GWAS for systemic sclerosis identifies six novel susceptibility loci including one in the Fcγ receptor region.

Here we report the largest Asian genome-wide association study (GWAS) for systemic sclerosis performed to date, based on data from Japanese subjects and comprising of 1428 cases and 112,599 controls. The lead SNP is in the FCGR/FCRL region, which shows a penetrating association in the Asian population, while a complete linkage disequilibrium SNP, rs10917688, is found in a cis-regulatory element for IRF8. IRF8 is also a significant locus in European GWAS for systemic sclerosis, but rs10917688 only shows an association in the presence of the risk allele of IRF8 in the Japanese population. Further analysis shows that rs10917688 is marked with H3K4me1 in primary B cells. A meta-analysis with a European GWAS detects 30 additional significant loci. Polygenic risk scores constructed with the effect sizes of the meta-analysis suggest the potential portability of genetic associations beyond populations. Prioritizing the top 5% of SNPs of IRF8 binding sites in B cells improves the fitting of the polygenic risk scores, underscoring the roles of B cells and IRF8 in the development of systemic sclerosis. The results also suggest that systemic sclerosis shares a common genetic architecture across populations.

Association between germline pathogenic variants and breast cancer risk in Japanese women: The HERPACC study.

Approximately 5%-10% of breast cancers are hereditary, caused by germline pathogenic variants (GPVs) in breast cancer predisposition genes. To date, most studies of the prevalence of GPVs and risk of breast cancer for each gene based on cases and noncancer controls have been conducted in Europe and the United States, and little information from Japanese populations is available. Furthermore, no studies considered confounding by established environmental factors and single-nucleotide polymorphisms (SNPs) identified in genome-wide association studies (GWAS) together in GPV evaluation. To evaluate the association between GPVs in nine established breast cancer predisposition genes including BRCA1/2 and breast cancer risk in Japanese women comprehensively, we conducted a case-control study within the Hospital-based Epidemiologic Research Program at Aichi Cancer Center (629 cases and 1153 controls). The associations between GPVs and the risk of breast cancer were assessed by odds ratios (OR) and 95% confidence intervals (CI) using logistic regression models adjusted for potential confounders. A total of 25 GPVs were detected among all cases (4.0%: 95% CI: 2.6-5.9), whereas four individuals carried GPVs in all controls (0.4%). The OR for breast cancer by all GPVs and by GPVs in BRCA1/2 was 12.2 (4.4-34.0, p = 1.74E-06) and 16.0 (4.2-60.9, p = 5.03E-0.5), respectively. A potential confounding with GPVs was observed for the GWAS-identified SNPs, whereas not for established environmental risk factors. In conclusion, GPVs increase the risk of breast cancer in Japanese women regardless of environmental factors and GWAS-identified SNPs. Future studies investigating interactions with environment and SNPs are warranted.

Siblings with MAN1B1-CDG Showing Novel Biochemical Profiles.

Congenital disorders of glycosylation (CDG), inherited metabolic diseases caused by defects in glycosylation, are characterized by a high frequency of intellectual disability (ID) and various clinical manifestations. Two siblings with ID, dysmorphic features, and epilepsy were examined using mass spectrometry of serum transferrin, which revealed a CDG type 2 pattern. Whole-exome sequencing showed that both patients were homozygous for a novel pathogenic variant of MAN1B1 (NM_016219.4:c.1837del) inherited from their healthy parents. We conducted a HPLC analysis of sialylated N-linked glycans released from total plasma proteins and characterized the α1,2-mannosidase I activity of the lymphocyte microsome fraction. The accumulation of monosialoglycans was observed in MAN1B1-deficient patients, indicating N-glycan-processing defects. The enzymatic activity of MAN1B1 was compromised in patient-derived lymphocytes. The present patients exhibited unique manifestations including early-onset epileptic encephalopathy and cerebral infarction. They also showed coagulation abnormalities and hypertransaminasemia. Neither sibling had truncal obesity, which is one of the characteristic features of MAN1B1-CDG.

