carried out de novo replication analysis of an East Asian population. Methods Association was tested using a logistic regression analysis. A meta-analysis was performed on the combined results using effect size and standard errors estimated for each study. Results In a combined meta-analysis of a discovery cohort (288 cases and 1139 controls), three hospital based sets in replication stage I (462 cases and 1745 controls), and an independent ethnic group in replication stage II (369 cases and 560 for controls), we identified a new locus associated with OF ( rs784288 in the MECOM gene) that showed genome-wide significance (p=3.59×10 −8 ; OR 1.39). RNA interference revealed that a MECOM knockdown suppresses osteoclastogenesis. Conclusions Our findings provide new insights into the genetic architecture underlying OF in East Asians.

We previously reported 2 osteoporosis-susceptibility genes--formiminotransferase N-terminal sub-domain containing gene (FONG) and thrombospondin, type 1, domain-containing 7A (THSD7A)--in which we identified two common single-nucleotide polymorphisms, rs7605378 (FONG) and rs12673692 (THSD7A). The former was associated with a predisposition to osteoporosis and the latter with bone mineral density. To further elucidate the importance of these polymorphisms in the pathogenesis of osteoporosis, we examined their association with the incidence of vertebral fracture. DNA extracted from the renal cortex of 2427 consecutive Japanese autopsies (1331 men, mean age: 79 years; 1096 women, mean age: 82 years) were examined in this study. The presence or absence of vertebral fracture during each subject's lifetime was determined by a thorough examination of the clinical records, as well as autopsy reports. After adjustments for sex and age at autopsy, logistic regression analysis revealed that homozygotes for the risk alleles of rs7605378 (A-allele) or rs12673629 (A-allele) possess an increased risk of vertebral fracture. The subjects simultaneously homozygous for both the risk alleles of rs7605378 (AA genotype) and rs12673629 (AA genotype) showed significantly higher risk of vertebral fracture (odds ratio 2.401, 95% confidence interval 1.305-4.416, P = 0.0048) than those who had at least one non-risk allele of either rs7605378 (AC/CC genotypes) or rs12673629 (AG/GG genotypes). The results suggest that Japanese subjects homozygous for the risk alleles of rs7605378 and rs12673629 have a higher risk of vertebral fracture.

Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and function as structural and regulatory molecules. PGs are composed of core proteins and glycosaminoglycan (GAG) side chains. The biosynthesis of GAGs starts with the linker region that consists of four sugar residues and is followed by repeating disaccharide units. By exome sequencing, we found that B3GALT6 encoding an enzyme involved in the biosynthesis of the GAG linker region is responsible for a severe skeletal dysplasia, spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMD-JL1). B3GALT6 loss-of-function mutations were found in individuals with SEMD-JL1 from seven families. In a subsequent candidate gene study based on the phenotypic similarity, we found that B3GALT6 is also responsible for a connective tissue disease, Ehlers-Danlos syndrome (progeroid form). Recessive loss-of-function mutations in B3GALT6 result in a spectrum of disorders affecting a broad range of skeletal and connective tissues characterized by lax skin, muscle hypotonia, joint dislocation, and spinal deformity. The pleiotropic phenotypes of the disorders indicate that B3GALT6 plays a critical role in a wide range of biological processes in various tissues, including skin, bone, cartilage, tendon, and ligament.

Axial spondylometaphyseal dysplasia (axial SMD) is a unique form of SMD characterized by dysplasia of axial skeleton and retinal dystrophy. Recently, C21orf2 has been identified as the first disease gene for axial SMD; however, the presence of genetic heterogeneity is known. In this study, we identified NEK1 as the second disease gene for axial SMD. By whole-exome sequencing in a patient with axial SMD, we identified compound heterozygous mutations of NEK1, c.3107C>G (p.S1036*) and c.3830A>C (p.D1277A), which co-segregated in the family. NEK1 mutations have previously been found in three types of short rib thoracic dystrophy, which have no retinal dystrophy. The skeletal phenotype of our patient was milder than those of previously reported cases with NEK1 mutations and those with axial SMD harboring C21orf2 mutations. Phenotypes associated with NEK1 mutations are variable and the phenotype-genotype corelation in skeletal ciliopathies is challenging.

