Mutations in MYO9B are associated with Charcot-Marie-Tooth disease type 2 neuropathies and isolated optic atrophy.

BACKGROUND AND PURPOSE: Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of disorders caused by mutations in at least 100 genes. However, approximately 60% of cases with axonal neuropathies (CMT2) still remain without a genetic diagnosis. We aimed at identifying novel disease genes responsible for CMT2. METHODS: We performed whole exome sequencing and targeted next generation sequencing panel analyses on a cohort of CMT2 families with evidence for autosomal recessive inheritance. We also performed functional studies to explore the pathogenetic role of selected variants. RESULTS: We identified rare, recessive variants in the MYO9B (myosin IX) gene in two families with CMT2. MYO9B has not yet been associated with a human disease. MYO9B is an unconventional single-headed processive myosin motor protein with signaling properties, and, consistent with this, our results indicate that a variant occurring in the MYO9B motor domain impairs protein expression level and motor activity. Interestingly, a Myo9b-null mouse has degenerating axons in sciatic nerves and optic nerves, indicating that MYO9B plays an essential role in both peripheral nervous system and central nervous system axons, respectively. The degeneration observed in the optic nerve prompted us to screen for MYO9B mutations in a cohort of patients with optic atrophy (OA). Consistent with this, we found compound heterozygous variants in one case with isolated OA. CONCLUSIONS: Novel or very rare variants in MYO9B are associated with CMT2 and isolated OA.

Effect of Genetic Factors, Age and Sex on Levels of Circulating Extracellular Vesicles and Platelets.

Extracellular vesicles (EVs) mediate cell interactions in biological processes, such as receptor activation or molecule transfer. Estimates of variation by age and sex have been limited by small sample size, and no report has assessed the contribution of genetic factors to levels of EVs. Here, we evaluated blood levels of 25 EV and 3 platelet traits in 974 individuals (933 genotyped) and reported the first genome-wide association study (GWAS) on levels of these traits. EV levels all decreased with age, whereas the trend for their surface markers was more heterogeneous. Platelets and CD31dim platelet EVs significantly increased in females compared to males, although CD31 expression on both platelets and platelet EVs decreased in females. Levels of the other EV subsets were similar between sexes. GWAS revealed three statistically significant genetic signals associated with EV levels in the F10 and GBP1 genes and in the intergenic region between LRIG1 and KBTBD8. These add to a signal in the 3'UTR of RHOF associated with CD31 expression on platelets that was previously found to be associated with other platelet traits. These findings suggest that EV formation is not a simple, constant adjunct of metabolism but is under both age-related and genetic control that can be independent of the regulation of the levels of the cells from which the EVs derive.

Clinical and laboratory predictors of monogenic very early onset inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. Treatment for patients who have a monogenic cause of their IBD, often the youngest children, known as very early onset IBD (VEO-IBD), can be different from standard treatment for polygenic cases. Yet, ascertainment of these patients is difficult. METHODS: We analyzed cases of VEO-IBD to understand the breadth of monogenic etiology and to identify clinical, laboratory, and flow cytometric correlates of this subpopulation. RESULTS: Genetic causes of very early onset inflammatory bowel disease are highly diverse ranging from pure epithelial defects to classic T cell defects. Flow cytometry, other than testing for chronic granulomatous disease, has a low sensitivity for monogenic etiologies. Poor growth was a clinical feature associated with monogenic causality. CONCLUSIONS: Genetic testing is, at this moment, the most robust method for the identification of monogenic cases of very early onset IBD.

Impaired Redox and Protein Homeostasis as Risk Factors and Therapeutic Targets in Toxin-Induced Biliary Atresia.