Functionally confirmed compound heterozygous ADAM17 missense loss-of-function variants cause neonatal inflammatory skin and bowel disease 1.

A disintegrin and metalloprotease 17 (ADAM17) is the major sheddase that processes more than 80 substrates, including tumour necrosis factor-α (TNFα). The homozygous genetic deficiency of ADAM17 causing a complete loss of ADAM17 expression was reported to be linked to neonatal inflammatory skin and bowel disease 1 (NISBD1). Here we report for the first time, a family with NISBD1 caused by functionally confirmed compound heterozygous missense variants of ADAM17, namely c.1699T>C (p.Cys567Arg) and c.1799G>A (p.Cys600Tyr). Both variants were detected in two siblings with clinical features of NISBD1, such as erythroderma with exudate in whole body, recurrent skin infection and sepsis and prolonged diarrhoea. In a cell-based assay using Adam10/17 double-knockout mouse embryonic fibroblasts (Adam10/17-/- mEFs) exogenously expressing each of these mutants, phorbol 12-myristate 13-acetate-stimulated shedding was strongly reduced compared with wild-type ADAM17. Thus, in vitro functional assays demonstrated that both missense variants cause the loss-of-function of ADAM17, resulting in the development of NISBD1. Our study further expands the spectrum of genetic pathology underlying ADAM17 in NISBD1 and establishes functional assay systems for its missense variants.

Genome-wide association meta-analysis identifies GP2 gene risk variants for pancreatic cancer.

Pancreatic cancer is the fourth leading cause of cancer-related deaths in Japan. To identify risk loci, we perform a meta-analysis of three genome-wide association studies comprising 2,039 pancreatic cancer patients and 32,592 controls in the Japanese population. Here, we identify 3 (13q12.2, 13q22.1, and 16p12.3) genome-wide significant loci (P < 5.0 × 10-8), of which 16p12.3 has not been reported in the Western population. The lead single nucleotide polymorphism (SNP) at 16p12.3 is rs78193826 (odds ratio = 1.46, 95% confidence interval = 1.29-1.66, P = 4.28 × 10-9), an Asian-specific, nonsynonymous glycoprotein 2 (GP2) gene variant. Associations between selected GP2 gene variants and pancreatic cancer are replicated in 10,822 additional cases and controls of East Asian origin. Functional analyses using cell lines provide supporting evidence of the effect of rs78193826 on KRAS activity. These findings suggest that GP2 gene variants are probably associated with pancreatic cancer susceptibility in populations of East Asian ancestry.

Claudin-6 is a single prognostic marker and functions as a tumor-promoting gene in a subgroup of intestinal type gastric cancer.

BACKGROUND: We aimed to identify novel tumor-promoting drivers highly expressed in gastric cancer (GC) that contribute to worsened prognosis in affected patients. METHODS: Genes whose expression was increased and correlated with worse prognosis in GC were screened using datasets from the Cancer Genome Atlas and Gene Expression Omnibus. We examined Claudin-6 (CLDN6) immunoreactivity in GC tissues and the effect of CLDN6 on cellular functions in GC cell lines. The mechanisms underlying GC-promoting function of CLDN6 were also investigated. RESULTS: CLDN6 was identified as a gene overexpressed in GC tumors as compared with adjacent non-tumorous tissues and whose increased expression was positively correlated with worse overall survival of GC patients, particularly those with Lauren's intestinal type GC, in data from multiple publicly available datasets. Additionally, membranous CLDN6 immunoreactivity detected in intestinal type GC tumors was correlated with worse overall survival. In CLDN6-expressing GC cells, silencing of CLDN6 inhibited cell proliferation and migration/invasion abilities, possibly via suppressing transcription of YAP1 and its downstream transcriptional targets at least in part. CONCLUSIONS: This study identified CLDN6 as a GC-promoting gene, suggesting that CLDN6 to be a possible single prognostic marker and promising therapeutic target for a subset of GC patients.