Osteosclerotic metaphyseal dysplasia (OSMD) is a rare skeletal dysplasia characterized by osteosclerotic metaphyses with osteopenic diaphyses of the long tubular bones. Our previous study identified a homozygous elongation mutation in leucine-rich repeat kinase 1 gene (LRRK1) in a patient with OSMD and showed that Lrrk1 knockout mice exhibited phenotypic similarity with OSMD. Here we report a second LRRK1 mutation in Indian sibs with OSMD. They had homozygous mutation (c.5971_5972insG) that produces an elongated mutant protein (p.A1991Gfs*31) similar to the first case. The sibs had normal stature, normal intelligence and recurrent fractures. The common radiographic feature was asymmetric and variable sclerosis of vertebral end plates, pelvic margin and metaphyses of tubular bones. One of the sibs had facial dysmorphisms, dentine abnormalities and acro-osteolysis. A comparison between the three OSMD cases with LRRK1 mutations with different ages suggested that the sclerotic lesions resolved with age. Our findings further support that LRRK1 would cause a subset of OSMD cases.

Desbuquois dysplasia (DBQD) is an autosomal recessive skeletal disorder characterized by growth retardation, joint laxity, short extremities, and progressive scoliosis. DBQD is classified into two types based on the presence (DBQD1) or absence (DBQD2) of characteristic hand abnormalities. CANT1 mutations have been reported in both DBQD1 and DBQD2. Recently, mutations in the gene encoding xylosyltransferase 1 (XYLT1) were identified in several families with DBQD2. In this study, we performed whole-exome sequencing in two Turkish families with DBQD2. We found a novel and a recurrent XYLT1 mutation in each family. The patients were homozygous for the mutations. Our results further support that XYLT1 is responsible for a major subset of DBQD2.

Spondyloepimetaphyseal dysplasias (SEMDs) comprise a heterogeneous group of autosomal-dominant and autosomal-recessive disorders. An apparent X-linked recessive (XLR) form of SEMD in a single Italian family was previously reported. We have been able to restudy this family together with a second family from Korea by segregating a severe SEMD in an X-linked pattern. Exome sequencing showed missense mutations in BGN c.439A>G (p.Lys147Glu) in the Korean family and c.776G>T (p.Gly259Val) in the Italian family; the c.439A>G (p.Lys147Glu) mutation was also identified in a further simplex SEMD case from India. Biglycan is an extracellular matrix proteoglycan that can bind transforming growth factor beta (TGF-β) and thus regulate its free concentration. In 3-dimensional simulation, both altered residues localized to the concave arc of leucine-rich repeat domains of biglycan that interact with TGF-β. The observation of recurrent BGN mutations in XLR SEMD individuals from different ethnic backgrounds allows us to define "XLR SEMD, BGN type" as a nosologic entity.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common spinal disorder that results from ectopic ossification of the posterior longitudinal ligament and causes intractable myelopathy and radiculopathy. In a previous genome-wide association study (GWAS), we found six loci associated with OPLL; however, susceptibility genes in these loci have not been identified yet. Here, we examined one of the GWAS loci and identified RSPO2 (encoding R-spondin 2) as a susceptibility gene for OPLL. R-spondin 2 is a secreted agonist of canonical Wnt-β-catenin signaling. RSPO2 was decreased in the early stage of chondrocyte differentiation. R-spondin 2 inhibited expression of genes encoding early chondrocyte differentiation markers by activating Wnt-β-catenin signaling. rs374810, the most significantly associated SNP in the GWAS locus in chromosomal region 8q23.1 was located in the chondrocyte promoter region of RSPO2. A transcription factor, CCAAT-enhancer-binding protein β (C/EBPβ), specifically bound to the RSPO2 core promoter region containing rs374810 and increased RSPO2 expression. The risk allele of rs374810 affected the binding of the promoter with C/EBPβ and decreased the RSPO2 transcription in vitro and in vivo. Our genetic and functional data indicate that RSPO2 is a susceptibility gene for OPLL.

Osteogenesis imperfecta (OI) is a heterogenous group of genetic disorders of bone fragility. OI type V is an autosomal-dominant disease characterized by calcification of the forearm interosseous membrane, radial head dislocation, a subphyseal metaphyseal radiodense line, and hyperplastic callus formation; the causative mutation involved in this disease has not been discovered yet. Using linkage analysis in a four-generation family and whole-exome sequencing, we identified a heterozygous mutation of c.−14C>T in the 5′-untranslated region of a gene encoding interferon-induced transmembrane protein 5 (IFITM5). It completely cosegregated with the disease in three families and occurred de novo in five simplex individuals. Transfection of wild-type and mutant IFITM5 constructs revealed that the mutation added five amino acids (Met-Ala-Leu-Glu-Pro) to the N terminus of IFITM5. Given that IFITM5 expression and protein localization is restricted to the skeletal tissue and IFITM5 involvement in bone formation, we conclude that this recurrent mutation would have a specific effect on IFITM5 function and thus cause OI type V.

