LHCGR and ALMS1 defects likely cooperate in the development of polycystic ovary syndrome indicated by double-mutant mice.

Polycystic ovary syndrome (PCOS) is a heterogeneous disorder with evidence of polygenetic components, and obesity may be a risk factor for hyperandrogenism. Previous studies have shown that LHCGR is enriched in the ovary and LHCGR deficiency causes infertility without typical PCOS phenotypes. ALMS1 is implicated in obesity and hyperandrogenism, the common phenotypes among PCOS patients. Through whole-exome sequencing of 22 PCOS families and targeted candidate gene sequencing of additional 65 sporadic PCOS patients, we identified potential causative mutations in LHCGR and ALMS1 in a sibling-pair PCOS family and three sporadic PCOS patients. The expression of LHCGRL638P in granulosa-like tumor cell line (KGN) cells promoted cyclic adenosine monophosphate production and granulosa cell proliferation, indicating that LHCGRL638P is an activating mutation. LhcgrL642P/L642P mice showed an irregular estrous cycle, reduced follicles with dynamic folliculogenesis, and increased testosterone (T), estradiol (E2), and dehydroepiandrosterone. Lhcgr+/L642PAlms1+/PB mice displayed increased T and E2 but decreased late secondary and preovulatory follicles. We showed that activating mutation of LHCGR likely plays important roles in the pathophysiology of PCOS involving abnormal reproductive physiology, whereas ALMS1 deficiency may promote anovulatory infertility via elevated androgens, suggesting that the disturbed LHCGR and ALMS1 cooperatively induce PCOS phenotypes, characterized as anovulation and hyperandrogenemia frequently observed in PCOS patients with obesity.

Rare Deleterious PARD3 Variants in the aPKC-Binding Region are Implicated in the Pathogenesis of Human Cranial Neural Tube Defects Via Disrupting Apical Tight Junction Formation.

Increasing evidence that mutation of planar cell polarity (PCP) genes contributes to human cranial neural tube defect (NTD) susceptibility prompted us to hypothesize that rare variants of genes in the core apical-basal polarity (ABP) pathway are risk factors for cranial NTDs. In this study, we screened for rare genomic variation of PARD3 in 138 cranial NTD cases and 274 controls. Overall, the rare deleterious variants of PARD3 were significantly associated with increased risk for cranial NTDs (11/138 vs.7/274, P < 0.05, OR = 3.3). These NTD-specific variants were significantly enriched in the aPKC-binding region (6/138 vs. 0/274, P < 0.01). The East Asian cohort in the ExAC database and another Chinese normal cohort further supported this association. Over-expression analysis in HEK293T and MDCK cells confirmed abnormal aPKC binding or interaction for two PARD3 variants (p.P913Q and p.D783G), resulting in defective tight junction formation via disrupted aPKC binding. Functional analysis in human neural progenitor cells and chick embryos revealed that PARD3 knockdown gave rise to abnormal cell polarity and compromised the polarization process of neuroepithelial tissue. Our studies suggest that rare deleterious variants of PARD3 in the aPKC-binding region contribute to human cranial NTDs, possibly by disrupting apical tight junction formation and subsequent polarization process of the neuroepithelium.

Extracellular Cl(-) regulates human SO4 (2-)/anion exchanger SLC26A1 by altering pH sensitivity of anion transport.

Genetic deficiency of the SLC26A1 anion exchanger in mice is known to be associated with hyposulfatemia and hyperoxaluria with nephrolithiasis, but many aspects of human SLC26A1 function remain to be explored. We report here the functional characterization of human SLC26A1, a 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS)-sensitive, electroneutral sodium-independent anion exchanger transporting sulfate, oxalate, bicarbonate, thiosulfate, and (with divergent properties) chloride. Human SLC26A1-mediated anion exchange differs from that of its rodent orthologs in its stimulation by alkaline pHo and inhibition by acidic pHo but not pHi and in its failure to transport glyoxylate. SLC26A1-mediated transport of sulfate and oxalate is highly dependent on allosteric activation by extracellular chloride or non-substrate anions. Extracellular chloride stimulates apparent V max of human SLC26A1-mediated sulfate uptake by conferring a 2-log decrease in sensitivity to inhibition by extracellular protons, without changing transporter affinity for extracellular sulfate. In contrast to SLC26A1-mediated sulfate transport, SLC26A1-associated chloride transport is activated by acid pHo, shows reduced sensitivity to DIDS, and exhibits cation dependence of its DIDS-insensitive component. Human SLC26A1 resembles SLC26 paralogs in its inhibition by phorbol ester activation of protein kinase C (PKC), which differs in its undiminished polypeptide abundance at or near the oocyte surface. Mutation of SLC26A1 residues corresponding to candidate anion binding site-associated residues in avian SLC26A5/prestin altered anion transport in patterns resembling those of prestin. However, rare SLC26A1 polymorphic variants from a patient with renal Fanconi Syndrome and from a patient with nephrolithiasis/calcinosis exhibited no loss-of-function phenotypes consistent with disease pathogenesis.