A 16q22.2-q23.1 deletion identified in a male infant with West syndrome.

In partial monosomy of the distal part of chromosome 16q, abnormal facial features, intellectual disability (ID), and feeding dysfunction are often reported. However, seizures are not typical and the majority of them were seizure-free. Here we present the case of a 16q22.2-q23.1 interstitial deletion identified in a male patient with severe ID, facial anomalies including forehead protrusions and flat nose bridge, patent ductus arteriosus, bilateral vocal cord atresia treated by tracheotomy, and West syndrome, which were developed 10 months after birth. Although phenobarbital, sodium valproate (VPA), and zonisamide were not effective as monotherapies or combination therapies, the patient's epileptic seizures and electroencephalogram anomalies disappeared following combined therapy with lamotrigine and VPA. Although WW Domain Containing Oxidoreductase (WWOX), which is known as a cause of autosomal recessive epileptic encephalopathy, was included within the 6.8-Mb deleted region which identified by targeted panel sequencing and validated by chromosomal microarray analysis, no pathogenic variants were detected in the other allele of WWOX. Therefore, it is possible that other genes within or outside of the long deleted region or their interactions may cause West syndrome in this patient.

Prognostic significance of GAD1 overexpression in patients with resected lung adenocarcinoma.

BACKGROUND AND OBJECTIVES: In a previous genome-wide screening, we identified hypermethylated CpG islands around glutamate decarboxylase 1 (GAD1) in lung adenocarcinoma (LADC). In this study, we aimed to investigate the methylation and expression status of GAD1 and its prognostic value in patients with LADC. METHODS: GAD1 methylation and mRNA expression status were analyzed using 33 tumorous and paired non-tumorous LADC samples and publicly available datasets. The prognostic value of GAD1 overexpression was investigated using publicly available datasets of mRNA levels and 162 cases of LADC by immunohistochemistry. RESULTS: The methylation and mRNA expression levels of GAD1, each having a positive correlation, were significantly higher in LADC tumors than in paired non-tumorous tissues. LADC patients with higher GAD1 mRNA expression showed significantly poorer prognosis for overall survival in publicly available datasets. Higher immunoreactivity of GAD1 was significantly associated with the pathological stage, pleural invasion, lymph vessel invasion, and poorer prognosis for cancer-specific and disease-free survival. Multivariate analysis revealed that GAD1 protein overexpression is an independent prognosticator for disease-free survival. CONCLUSIONS: GAD1 mRNA and protein expression levels were significant prognostic factors in LADC, suggesting that they might be useful biomarkers to stratify patients with worse clinical outcomes after resection.

ADAM17 selectively activates the IL-6 trans-signaling/ERK MAPK axis in KRAS-addicted lung cancer.

Oncogenic KRAS mutations are major drivers of lung adenocarcinoma (LAC), yet the direct therapeutic targeting of KRAS has been problematic. Here, we reveal an obligate requirement by oncogenic KRAS for the ADAM17 protease in LAC In genetically engineered and xenograft (human cell line and patient-derived) KrasG12D-driven LAC models, the specific blockade of ADAM17, including with a non-toxic prodomain inhibitor, suppressed tumor burden by reducing cellular proliferation. The pro-tumorigenic activity of ADAM17 was dependent upon its threonine phosphorylation by p38 MAPK, along with the preferential shedding of the ADAM17 substrate, IL-6R, to release soluble IL-6R that drives IL-6 trans-signaling via the ERK1/2 MAPK pathway. The requirement for ADAM17 in KrasG12D-driven LAC was independent of bone marrow-derived immune cells. Furthermore, in KRAS mutant human LAC, there was a significant positive correlation between augmented phospho-ADAM17 levels, observed primarily in epithelial rather than immune cells, and activation of ERK and p38 MAPK pathways. Collectively, these findings identify ADAM17 as a druggable target for oncogenic KRAS-driven LAC and provide the rationale to employ ADAM17-based therapeutic strategies for targeting KRAS mutant cancers.