BACKGROUND & AIMS: Extrahepatic biliary atresia (BA) is a pediatric liver disease with no approved medical therapy. Recent studies using human samples and experimental modeling suggest that glutathione redox metabolism and heterogeneity play a role in disease pathogenesis. We sought to dissect the mechanistic basis of liver redox variation and explore how other stress responses affect cholangiocyte injury in BA. METHODS: We performed quantitative in situ hepatic glutathione redox mapping in zebrafish larvae carrying targeted mutations in glutathione metabolism genes and correlated these findings with sensitivity to the plant-derived BA-linked toxin biliatresone. We also determined whether genetic disruption of HSP90 protein quality control pathway genes implicated in human BA altered biliatresone toxicity in zebrafish and human cholangiocytes. An in vivo screening of a known drug library was performed to identify novel modifiers of cholangiocyte injury in the zebrafish experimental BA model, with subsequent validation. RESULTS: Glutathione metabolism gene mutations caused regionally distinct changes in the redox potential of cholangiocytes that differentially sensitized them to biliatresone. Disruption of human BA-implicated HSP90 pathway genes sensitized zebrafish and human cholangiocytes to biliatresone-induced injury independent of glutathione. Phosphodiesterase-5 inhibitors and other cyclic guanosine monophosphate signaling activators worked synergistically with the glutathione precursor N-acetylcysteine in preventing biliatresone-induced injury in zebrafish and human cholangiocytes. Phosphodiesterase-5 inhibitors enhanced proteasomal degradation and required intact HSP90 chaperone. CONCLUSION: Regional variation in glutathione metabolism underlies sensitivity to the biliary toxin biliatresone and may account for the reported association between BA transplant-free survival and glutathione metabolism gene expression. Human BA can be causatively linked to genetic modulation of protein quality control. Combined treatment with N-acetylcysteine and cyclic guanosine monophosphate signaling enhancers warrants further investigation as therapy for BA.

A genome-wide association study identifies a susceptibility locus for biliary atresia on 2p16.1 within the gene EFEMP1.

Biliary atresia (BA) is a rare pediatric cholangiopathy characterized by fibrosclerosing obliteration of the extrahepatic bile ducts, leading to cholestasis, fibrosis, cirrhosis, and eventual liver failure. The etiology of BA remains unknown, although environmental, inflammatory, infectious, and genetic risk factors have been proposed. We performed a genome-wide association study (GWAS) in a European-American cohort of 343 isolated BA patients and 1716 controls to identify genetic loci associated with BA. A second GWAS was performed in an independent European-American cohort of 156 patients with BA and other extrahepatic anomalies and 212 controls to confirm the identified candidate BA-associated SNPs. Meta-analysis revealed three genome-wide significant BA-associated SNPs on 2p16.1 (rs10865291, rs6761893, and rs727878; P < 5 ×10-8), located within the fifth intron of the EFEMP1 gene, which encodes a secreted extracellular protein implicated in extracellular matrix remodeling, cell proliferation, and organogenesis. RNA expression analysis showed an increase in EFEMP1 transcripts from human liver specimens isolated from patients with either BA or other cholestatic diseases when compared to normal control liver samples. Immunohistochemistry demonstrated that EFEMP1 is expressed in cholangiocytes and vascular smooth muscle cells in liver specimens from patients with BA and other cholestatic diseases, but it is absent from cholangiocytes in normal control liver samples. Efemp1 transcripts had higher expression in cholangiocytes and portal fibroblasts as compared with other cell types in normal rat liver. The identification of a novel BA-associated locus, and implication of EFEMP1 as a new BA candidate susceptibility gene, could provide new insights to understanding the mechanisms underlying this severe pediatric disorder.

Neural crest-derived tumor neuroblastoma and melanoma share 1p13.2 as susceptibility locus that shows a long-range interaction with the SLC16A1 gene.

Neuroblastoma (NB) and malignant cutaneous melanoma (CMM) are neural crest cells (NCC)-derived tumors and may have a shared genetic basis, but this has not been investigated systematically by genome-wide association studies (GWAS). We took a three-staged approach to conduct cross-disease meta-analysis of GWAS for NB and CMM (2101 NB cases and 4202 controls; 12 874 CMM cases and 23 203 controls) to identify shared loci. Findings were replicated in 1403 NB cases and 1403 controls of European ancestry and in 636 NB, 508 CMM cases and 2066 controls of Italian origin. We found a cross-association at locus 1p13.2 (rs2153977, odds ratio = 0.91, P = 5.36 × 10-8). We also detected a suggestive (P < 10-7) NB-CMM cross-association at 2q37.1 with opposite effect on cancer risk. Pathway analysis of 110 NB-CMM risk loci with P < 10-4 demonstrated enrichment of biological processes such as cell migration, cell cycle, metabolism and immune response, which are essential of human NCC development, underlying both tumors. In vitro and in silico analyses indicated that the rs2153977-T protective allele, located in an NB and CMM enhancer, decreased expression of SLC16A1 via long-range loop formation and altered a T-box protein binding site. Upon depletion of SLC16A1, we observed a decrease of cellular proliferation and invasion in both NB and CMM cell lines, suggesting its role as oncogene. This is the largest study to date examining pleiotropy across two NC cell-derived tumors identifying 1p13.2 as common susceptibility locus for NB and CMM risk. We demonstrate that combining genome-wide association studies results across cancers with same origins can identify new loci common to neuroblastoma and melanoma arising from tissues which originate from neural crest cells. Our results also show 1p13.2 confer risk to neuroblastoma and melanoma by regulating SLC16A1.