Congenital chloride-losing diarrhea in a Mexican child with the novel homozygous SLC26A3 mutation G393W.

Congenital chloride diarrhea is an autosomal recessive disease caused by mutations in the intestinal lumenal membrane Cl(-)/HCO(-) 3 exchanger, SLC26A3. We report here the novel SLC26A3 mutation G393W in a Mexican child, the first such report in a patient from Central America. SLC26A3 G393W expression in Xenopus oocytes exhibits a mild hypomorphic phenotype, with normal surface expression and moderately reduced anion transport function. However, expression of HA-SLC26A3 in HEK-293 cells reveals intracellular retention and greatly decreased steady-state levels of the mutant polypeptide, in contrast to peripheral membrane expression of the wildtype protein. Whereas wildtype HA-SLC26A3 is apically localized in polarized monolayers of filter-grown MDCK cells and Caco2 cells, mutant HA-SLC26A3 G393W exhibits decreased total polypeptide abundance, with reduced or absent surface expression and sparse punctate (or absent) intracellular distribution. The WT protein is similarly localized in LLC-PK1 cells, but the mutant fails to accumulate to detectable levels. We conclude that the chloride-losing diarrhea phenotype associated with homozygous expression of SLC26A3 G393W likely reflects lack of apical surface expression in enterocytes, secondary to combined abnormalities in polypeptide trafficking and stability. Future progress in development of general or target-specific folding chaperonins and correctors may hold promise for pharmacological rescue of this and similar genetic defects in membrane protein targeting.

Somatic mutations in cerebral cortical malformations.

BACKGROUND: Although there is increasing recognition of the role of somatic mutations in genetic disorders, the prevalence of somatic mutations in neurodevelopmental disease and the optimal techniques to detect somatic mosaicism have not been systematically evaluated. METHODS: Using a customized panel of known and candidate genes associated with brain malformations, we applied targeted high-coverage sequencing (depth, ≥200×) to leukocyte-derived DNA samples from 158 persons with brain malformations, including the double-cortex syndrome (subcortical band heterotopia, 30 persons), polymicrogyria with megalencephaly (20), periventricular nodular heterotopia (61), and pachygyria (47). We validated candidate mutations with the use of Sanger sequencing and, for variants present at unequal read depths, subcloning followed by colony sequencing. RESULTS: Validated, causal mutations were found in 27 persons (17%; range, 10 to 30% for each phenotype). Mutations were somatic in 8 of the 27 (30%), predominantly in persons with the double-cortex syndrome (in whom we found mutations in DCX and LIS1), persons with periventricular nodular heterotopia (FLNA), and persons with pachygyria (TUBB2B). Of the somatic mutations we detected, 5 (63%) were undetectable with the use of traditional Sanger sequencing but were validated through subcloning and subsequent sequencing of the subcloned DNA. We found potentially causal mutations in the candidate genes DYNC1H1, KIF5C, and other kinesin genes in persons with pachygyria. CONCLUSIONS: Targeted sequencing was found to be useful for detecting somatic mutations in patients with brain malformations. High-coverage sequencing panels provide an important complement to whole-exome and whole-genome sequencing in the evaluation of somatic mutations in neuropsychiatric disease. (Funded by the National Institute of Neurological Disorders and Stroke and others.).

Detection of copy number variants reveals association of cilia genes with neural tube defects.