Congenital scoliosis (CS) is a common vertebral malformation with incidence of up to 1 of 1000 births worldwide. Recently, TBX6 has been reported as the first disease gene for CS: about 10% of CS patients are compound heterozygotes of rare null mutations and a common haplotype composed by 3 SNPs in TBX6. Lefebvre et al in this journal reported that 2 patients with spondylocostal dysostosis (SCD), a rare skeletal dysplasia affecting spine and ribs also have TBX6 mutations: 1 carried the microdeletion and a rare missense variant, and another 2 rare missense variants. We investigated the pathogenicity of the 3 missense variants in SCD by a luciferase assay. The results were negative for the proposal of Lefebvre et al. We consider these 2 SCD patients are more probably compound heterozygotes of null mutations and a common risk haplotype just as CS patients with TBX6 mutations.

Spondyloepimetaphyseal dysplasia with joint laxity, leptodactylic type (lepto-SEMDJL, aka SEMDJL, Hall type), is an autosomal dominant skeletal disorder that, in spite of being relatively common among skeletal dysplasias, has eluded molecular elucidation so far. We used whole-exome sequencing of five unrelated individuals with lepto-SEMDJL to identify mutations in KIF22 as the cause of this skeletal condition. Missense mutations affecting one of two adjacent amino acids in the motor domain of KIF22 were present in 20 familial cases from eight families and in 12 other sporadic cases. The skeletal and connective tissue phenotype produced by these specific mutations point to functions of KIF22 beyond those previously ascribed functions involving chromosome segregation. Although we have found Kif22 to be strongly upregulated at the growth plate, the precise pathogenetic mechanisms remain to be elucidated.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motor neurons. Several susceptibility genes for ALS have been reported; however, ALS etiology and pathogenesis remain largely unknown. To identify further ALS-susceptibility genes, we conducted a large-scale case-control association study using gene-based tag single-nucleotide polymorphisms (SNPs). A functional SNP (rs2275294) was found to be significantly associated with ALS through a stepwise screening approach (combined P= 9.3 × 10(-10), odds ratio = 1.32). The SNP was located in an enhancer region of ZNF512B, a transcription factor of unknown biological function, and the susceptibility allele showed decreased activity and decreased binding to nuclear proteins. ZNF512B over-expression increased transforming growth factor-β (TGF-β) signaling, while knockdown had the opposite effect. ZNF512B expression was increased in the anterior horn motor neurons of the spinal cord of ALS patients when compared with controls. Our results strongly suggest that ZNF512B is an important positive regulator of TGF-β signaling and that decreased ZNF512B expression increases susceptibility to ALS.

WOS: 000286501500003, PubMed ID: 21194678, Microphthalmia with limb anomalies (MLA) is a rare autosomal-recessive disorder, presenting with anophthalmia or microphthalmia and hand and/or foot malformation. We mapped the MLA locus to 14q24 and successfully identified three homozygous (one nonsense and two splice site) mutations in the SPARC (secreted protein acidic and rich in cysteine)-related modular calcium binding 1 (SMOC1) in three families. Smoc1 is expressed in the developing optic stalk, ventral optic cup, and limbs of mouse embryos. Smoc1 null mice recapitulated MLA phenotypes, including aplasia or hypoplasia of optic nerves, hypoplastic fibula and bowed tibia, and syndactyly in limbs. A thinned and irregular ganglion cell layer and atrophy of the anteroventral part of the retina were also observed. Soft tissue syndactyly, resulting from inhibited apoptosis, was related to disturbed expression of genes involved in BMP signaling in the interdigital mesenchyme. Our findings indicate that SMOC1/Smoc1 is essential for ocular and limb development in both humans and mice., Ministry of Health, Labour and WelfareMinistry of Health, Labour and Welfare, Japan; Japan Science and Technology AgencyJapan Science & Technology Agency (JST); Japan Society for the Promotion of ScienceMinistry of Education, Culture, Sports, Science and Technology, Japan (MEXT)Japan Society for the Promotion of Science, We would like to thank the patients and their families for their participation in this study. We thank Yoshiko Takahashi (Nara Institute of Science and Technology) and Atsushi Yamada (Showa University) for providing the Bmp2 and Sox9 probes; Elizabeth J. Robertson (University of Oxford) and Makoto Ishibashi (Kyoto University) for the Bmp7 probe; Robert E. Maxson, Jr. (University of Southern California Keck School of Medicine) for the Msx2 probe; Tomonori Hirose, Kazunori Akimoto, and Kazunori Sasaki (Yokohama City University) for providing useful information about mouse breeding, taking photos on a stereo microscope, and mRNA quantification; and Kohei Shiota and Sumiko Kimura (Kyoto University) for helpful comments about NB staining and limb anomalies. This work was supported by research grants from the Ministry of Health, Labour and Welfare (T. Furuichi, N. Miyake, N. Matsumoto, and H.S.) and the Japan Science and Technology Agency (N. Matsumoto), a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (T. Furuichi and N. Matsumoto), and a Grant-in-Aid for Young Scientist from the Japan Society for the Promotion of Science (K.N., H.D., N. Miyake, and H.S.). This work has been carried out at the Advanced Medical Research Center of Yokohama City University.