Common variants upstream of MLF1 at 3q25 and within CPZ at 4p16 associated with neuroblastoma.

Neuroblastoma is a cancer of the developing sympathetic nervous system that most commonly presents in young children and accounts for approximately 12% of pediatric oncology deaths. Here, we report on a genome-wide association study (GWAS) in a discovery cohort or 2,101 cases and 4,202 controls of European ancestry. We identify two new association signals at 3q25 and 4p16 that replicated robustly in multiple independent cohorts comprising 1,163 cases and 4,396 controls (3q25: rs6441201 combined P = 1.2x10-11, Odds Ratio 1.23, 95% CI:1.16-1.31; 4p16: rs3796727 combined P = 1.26x10-12, Odds Ratio 1.30, 95% CI: 1.21-1.40). The 4p16 signal maps within the carboxypeptidase Z (CPZ) gene. The 3q25 signal resides within the arginine/serine-rich coiled-coil 1 (RSRC1) gene and upstream of the myeloid leukemia factor 1 (MLF1) gene. Increased expression of MLF1 was observed in neuroblastoma cells homozygous for the rs6441201 risk allele (P = 0.02), and significant growth inhibition was observed upon depletion of MLF1 (P < 0.0001) in neuroblastoma cells. Taken together, we show that common DNA variants within CPZ at 4p16 and upstream of MLF1 at 3q25 influence neuroblastoma susceptibility and MLF1 likely plays an important role in neuroblastoma tumorigenesis.

Expanding the spectrum of genes responsible for hereditary motor neuropathies.

BACKGROUND: Inherited peripheral neuropathies (IPNs) represent a broad group of genetically and clinically heterogeneous disorders, including axonal Charcot-Marie-Tooth type 2 (CMT2) and hereditary motor neuropathy (HMN). Approximately 60%-70% of cases with HMN/CMT2 still remain without a genetic diagnosis. Interestingly, mutations in HMN/CMT2 genes may also be responsible for motor neuron disorders or other neuromuscular diseases, suggesting a broad phenotypic spectrum of clinically and genetically related conditions. Thus, it is of paramount importance to identify novel causative variants in HMN/CMT2 patients to better predict clinical outcome and progression. METHODS: We designed a collaborative study for the identification of variants responsible for HMN/CMT2. We collected 15 HMN/CMT2 families with evidence for autosomal recessive inheritance, who had tested negative for mutations in 94 known IPN genes, who underwent whole-exome sequencing (WES) analyses. Candidate genes identified by WES were sequenced in an additional cohort of 167 familial or sporadic HMN/CMT2 patients using next-generation sequencing (NGS) panel analysis. RESULTS: Bioinformatic analyses led to the identification of novel or very rare variants in genes, which have not been previously associated with HMN/CMT2 (ARHGEF28, KBTBD13, AGRN and GNE); in genes previously associated with HMN/CMT2 but in combination with different clinical phenotypes (VRK1 and PNKP), and in the SIGMAR1 gene, which has been linked to HMN/CMT2 in only a few cases. These findings were further validated by Sanger sequencing, segregation analyses and functional studies. CONCLUSIONS: These results demonstrate the broad spectrum of clinical phenotypes that can be associated with a specific disease gene, as well as the complexity of the pathogenesis of neuromuscular disorders.

An Exploratory Association Analysis of the Insulin Gene Region With Diabetes Mellitus in Two Dog Breeds.

Samoyeds and Australian Terriers are the 2 dog breeds at highest risk (>10-fold) for diabetes mellitus in the United States. It is unknown if the insulin (INS) gene is involved in the pathophysiology of diabetes in Samoyeds and Australian Terriers. It was hypothesized that the INS gene region provides a common genetic causality for diabetes in Samoyeds and Australian Terriers. We conducted a 2-stage genetic association study involving both breeds. In the discovery stage (Stage 1), Samoyeds with and without diabetes were compared in the frequencies of 447 tagging single-nucleotide polymorphisms (SNPs) within 2.5 megabases (Mb) up- and downstream of the INS gene on the Illumina CanineHD BeadChip. SNPs yielding a P-value < 0.005 were selected for further follow-up. In the validation stage (Stage 2), Australian Terriers with and without diabetes were compared in the SNPs genotyped by the Affymetrix GeneChip Canine Genome 2.0 Array and within 1 Mb up- and downstream of the selected SNPs from Stage 1. Two SNPs that were in high linkage disequilibrium (LD, r2 = 0.7) were selected from Stage 1. In Stage 2, among the 76 SNPs examined, 5 were significantly associated with diabetes after Bonferroni's correction for multiple comparisons. Three of these 5 SNPs were in complete LD (r2 = 1 for all associations) and the 2 remaining SNPs were in moderate LD (r2 = 0.4). In conclusion, an association between the INS gene region and diabetes was suggested in 2 dog breeds of different clades. This region could have importance in diabetes in other breeds or in canine diabetes at large.