BACKGROUND: Neural tube defects (NTDs) are one of the most common birth defects caused by a combination of genetic and environmental factors. Currently, little is known about the genetic basis of NTDs although up to 70% of human NTDs were reported to be attributed to genetic factors. Here we performed genome-wide copy number variants (CNVs) detection in a cohort of Chinese NTD patients in order to exam the potential role of CNVs in the pathogenesis of NTDs. METHODS: The genomic DNA from eighty-five NTD cases and seventy-five matched normal controls were subjected for whole genome CNVs analysis. Non-DGV (the Database of Genomic Variants) CNVs from each group were further analyzed for their associations with NTDs. Gene content in non-DGV CNVs as well as participating pathways were examined. RESULTS: Fifty-five and twenty-six non-DGV CNVs were detected in cases and controls respectively. Among them, forty and nineteen CNVs involve genes (genic CNV). Significantly more non-DGV CNVs and non-DGV genic CNVs were detected in NTD patients than in control (41.2% vs. 25.3%, p<0.05 and 37.6% vs. 20%, p<0.05). Non-DGV genic CNVs are associated with a 2.65-fold increased risk for NTDs (95% CI: 1.24-5.87). Interestingly, there are 41 cilia genes involved in non-DGV CNVs from NTD patients which is significantly enriched in cases compared with that in controls (24.7% vs. 9.3%, p<0.05), corresponding with a 3.19-fold increased risk for NTDs (95% CI: 1.27-8.01). Pathway analyses further suggested that two ciliogenesis pathways, tight junction and protein kinase A signaling, are top canonical pathways implicated in NTD-specific CNVs, and these two novel pathways interact with known NTD pathways. CONCLUSIONS: Evidence from the genome-wide CNV study suggests that genic CNVs, particularly ciliogenic CNVs are associated with NTDs and two ciliogenesis pathways, tight junction and protein kinase A signaling, are potential pathways involved in NTD pathogenesis.

Homozygous PLCB1 deletion associated with malignant migrating partial seizures in infancy.

Malignant migrating partial seizures in infancy (MMPEI) is an early onset epileptic encephalopathy with few known etiologies. We sought to identify a novel cause of MMPEI in a child with MMPEI whose healthy parents were consanguineous. We used array comparative genomic hybridization (CGH) to identify copy number variants genome-wide and long-range polymerase chain reaction to further delineate the breakpoints of a deletion found by CGH. The proband had an inherited homozygous deletion of chromosome 20p13, disrupting the promoter region and first three coding exons of the gene PLCB1. Additional MMPEI cases were screened for similar deletions or mutations in PLCB1 but did not harbor mutations. Our results suggest that loss of PLCß1 function is one cause of MMPEI, consistent with prior studies in a Plcb1 knockout mouse model that develops early onset epilepsy. We provide novel insight into the molecular mechanisms underlying MMPEI and further implicate PLCB1 as a candidate gene for severe childhood epilepsies. This work highlights the importance of pursuing genetic etiologies for severe early onset epilepsy syndromes.

Identification of novel rare mutations of DACT1 in human neural tube defects.

Neural tube defects (NTDs) constitute the second most frequent cause of human congenital abnormalities. Complex multigenetic causes have been suggested to contribute to NTDs. The planar cell polarity (PCP) pathway plays a critical role in neural tube closure in model organisms and in human. Knockout of Dact1 (Dapper, Frodo) leads to deregulated PCP signaling with defective neural tube in mice. Here, we report that five missense heterozygote mutations of the DACT1 gene are specifically identified in 167 stillborn or miscarried Han Chinese fetuses with neural tube defects. Our biochemical analyses revealed that among the five mutations, N356K and R45W show loss-of-function or reduced activities in inducing Dishevelled2 (DVL2) degradation and inhibiting jun-N-terminal kinase (JNK) phosphorylation, implicating mutated DACT1 as a risk factor for human NTDs. Our findings, together with early reports, suggest that rare mutations of the PCP-related genes may constitute a great contribution to human NTDs.

Global DNA hypomethylation is associated with NTD-affected pregnancy: A case-control study.