Novel compound heterozygous CDH23 variants in a patient with Usher syndrome type I.

Usher syndrome type I (USH1) is characterized by congenital, bilateral, profound sensorineural hearing loss, vestibular areflexia, and adolescent-onset retinitis pigmentosa. Here, we report a 12-year-old female patient with typical USH1. Targeted panel sequencing revealed compound heterozygous variants of the Cadherin 23 (CDH23) gene, which confirmed the USH1 diagnosis. A novel NM_022124.5:c.130G>A/p.(Glu44Lys) was identified, expanding the mutation spectrum of CDH23.

Primary microcephaly caused by novel compound heterozygous mutations in ASPM.

Autosomal recessive primary microcephaly (microcephaly primary hereditary, MCPH) is a genetically heterogeneous rare developmental disorder that is characterized by prenatal onset of abnormal brain growth, which leads to intellectual disability of variable severity. We report a 5-year-old male who presented with a severe form of primary microcephaly. Targeted panel sequencing revealed compound heterozygous truncating mutations of the abnormal spindle-like microcephaly-associated (ASPM) gene, which confirmed the MCPH5 diagnosis. A novel NM_018136.4: c.9742_9745del (p.Lys3248Serfs*13) deletion mutation was identified.

A rare male patient with classic Rett syndrome caused by MeCP2_e1 mutation.

Rett syndrome (RTT) is a severe neurodevelopmental disorder typically affecting females. It is mainly caused by loss-of-function mutations that affect the coding sequence of exon 3 or 4 of methyl-CpG-binding protein 2 (MECP2). Severe neonatal encephalopathy resulting in death before the age of 2 years is the most common phenotype observed in males affected by a pathogenic MECP2 variant. Mutations in MECP2 exon 1 affecting the MeCP2_e1 isoform are relatively rare causes of RTT in females, and only one case of a male patient with MECP2-related severe neonatal encephalopathy caused by a mutation in MECP2 exon 1 has been reported. This is the first reported case of a male with classic RTT caused by a 5-bp duplication in the open-reading frame of MECP2 exon 1 (NM_001110792.1:c.23_27dup) that introduced a premature stop codon [p.(Ser10Argfs*36)] in the MeCP2_e1 isoform, which has been reported in one female patient with classic RTT. Therefore, both males and females displaying at least some type of MeCP2_e1 mutation may exhibit the classic RTT phenotype.

KH-type splicing regulatory protein is involved in esophageal squamous cell carcinoma progression.

KH-type splicing regulatory protein (KHSRP) is a multifunctional RNA-binding protein, which is involved in several post-transcriptional aspects of RNA metabolism, including microRNA (miRNA) biogenesis. It affects distinct cell functions in different tissues and can have an impact on various pathological conditions. In the present study, we investigated the oncogenic functions of KHSRP and their underlying mechanisms in the pathogenesis of esophageal squamous cell carcinoma (ESCC). KHSRP expression levels were elevated in ESCC tumors when compared with those in non-tumorous tissues by immunohistochemistry, and cytoplasmic KHSRP overexpression was found to be an independent prognosticator for worse overall survival in a cohort of 104 patients with ESCC. KHSRP knockdown inhibited growth, migration, and invasion of ESCC cells. KHSRP knockdown also inhibited the maturation of cancer-associated miRNAs, such as miR-21, miR-130b, and miR-301, and induced the expression of their target mRNAs, such as BMP6, PDCD4, and TIMP3, resulting in the inhibition of epithelial-to-mesenchymal transition. Our findings uncover a novel oncogenic function of KHSRP in esophageal tumorigenesis and implicate its use as a marker for prognostic evaluation and as a putative therapeutic target in ESCC.