Fine mapping of 2q35 high-risk neuroblastoma locus reveals independent functional risk variants and suggests full-length BARD1 as tumor-suppressor.

A previous genome-wide association study (GWAS) identified common variation at the BARD1 locus as being highly associated with susceptibility to high-risk neuroblastoma, but the mechanisms underlying this association have been not extensively investigated. Here, we performed a fine mapping analysis of BARD1 locus (2q35) using GWAS data from 556 high-risk neuroblastoma patients and 2,575 controls of European-American ancestry, and identified two independent genome-wide neuroblastoma-associated loci. Functional single-nucleotide polymorphism (SNP) prioritization identified two causative variants that independently contributed to neuroblastoma risk, and each replicated robustly in multiple independent cohorts comprising 445 high-risk cases and 3,170 controls (rs17489363: combined p = 1.07 × 10-31 , OR:1.79, 95% CI:1.62-1.98 and rs1048108: combined p = 7.27 × 10-14 , OR:0.65, 95% CI:0.58-0.73). Particularly, the T risk allele of rs17489363 in the canonical promoter region of full-length BARD1 altered binding site of the transcription factor HSF1 and correlated with low expression of full-length BARD1 mRNA and protein. Low-level expression of full-length BARD1 associated with advanced neuroblastoma. In human neuroblastoma cells, attenuating full-length BARD1 increased proliferation and invasion capacity. In conclusion, we have identified two potentially causative SNPs at the BARD1 locus associated with predisposition to high-risk neuroblastoma, and have shown that full-length BARD1 may act as tumor suppressor.

Copy-Number Variation of the Glucose Transporter Gene SLC2A3 and Congenital Heart Defects in the 22q11.2 Deletion Syndrome.

The 22q11.2 deletion syndrome (22q11DS; velocardiofacial/DiGeorge syndrome; VCFS/DGS) is the most common microdeletion syndrome and the phenotypic presentation is highly variable. Approximately 65% of individuals with 22q11DS have a congenital heart defect (CHD), mostly of the conotruncal type, and/or an aortic arch defect. The etiology of this phenotypic variability is not currently known. We hypothesized that copy-number variants (CNVs) outside the 22q11.2 deleted region might increase the risk of being born with a CHD in this sensitized population. Genotyping with Affymetrix SNP Array 6.0 was performed on two groups of subjects with 22q11DS separated by time of ascertainment and processing. CNV analysis was completed on a total of 949 subjects (cohort 1, n = 562; cohort 2, n = 387), 603 with CHDs (cohort 1, n = 363; cohort 2, n = 240) and 346 with normal cardiac anatomy (cohort 1, n = 199; cohort 2, n = 147). Our analysis revealed that a duplication of SLC2A3 was the most frequent CNV identified in the first cohort. It was present in 18 subjects with CHDs and 1 subject without (p = 3.12 × 10(-3), two-tailed Fisher's exact test). In the second cohort, the SLC2A3 duplication was also significantly enriched in subjects with CHDs (p = 3.30 × 10(-2), two-tailed Fisher's exact test). The SLC2A3 duplication was the most frequent CNV detected and the only significant finding in our combined analysis (p = 2.68 × 10(-4), two-tailed Fisher's exact test), indicating that the SLC2A3 duplication might serve as a genetic modifier of CHDs and/or aortic arch anomalies in individuals with 22q11DS.

Subphenotype meta-analysis of testicular cancer genome-wide association study data suggests a role for RBFOX family genes in cryptorchidism susceptibility.