BACKGROUND: Neural tube defects are severe, common birth defects that result from failure of neural tube closure. They are considered to be a multifactorial disorder, and our knowledge of causal mechanisms remains limited. We hypothesized that abnormal DNA methylation occurs in NTD-affected fetuses. The correlations of global DNA methylation levels with complexity of NTDs and known risk factors of NTDs, MTHFR genotype and fever, were analyzed. METHODS: A hospital-based case-control study was performed. Epidemiologic data, pathologic diagnosis, and methylenetetrahydrofolate reductase (MTHFR) genotype analysis were completed. Array comparative genomic hybridization was used to exclude cytogenetic abnormalities. Global DNA methylation statuses were determined for both brain and skin tissue. RESULTS: Sixty-five NTD-affected fetuses and 65 normal controls matched for gestational and maternal ages were collected. In brain tissue, global DNA methylation levels were significantly decreased in cases compared with controls (4.12 vs. 4.99%; p < 0.001). DNA hypomethylation (<4.35%) resulted in a significant 5.736-fold increased risk for NTDs (95% confidence interval, 1.731-19.009; p = 0.004). Nonisolated NTDs had lower levels of global DNA methylation than did isolated NTDs (3.77 vs. 4.70%; p = 0.022). After stratifying subjects by MTHFR genotype, we observed a skewed distribution of global DNA methylation levels. For genotype C/C, global DNA methylation status was the same in the two groups (4.51 vs. 4.72%; p = 0.687). For T/T, cases had significantly lower global methylation levels than did controls (5.23 vs. 3.79%; p < 0.001). CONCLUSIONS: Global DNA hypomethylation in fetal brain tissue was associated with NTD-affected pregnancy. DNA methylation levels were correlated with NTD complexity. The MTHFR genotype contributed to global DNA hypomethylation. Birth Defects Research (Part A), 2010. (c) 2010 Wiley-Liss, Inc.

Audiologic phenotype and progression in GJB2 (Connexin 26) hearing loss.

OBJECTIVES: To document the audiologic phenotype of children with biallelic GJB2 (connexin 26) mutations, and to correlate it with the genotype. DESIGN: Prospective, observational study. SETTING: Tertiary care children's hospital. PATIENTS: Infants and children with sensorineural hearing loss (SNHL). INTERVENTION: Sequencing of the GJB2 (connexin 26) gene. MAIN OUTCOME MEASURES: Degree and progression of SNHL. RESULTS: From December 1, 1998, through November 30, 2006, 126 children with biallelic GJB2 mutations were identified. Of the 30 different mutations identified, 13 (43%) were truncating and 17 (57%) were nontruncating; 62 patients had 2 truncating, 30 had 1 truncating and 1 nontruncating, and 17 had 2 nontruncating mutations. Eighty-four patients (67%) initially had measurable hearing in the mild to severe range in at least 1 of 4 frequencies (500, 1000, 2000, or 4000 Hz). Of these 84 patients with residual hearing, 47 (56%) had some degree of progressive hearing loss. Patients with 2 truncating mutations had significantly worse hearing compared with all other groups. Patients who had 1 or 2 copies of either an M34T or a V37I allele had the mildest hearing loss. CONCLUSIONS: Hearing loss owing to GJB2 mutations ranges from mild to profound and is usually congenital. More than 50% of patients will experience some hearing loss progression, generally gradually but occasionally precipitously. Hearing loss severity may be influenced by genetic factors, such as the degree of preserved protein function in nontruncating mutations, whereas hearing loss progression may be dependent on factors other than the connexin 26 protein. Genetic counseling for patients with GJB2 mutations should include the variable audiologic phenotype and the possibility of progression.

Distinct and novel SLC26A4/Pendrin mutations in Chinese and U.S. patients with nonsyndromic hearing loss.