Construction of a combinatorial pipeline using two somatic variant calling methods for whole exome sequence data of gastric cancer.

High-throughput next-generation sequencing is a powerful tool to identify the genotypic landscapes of somatic variants and therapeutic targets in various cancers including gastric cancer, forming the basis for personalized medicine in the clinical setting. Although the advent of many computational algorithms leads to higher accuracy in somatic variant calling, no standard method exists due to the limitations of each method. Here, we constructed a new pipeline. We combined two different somatic variant callers with different algorithms, Strelka and VarScan 2, and evaluated performance using whole exome sequencing data obtained from 19 Japanese cases with gastric cancer (GC); then, we characterized these tumors based on identified driver molecular alterations. More single nucleotide variants (SNVs) and small insertions/deletions were detected by Strelka and VarScan 2, respectively. SNVs detected by both tools showed higher accuracy for estimating somatic variants compared with those detected by only one of the two tools and accurately showed the mutation signature and mutations of driver genes reported for GC. Our combinatorial pipeline may have an advantage in detection of somatic mutations in GC and may be useful for further genomic characterization of Japanese patients with GC to improve the efficacy of GC treatments. J. Med. Invest. 64: 233-240, August, 2017.

Novel CLCN7 compound heterozygous mutations in intermediate autosomal recessive osteopetrosis.

Osteopetrosis is a heritable disorder of the skeleton that is characterized by increased bone density on radiographs caused by defects in osteoclast formation and function. Mutations in >10 genes are identified as causative for this clinically and genetically heterogeneous disease in humans. We report two novel missense variations in a compound heterozygous state in the CLCN7 gene, detected through targeted exome sequencing, in a 15-year-old Japanese female with intermediate autosomal recessive osteopetrosis.

The first Japanese patient with mandibular hypoplasia, deafness, progeroid features and lipodystrophy diagnosed via POLD1 mutation detection.

Mandibular hypoplasia, deafness, progeroid features and lipodystrophy (MDPL) syndrome is a rare autosomal dominant disorder caused by heterozygous POLD1 mutations. To date, 13 patients affected by POLD1 mutation-caused MDPL have been described. We report a clinically undiagnosed 11-year-old male who noted joint contractures at 6 years of age. Targeted exome sequencing identified a known POLD1 mutation [NM_002691.3:c.1812_1814del, p.(Ser605del)] that diagnosed him as the first Japanese/East Asian MDPL case.

A case with concurrent duplication, triplication, and uniparental isodisomy at 1q42.12-qter supporting microhomology-mediated break-induced replication model for replicative rearrangements.

BACKGROUND: Complex genomic rearrangements (CGRs) consisting of interstitial triplications in conjunction with uniparental isodisomy (isoUPD) have rarely been reported in patients with multiple congenital anomalies (MCA)/intellectual disability (ID). One-ended DNA break repair coupled with microhomology-mediated break-induced replication (MMBIR) has been recently proposed as a possible mechanism giving rise to interstitial copy number gains and distal isoUPD, although only a few cases providing supportive evidence in human congenital diseases with MCA have been documented. CASE PRESENTATION: Here, we report on the chromosomal microarray (CMA)-based identification of the first known case with concurrent interstitial duplication at 1q42.12-q42.2 and triplication at 1q42.2-q43 followed by isoUPD for the remainder of chromosome 1q (at 1q43-qter). In distal 1q duplication/triplication overlapping with 1q42.12-q43, variable clinical features have been reported, and our 25-year-old patient with MCA/ID presented with some of these frequently described features. Further analyses including the precise mapping of breakpoint junctions within the CGR in a sequence level suggested that the CGR found in association with isoUPD in our case is a triplication with flanking duplications, characterized as a triplication with a particularly long duplication-inverted triplication-duplication (DUP-TRP/INV-DUP) structure. Because microhomology was observed in both junctions between the triplicated region and the flanking duplicated regions, our case provides supportive evidence for recently proposed replication-based mechanisms, such as MMBIR, underlying the formation of CGRs + isoUPD implicated in chromosomal disorders. CONCLUSIONS: To the best of our knowledge, this is the first case of CGRs + isoUPD observed in 1q and having DUP-TRP/INV-DUP structure with a long proximal duplication, which supports MMBIR-based model for genomic rearrangements. Molecular cytogenetic analyses using CMA containing single-nucleotide polymorphism probes with further analyses of the breakpoint junctions are recommended in cases suspected of having complex chromosomal abnormalities based on discrepancies between clinical and conventional cytogenetic findings.