STUDY QUESTION: Can subphenotype analysis of genome-wide association study (GWAS) data from subjects with testicular germ cell tumor (TGCT) provide insight into cryptorchidism (undescended testis, UDT) susceptibility? SUMMARY ANSWER: Suggestive intragenic GWAS signals common to UDT, TGCT case-case and TGCT case-control analyses occur in genes encoding RBFOX RNA-binding proteins (RBPs) and their neurodevelopmental targets. WHAT IS KNOWN ALREADY: UDT is a strong risk factor for TGCT, but while genetic risk factors for TGCT are well-known, genetic susceptibility to UDT is poorly understood and appears to be more complex. STUDY DESIGN, SIZE, DURATION: We performed a secondary subphenotype analysis of existing GWAS data from the Testicular Cancer Consortium (TECAC) and compared these results with our previously published UDT GWAS data, and with data previously acquired from studies of the fetal rat gubernaculum. PARTICIPANTS/MATERIALS, SETTING, METHODS: Studies from the National Cancer Institute (NCI), United Kingdom (UK) and University of Pennsylvania (Penn) that enrolled white subjects were the source of the TGCT GWAS data. We completed UDT subphenotype case-case (TGCT/UDT vs TGCT/non-UDT) and case-control (TGCT/UDT vs control), collectively referred to as 'TECAC' analyses, followed by a meta-analysis comprising 129 TGCT/UDT cases, 1771 TGCT/non-UDT cases, and 3967 unaffected controls. We reanalyzed our UDT GWAS results comprising 844 cases and 2718 controls by mapping suggestive UDT and TECAC signals (defined as P < 0.001) to genes using Ingenuity Pathway Analysis (IPA®). We compared associated pathways and enriched gene categories common to all analyses after Benjamini-Hochberg multiple testing correction, and analyzed transcript levels and protein expression using qRT-PCR and rat fetal gubernaculum confocal imaging, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: We found suggestive signals within 19 genes common to all three analyses, including RBFOX1 and RBFOX3, neurodevelopmental paralogs that encode RBPs targeting (U)GCATG-containing transcripts. Ten of the 19 genes participate in neurodevelopment and/or contribute to risk of neurodevelopmental disorders. Experimentally predicted RBFOX gene targets were strongly overrepresented among suggestive intragenic signals for the UDT (117 of 628 (19%), P = 3.5 × 10-24), TECAC case-case (129 of 711 (18%), P = 2.5 × 10-27) and TECAC case-control (117 of 679 (17%), P = 2 × 10-21) analyses, and a majority of the genes common to all three analyses (12 of 19 (63%), P = 3 × 10-9) are predicted RBFOX targets. Rbfox1, Rbfox2 and their encoded proteins are expressed in the rat fetal gubernaculum. Predicted RBFOX targets are also enriched among transcripts differentially regulated in the fetal gubernaculum during normal development (P = 3 × 10-31), in response to in vitro hormonal stimulation (P = 5 × 10-45) and in the cryptorchid LE/orl rat (P = 2 × 10-42). LARGE SCALE DATA: GWAS data included in this study are available in the database of Genotypes and Phenotypes (dbGaP accession numbers phs000986.v1.p1 and phs001349.v1p1). LIMITATIONS, REASONS FOR CAUTION: These GWAS data did not reach genome-wide significance for any individual analysis. UDT appears to have a complex etiology that also includes environmental factors, and such complexity may require much larger sample sizes than are currently available. The current methodology may also introduce bias that favors false discovery of larger genes. WIDER IMPLICATIONS OF THE FINDINGS: Common suggestive intragenic GWAS signals suggest that RBFOX paralogs and other neurodevelopmental genes are potential UDT risk candidates, and potential TGCT susceptibility modifiers. Enrichment of predicted RBFOX targets among differentially expressed transcripts in the fetal gubernaculum additionally suggests a role for this RBP family in regulation of testicular descent. As RBFOX proteins regulate alternative splicing of Calca to generate calcitonin gene-related peptide, a protein linked to development and function of the gubernaculum, additional studies that address the role of these proteins in UDT are warranted. STUDY FUNDING/COMPETING INTEREST(S): The Eunice Kennedy Shriver National Institute for Child Health and Human Development (R01HD060769); National Center for Research Resources (P20RR20173), National Institute of General Medical Sciences (P20GM103464), Nemours Biomedical Research, the Testicular Cancer Consortium (U01CA164947), the Intramural Research Program of the NCI, a support services contract HHSN26120130003C with IMS, Inc., the Abramson Cancer Center at Penn, National Cancer Institute (CA114478), the Institute of Cancer Research, UK and the Wellcome Trust Case-Control Consortium (WTCCC) 2. None of the authors reports a conflict of interest.

Integrative genomics identifies LMO1 as a neuroblastoma oncogene.

Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 × 10(-16), odds ratio of risk allele = 1.34 (95% confidence interval 1.25-1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P < 0.0001) and survival (P = 0.041). The germline single nucleotide polymorphism (SNP) risk alleles and somatic copy number gains were associated with increased LMO1 expression in neuroblastoma cell lines and primary tumours, consistent with a gain-of-function role in tumorigenesis. Short hairpin RNA (shRNA)-mediated depletion of LMO1 inhibited growth of neuroblastoma cells with high LMO1 expression, whereas forced expression of LMO1 in neuroblastoma cells with low LMO1 expression enhanced proliferation. These data show that common polymorphisms at the LMO1 locus are strongly associated with susceptibility to developing neuroblastoma, but also may influence the likelihood of further somatic alterations at this locus, leading to malignant progression.

Recessive and dominant mutations in COL12A1 cause a novel EDS/myopathy overlap syndrome in humans and mice.

Collagen VI-related myopathies are disorders of connective tissue presenting with an overlap phenotype combining clinical involvement from the muscle and from the connective tissue. Not all patients displaying related overlap phenotypes between muscle and connective tissue have mutations in collagen VI. Here, we report a homozygous recessive loss of function mutation and a de novo dominant mutation in collagen XII (COL12A1) as underlying a novel overlap syndrome involving muscle and connective tissue. Two siblings homozygous for a loss of function mutation showed widespread joint hyperlaxity combined with weakness precluding independent ambulation, while the patient with the de novo missense mutation was more mildly affected, showing improvement including the acquisition of walking. A mouse model with inactivation of the Col12a1 gene showed decreased grip strength, a delay in fiber-type transition and a deficiency in passive force generation while the muscle seems more resistant to eccentric contraction induced force drop, indicating a role for a matrix-based passive force-transducing elastic element in the generation of the weakness. This new muscle connective tissue overlap syndrome expands on the emerging importance of the muscle extracellular matrix in the pathogenesis of muscle disease.

Rare copy number variants and congenital heart defects in the 22q11.2 deletion syndrome.

The 22q11.2 deletion syndrome (22q11DS; velocardiofacial/DiGeorge syndrome; VCFS/DGS; MIM #192430; 188400) is the most common microdeletion syndrome. The phenotypic presentation of 22q11DS is highly variable; approximately 60-75 % of 22q11DS patients have been reported to have a congenital heart defect (CHD), mostly of the conotruncal type, and/or aortic arch defect. The etiology of the cardiac phenotypic variability is not currently known for the majority of patients. We hypothesized that rare copy number variants (CNVs) outside the 22q11.2 deleted region may modify the risk of being born with a CHD in this sensitized population. Rare CNV analysis was performed using Affymetrix SNP Array 6.0 data from 946 22q11DS subjects with CHDs (n = 607) or with normal cardiac anatomy (n = 339). Although there was no significant difference in the overall burden of rare CNVs, an overabundance of CNVs affecting cardiac-related genes was detected in 22q11DS individuals with CHDs. When the rare CNVs were examined with regard to gene interactions, specific cardiac networks, such as Wnt signaling, appear to be overrepresented in 22q11DS CHD cases but not 22q11DS controls with a normal heart. Collectively, these data suggest that CNVs outside the 22q11.2 region may contain genes that modify risk for CHDs in some 22q11DS patients.

Exome sequencing analysis reveals variants in primary immunodeficiency genes in patients with very early onset inflammatory bowel disease.

BACKGROUND & AIMS: Very early onset inflammatory bowel disease (VEO-IBD), IBD diagnosed at 5 years of age or younger, frequently presents with a different and more severe phenotype than older-onset IBD. We investigated whether patients with VEO-IBD carry rare or novel variants in genes associated with immunodeficiencies that might contribute to disease development. METHODS: Patients with VEO-IBD and parents (when available) were recruited from the Children's Hospital of Philadelphia from March 2013 through July 2014. We analyzed DNA from 125 patients with VEO-IBD (age, 3 wk to 4 y) and 19 parents, 4 of whom also had IBD. Exome capture was performed by Agilent SureSelect V4, and sequencing was performed using the Illumina HiSeq platform. Alignment to human genome GRCh37 was achieved followed by postprocessing and variant calling. After functional annotation, candidate variants were analyzed for change in protein function, minor allele frequency less than 0.1%, and scaled combined annotation-dependent depletion scores of 10 or less. We focused on genes associated with primary immunodeficiencies and related pathways. An additional 210 exome samples from patients with pediatric IBD (n = 45) or adult-onset Crohn's disease (n = 20) and healthy individuals (controls, n = 145) were obtained from the University of Kiel, Germany, and used as control groups. RESULTS: Four hundred genes and regions associated with primary immunodeficiency, covering approximately 6500 coding exons totaling more than 1 Mbp of coding sequence, were selected from the whole-exome data. Our analysis showed novel and rare variants within these genes that could contribute to the development of VEO-IBD, including rare heterozygous missense variants in IL10RA and previously unidentified variants in MSH5 and CD19. CONCLUSIONS: In an exome sequence analysis of patients with VEO-IBD and their parents, we identified variants in genes that regulate B- and T-cell functions and could contribute to pathogenesis. Our analysis could lead to the identification of previously unidentified IBD-associated variants.