Background Desbuquois dysplasia (DD) is a recessively inherited condition characterised by short stature, generalised skeletal dysplasia and advanced bone maturation. DD is both clinically and radiographically heterogeneous, and two subtypes have been distinguished based on the presence (type 1) or absence (type 2) of an accessory metacarpal bone. In addition, an apparently distinct variant without additional metacarpal bone but with short metacarpals and long phalanges (Kim variant) has been described recently. Mutations in the gene that encodes for CANT1 (calcium-activated nucleotidase 1) have been identified in a subset of patients with DD type 1. Methods A series of 11 subjects with DD from eight families (one type 1, two type 2, five Kim variant) were examined for CANT1 mutations by direct sequencing of all coding exons and their flanking introns. Results Eight distinct mutations were identified in seven families (one type 1, one type 2 and all 5 Kim variant): three were nonsense and five were missense. All missense mutations occurred at highly conserved amino acids in the nucleotidase conserved regions of CANT1. Measurement of nucleotidase activity in vitro showed that the missense mutations were all associated with loss-of-function. Conclusion The clinical-radiographic spectrum produced by CANT1 mutations must be extended to include DD type 2 and Kim variant. While presence or absence of an additional metacarpal ossification centre has been used to distinguish subtypes of DD, this sign is not a distinctive criterion to predict the molecular basis in DD.

Ehlers-Danlos syndrome (EDS) is a heterogeneous connective tissue disorder involving skin and joint laxity and tissue fragility. A new type of EDS, similar to kyphoscoliosis type but without lysyl hydroxylase deficiency, has been investigated. We have identified a homozygous CHST14 (carbohydrate sulfotransferase 14) mutation in the two familial cases and compound heterozygous mutations in four sporadic cases. CHST14 encodes dermatan 4-O-sulfotransferase 1 (D4ST1), which transfers active sulfate from 3′-phosphoadenosine 5′-phosphosulfate to position 4 of the N-acetyl-D-galactosamine (GalNAc) residues of dermatan sulfate (DS). Transfection experiments of mutants and enzyme assays using fibroblast lysates of patients showed the loss of D4ST1 activity. CHST14 mutations altered the glycosaminoglycan (GAG) components in patients' fibroblasts. Interestingly, DS of decorin proteoglycan, a key regulator of collagen fibril assembly, was completely lost and replaced by chondroitin sulfate (CS) in the patients' fibroblasts, leading to decreased flexibility of GAG chains. The loss of the decorin DS proteoglycan due to CHST14 mutations may preclude proper collagen bundle formation or maintenance of collagen bundles while the sizes and shapes of collagen fibrils are unchanged as observed in the patients' dermal tissues. These findings indicate the important role of decorin DS in the extracellular matrix and a novel pathomechanism in EDS. Hum Mutat 31:1–9, 2010. © 2010 Wiley-Liss, Inc.

We present the clinical and radiological findings of seven patients with a seemingly new variant of Desbuquois dysplasia (DBQD) and emphasize the radiographic findings in the hand. All cases showed remarkably accelerated carpal bone ages in childhood, but none of the patients had an accessory ossification center distal to the second metacarpal, or thumb anomalies, instead, there was shortness of one or all metacarpals, with elongated appearance of phalanges, resulting in nearly equal length of the second to fifth fingers. The two sibs followed for 20 years showed narrowing and fusion of the intercarpal joints with age and ultimately, precocious degenerative arthritis. The changes in the feet were similar to those of the hands, with advanced tarsal bone ages, shortness of the metatarsals and elongation of the second and third toes. Other radiographic findings were narrowness of the intervertebral disc spaces resulting in precocious degenerative spondylosis and progressive scoliosis. The femoral neck was short and thick and showed a persistent enlargement of the lesser trochanter with a high-riding, bulbous greater trochanter that became more prominent with age. Molecular testing of the diastrophic dysplasia sulfate transporter (DTDST) gene was performed on six patients and no mutations were detected. This radiographic and clinical observation further adds to the evidence that there may be subtypes of DBQD. Long-term follow-up showed that severe precocious osteoarthritis of the hand and spine is a major manifestation of this specific variant.