A 590 kb deletion caused by non-allelic homologous recombination between two LINE-1 elements in a patient with mesomelia-synostosis syndrome.

Mesomelia-synostoses syndrome (MSS) is a rare, autosomal-dominant, syndromal osteochondrodysplasia characterized by mesomelic limb shortening, acral synostoses, and multiple congenital malformations due to a non-recurrent deletion at 8q13 that always encompasses two coding-genes, SULF1 and SLCO5A1. To date, five unrelated patients have been reported worldwide, and MMS was previously proposed to not be a genomic disorder associated with deletions recurring from non-allelic homologous recombination (NAHR) in at least two analyzed cases. We conducted targeted gene panel sequencing and subsequent array-based copy number analysis in an 11-year-old undiagnosed Japanese female patient with multiple congenital anomalies that included mesomelic limb shortening and detected a novel 590 Kb deletion at 8q13 encompassing the same gene set as reported previously, resulting in the diagnosis of MSS. Breakpoint sequences of the deleted region in our case demonstrated the first LINE-1s (L1s)-mediated unequal NAHR event utilizing two distant L1 elements as homology substrates in this disease, which may represent a novel causative mechanism of the 8q13 deletion, expanding the range of mechanisms involved in the chromosomal rearrangements responsible for MSS.

Genome-first approach diagnosed Cabezas syndrome via novel CUL4B mutation detection.

Cabezas syndrome is a syndromic form of X-linked intellectual disability primarily characterized by a short stature, hypogonadism and abnormal gait, with other variable features resulting from mutations in the CUL4B gene. Here, we report a clinically undiagnosed 5-year-old male with severe intellectual disability. A genome-first approach using targeted exome sequencing identified a novel nonsense mutation [NM_003588.3:c.2698G>T, p.(Glu900*)] in the last coding exon of CUL4B, thus diagnosing this patient with Cabezas syndrome.

Frequent silencing of the candidate tumor suppressor TRIM58 by promoter methylation in early-stage lung adenocarcinoma.

In this study, we aimed to identify novel drivers that would be epigenetically altered through aberrant methylation in early-stage lung adenocarcinoma (LADC), regardless of the presence or absence of tobacco smoking-induced epigenetic field defects. Through genome-wide screening for aberrantly methylated CpG islands (CGIs) in 12 clinically uniform, stage-I LADC cases affecting six non-smokers and six smokers, we identified candidate tumor-suppressor genes (TSGs) inactivated by hypermethylation. Through systematic expression analyses of those candidates in panels of additional tumor samples and cell lines treated or not treated with 5-aza-deoxycitidine followed by validation analyses of cancer-specific silencing by CGI hypermethylation using a public database, we identified TRIM58 as the most prominent candidate for TSG. TRIM58 was robustly silenced by hypermethylation even in early-stage primary LADC, and the restoration of TRIM58 expression in LADC cell lines inhibited cell growth in vitro and in vivo in anchorage-dependent and -independent manners. Our findings suggest that aberrant inactivation of TRIM58 consequent to CGI hypermethylation might stimulate the early carcinogenesis of LADC regardless of smoking status; furthermore, TRIM58 methylation might be a possible early diagnostic and epigenetic therapeutic target in LADC.