Polygenic inheritance of cryptorchidism susceptibility in the LE/orl rat.

STUDY HYPOTHESIS: Susceptibility to inherited cryptorchidism in the LE/orl rat may be associated with genetic loci that influence developmental patterning of the gubernaculum by the fetal testis. STUDY FINDING: Cryptorchidism in the LE/orl rat is associated with a unique combination of homozygous minor alleles at multiple loci, and the encoded proteins are co-localized with androgen receptor (AR) and Leydig cells in fetal gubernaculum and testis, respectively. WHAT IS KNOWN ALREADY: Prior studies have shown aberrant perinatal gubernacular migration, muscle patterning defects and reduced fetal testicular testosterone in the LE/orl strain. In addition, altered expression of androgen-responsive, cytoskeletal and muscle-related transcripts in the LE/orl fetal gubernaculum suggest a role for defective AR signaling in cryptorchidism susceptibility. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: The long-term LE/orl colony and short-term colonies of outbred Crl:LE and Crl:SD, and inbred WKY/Ncrl rats were maintained for studies. Animals were intercrossed (LE/orl X WKY/Ncrl), and obligate heterozygotes were reciprocally backcrossed to LE/orl rats to generate 54 F2 males used for genotyping and/or linkage analysis. At least five fetuses per gestational time point from two or more litters were used for quantitative real-time RT-PCR (qRT-PCR) and freshly harvested embryonic (E) day 17 gubernaculum was used to generate conditionally immortalized cell lines. We completed genotyping and gene expression analyses using genome-wide microsatellite markers and single nucleotide polymorphism (SNP) arrays, PCR amplification, direct sequencing, restriction enzyme digest with fragment analysis, whole genome sequencing (WGS), and qRT-PCR. Linkage analysis was performed in Haploview with multiple testing correction, and qRT-PCR data were analyzed using ANOVA after log transformation. Imaging was performed using custom and commercial antibodies directed at candidate proteins in gubernaculum and testis tissues, and gubernaculum cell lines. MAIN RESULTS AND THE ROLE OF CHANCE: LE/orl rats showed reduced fertility and fecundity, and higher risk of perinatal death as compared with Crl:LE rats, but there were no differences in breeding outcomes between normal and unilaterally cryptorchid males. Linkage analysis identified multiple peaks, and with selective breeding of outbred Crl:LE and Crl:SD strains for alleles within two of the most significant (P < 0.003) peaks on chromosomes 6 and 16, we were able to generate a non-LE/orl cryptorchid rat. Associated loci contain potentially functional minor alleles (0.25-0.36 in tested rat strains) including an exonic deletion in Syne2, a large intronic insertion in Ncoa4 (an AR coactivator) and potentially deleterious variants in Solh/Capn15, Ankrd28, and Hsd17b2. Existing WGS data indicate that homozygosity for these combined alleles does not occur in any other sequenced rat strain. We observed a modifying effect of the Syne2(del) allele on expression of other candidate genes, particularly Ncoa4, and for muscle and hormone-responsive transcripts. The selected candidate genes/proteins are highly expressed, androgen-responsive and/or co-localized with developing muscle and AR in fetal gubernaculum, and co-localized with Leydig cells in fetal testis. LIMITATIONS, REASONS FOR CAUTION: The present study identified multiple cryptorchidism-associated linkage peaks in the LE/orl rat, containing potentially causal alleles. These are strong candidate susceptibility loci, but further studies are needed to demonstrate functional relevance to the phenotype. WIDER IMPLICATIONS OF THE FINDINGS: Association data from both human and rat models of spontaneous, nonsyndromic cryptorchidism support a polygenic etiology of the disease. Both the present study and a human genome-wide association study suggest that common variants with weak effects contribute to susceptibility, and may exist in genes encoding proteins that participate in AR signaling in the developing gubernaculum. These findings have potential implications for the gene-environment interaction in the etiology of cryptorchidism. LARGE SCALE DATA: Sequences were deposited in the Rat Genome Database (RGD, http://rgd.mcw.edu/). STUDY FUNDING AND COMPETING INTERESTS: This work was supported by: R01HD060769 from the Eunice Kennedy Shriver National Institute for Child Health and Human Development (NICHD), 2P20GM103446 and P20GM103464 from the National Institute of General Medical Sciences (NIGMS), and Nemours Biomedical Research. The authors have no competing interests to declare.