Mutations of the human SLC26A4/PDS gene constitute the most common cause of syndromic and nonsyndromic hearing loss. Definition of the SLC26A4 mutation spectrum among different populations with sensorineural hearing loss is important for development of optimal genetic screening services for congenital hearing impairment. We screened for SLC26A4 mutations among Chinese and U.S. subjects with hearing loss, using denaturing HPLC (DHPLC) and direct DNA sequencing. Fifty-two of 55 Chinese subjects with deafness accompanied by enlargement of the vestibular aqueduct (EVA) exhibited at least one mutant SLC26A4 allele, whereas SLC26A4 mutations were found in only 2 of 116 deaf Chinese patients without EVA. The spectrum of SLC26A4 mutations differed among Chinese and U.S. subjects and included 10 previously unreported SLC26A4 variants: 4 in the Chinese population (p.E303Q, p.X329, p.X467, p.X573) and 6 in the U.S. population (p.V250A, p.D266N, p.F354S, p.D697A, p.K715N, p.E737D). Among the seven novel in-frame missense mutations, five encoded SLC26A4 proteins with substantially reduced Cl(-)/anion exchange activity as expressed and measured in Xenopus oocytes, but four of these were sufficiently active to allow study of anion selectivity. The only mutant polypeptide exhibiting complete loss of anion exchange function, p.E303Q, was expressed at or near the oocyte surface at near-wild-type levels. Two variants, p.F354S and p.E737D, displayed selective reduction in relative rate of Cl(-)/HCO(3)(-) exchange compared with similarly measured rates of Cl(-)/Cl(-) and Cl(-)/I(-) exchange. Our data show that mutation analysis of the SLC26A4 gene is of high diagnostic yield among subjects with deafness and bilateral EVA in both China and the U.S. However, the pathogenicity of monoallelic SLC26A4 gene variants in patients with hearing loss remains unclear in many instances.

[Diagnostic methods and clinic application for mtDNA A1555G and GJB2 and SLC26A4 genes in deaf patients].

OBJECTIVE: To establish the method of clinic genetic testing for common deaf genes such as mtDNA nt1555, GJB2 gene and SLC26A4 (Pendrin's syndrome gene, PDS) gene. METHODS: Three hundred and sixty seven sporadic patients with hearing loss from out-patient department of General Hospital of Chinese People's Liberation Army, 60 patients with history of maternal inherited hearing loss from 27 family, 20 congenital deaf patients from special educational school for deaf and dumb, 3 deaf patients with enlarged vestibular aqueduct (EVA) confirmed by CT scan, 50 control individuals with normal bone conductive hearing were analyzed. The genetic testing kit for mtDNA A1555G mutation was used to detect mtDNA A1555G mutation. The whole gene sequencing were accomplished in 20 congenital deaf patients. In 3 patients with EVA, fragments covering all exons of PDS gene were analyzed by denatured high productive liquid chromatogram and special exons were sequenced when DHPLC showed abnormal wave patterns of amplicons covering these exons. RESULTS: Fifty nine patients from 26 family and 5 sporadic patients were found to carry mtDNA A1555G mutation. Among 20 congenital deaf patients, 2 cases were found to have homozygous GJB2 235 del C mutation, 1 case had compound 235del C and 299-300 del AT mutation. Other 2 cases carried heterozygous 109 A-G mutation. Among 3 patients with EVA, 1 case was found to have heterozygous PDS G316X mutation and other 2 cases had homozygous 919-2 A-G mutation. CONCLUSIONS Genetic testing for deafness is feasible procedure with remarkable clinic significance.

High intensity ras signaling induces premature senescence by activating p38 pathway in primary human fibroblasts.

Although oncogenic ras plays a pivotal role in neoplastic transformation, it triggers an anti-oncogenic defense mechanism known as premature senescence in normal cells. In this study, we investigated the induction of cellular responses by different expression levels of oncogenic ras in primary human fibroblasts. We found that a moderate, severalfold increase in ras expression promoted cell growth. Further elevation of ras expression initially enhanced proliferation but eventually induced p16INK4A expression and senescence. The induction of these opposing cellular responses by ras signals of different intensity was achieved through differential activation of the MAPK pathways that mediated these responses. Whereas moderate ras activities only stimulated the mitogenic MEK-ERK pathway, high intensity ras signals induced MEK and ERK to higher levels, leading to stimulation of the MKK3/6-p38 pathway, which had been shown previously to act downstream of Ras-MEK to trigger the senescence response. Thus, these studies have revealed a mechanism for the differential effects of ras on cell proliferation. Furthermore, moderate ras activity mediated transformation in cooperation with E6E7 and hTERT, suggesting that a moderate intensity ras signal can provide sufficient oncogenic activities for tumorigenesis. This result also implies that the ability of ras to promote proliferation and oncogenic transformation can be uncoupled with that to induce senescence in cell culture and that the development of tumors with relatively low ras activities may not need to acquire genetic alterations that bypass premature senescence.