Compound heterozygous mutations in NEK8 in siblings with end-stage renal disease with hepatic and cardiac anomalies.

We studied two brothers who presented in the newborn period with cardiac, renal, and hepatic anomalies that were initially suggestive of ALGS, although no mutations in JAG1 or NOTCH2 were identified. Exome sequencing demonstrated compound heterozygous mutations in the NEK8 gene (Never in mitosis A-related Kinase 8), a ciliary kinase indispensable for cardiac and renal development based on murine studies. The mutations included a c.2069_2070insC variant (p.Ter693LeufsTer86), and a c.1043C>T variant (p.Thr348Met) in the highly conserved RCC1 (Regulation of Chromosome Condensation 1) domain. The RCC1 domain is crucial for localization of the NEK8 protein to the centrosomes and cilia. Mutations in NEK8 have been previously reported in three fetuses (from a single family) with renal-hepatic-pancreatic dysplasia 2 (RHPD2), similar to Ivemark syndrome, and in three individuals with nephronophthisis (NPHP9). This is the third report of disease-causing mutations in the NEK8 gene in humans and only the second describing multi-organ involvement. The clinical features we describe differ from those in the previously published report in that (1) a pancreatic phenotype was not observed in the individuals reported here, (2) there were more prominent cardiac findings, (consistent with observations in murine models), and (3) we observed bile duct hypoplasia rather than ductal plate malformation. The patients reported here expand our understanding of the NEK8-associated phenotype. Our findings highlight the variable phenotypic expressivity and the spectrum of clinical manifestations due to mutations in the NEK8 gene.

Genomic copy number variation association study in Caucasian patients with nonsyndromic cryptorchidism.

BACKGROUND: Copy number variation (CNV) is a potential contributing factor to many genetic diseases. Here we investigated the potential association of CNV with nonsyndromic cryptorchidism, the most common male congenital genitourinary defect, in a Caucasian population. METHODS: Genome wide genotyping were performed in 559 cases and 1772 controls (Group 1) using Illumina HumanHap550 v1, HumanHap550 v3 or Human610-Quad platforms and in 353 cases and 1149 controls (Group 2) using the Illumina Human OmniExpress 12v1 or Human OmniExpress 12v1-1. Signal intensity data including log R ratio (LRR) and B allele frequency (BAF) for each single nucleotide polymorphism (SNP) were used for CNV detection using PennCNV software. After sample quality control, gene- and CNV-based association tests were performed using cleaned data from Group 1 (493 cases and 1586 controls) and Group 2 (307 cases and 1102 controls) using ParseCNV software. Meta-analysis was performed using gene-based test results as input to identify significant genes, and CNVs in or around significant genes were identified in CNV-based association test results. Called CNVs passing quality control and signal intensity visualization examination were considered for validation using TaqMan CNV assays and QuantStudio® 3D Digital PCR System. RESULTS: The meta-analysis identified 373 genome wide significant (p < 5X10-4) genes/loci including 49 genes/loci with deletions and 324 with duplications. Among them, 17 genes with deletion and 1 gene with duplication were identified in CNV-based association results in both Group 1 and Group 2. Only 2 genes (NUCB2 and UPF2) containing deletions passed CNV quality control in both groups and signal intensity visualization examination, but laboratory validation failed to verify these deletions. CONCLUSIONS: Our data do not support that structural variation is a major cause of nonsyndromic cryptorchidism.

IQ and hemizygosity for the Val158 Met functional polymorphism of COMT in 22q11DS.

22q11.2 Deletion Syndrome (22q11DS) is a multisystem disorder caused by a hemizygous deletion within 22q11.2. Patients with the deletion display a wide range of cognitive deficits. The gene catechol-O-methyl-transferase (COMT) resides in the typically deleted region of 22q11.2 and is rendered hemizygous in individuals affected by the 22q11DS. COMT is a critical enzyme in the degradation of catecholamine neurotransmitters in the brain. A functional polymorphism, Val158 Met, has been associated with a variety of neurocognitive outcomes. In this study, 159 patients with 22q11DS were analyzed for a potential association between intelligence quotient (IQ) and COMT genotype. We performed a univariate analysis for overall influence and modified our analysis to focus on possible differences between average, borderline, and intellectually impaired patients. No correlation between COMT genotype and IQ performance was found. © 2016 Wiley Periodicals, Inc.