Novel mutations in VANGL1 in neural tube defects

Neural tube defects (NTDs) are severe congenital malformations caused by failure of the neural tube to close during neurulation. Their etiology is complex involving both environmental and genetic factors. We have recently reported three mutations in the planar cell polarity gene VANGL1 associated with NTDs. The aim of the present study was to define the role of VANGL1 genetic variants in the development of NTDs in a large cohort of various ethnic origins. We identified five novel missense variants in VANGL1, p.Ser83Leu, p.Phe153Ser, p.Arg181Gln, p.Leu202Phe and p.Ala404Ser, occurring in sporadic and familial cases of spinal dysraphisms. All five variants affect evolutionary conserved residues and are absent from all controls analyzed. This study provides further evidence supporting the role of VANGL1 as a risk factor in the development of spinal NTDs. © 2009 Wiley‐Liss, Inc.     

Contribution of VANGL2 mutations to isolated neural tube defects

Kibar Z, Salem S, Bosoi CM, Pauwels E, De Marco P, Merello E, Bassuk AG, Capra V, Gros P. Contribution of VANGL2 mutations to isolated neural tube defects. Vangl2 was identified as the gene defective in the Looptail (Lp) mouse model for neural tube defects (NTDs). This gene forms part of the planar cell polarity (PCP) pathway, also called the non‐canonical Frizzled/Dishevelled pathway, which mediates the morphogenetic process of convergent extension essential for proper gastrulation and neural tube formation in vertebrates. Genetic defects in PCP signaling have strongly been associated with NTDs in mouse models. To assess the role of VANGL2 in the complex etiology of NTDs in humans, we resequenced this gene in a large multi‐ethnic cohort of 673 familial and sporadic NTD patients, including 453 open spina bifida and 202 closed spinal NTD cases. Six novel rare missense mutations were identified in seven patients, five of which were affected with closed spinal NTDs. This suggests that VANGL2 mutations may predispose to NTDs in approximately 2.5% of closed spinal NTDs (5 in 202), at a frequency that is significantly different from that of 0.4% (2 in 453) detected in open spina bifida patients (p = 0.027). Our findings strongly implicate VANGL2 in the genetic causation of spinal NTDs in a subset of patients and provide additional evidence for a pathogenic role of PCP signaling in these malformations.     

Loss of RAD9B impairs early neural development and contributes to the risk for human spina bifida

DNA damage response (DDR) genes orchestrating the network of DNA repair, cell cycle control, are essential for the rapid proliferation of neural progenitor cells. To date, the potential association between specific DDR genes and the risk of human neural tube defects (NTDs) has not been investigated. Using whole‐genome sequencing and targeted sequencing, we identified significant enrichment of rare deleterious RAD9B variants in spina bifida cases compared to controls (8/409 vs. 0/298; p = .0241). Among the eight identified variants, the two frameshift mutants and p.Gln146Glu affected RAD9B nuclear localization. The two frameshift mutants also decreased the protein level of RAD9B. p.Ser354Gly, as well as the two frameshifts, affected the cell proliferation rate. Finally, p.Ser354Gly, p.Ser10Gly, p.Ile112Met, p.Gln146Glu, and the two frameshift variants showed a decreased ability for activating JNK phosphorylation. RAD9B knockdowns in human embryonic stem cells profoundly affected early differentiation through impairing PAX6 and OCT4 expression. RAD9B deficiency impeded in vitro formation of neural organoids, a 3D cell culture model for human neural development. Furthermore, the RNA‐seq data revealed that loss of RAD9B dysregulates cell adhesion genes during organoid formation. These results represent the first demonstration of a DDR gene as an NTD risk factor in humans.     

Functional,structural, and genetic evaluation of 20 CDKN2A germ line mutations identified in melanoma‐prone families or patients

Germline mutations of the CDKN2A gene are found in melanoma‐prone families and individuals with multiple sporadic melanomas. The encoded protein, p16^INK4A, comprises four ankyrin‐type repeats, and the mutations, most of which are missense and occur throughout the entire coding region, can disrupt the conformation of these structural motifs as well as the association of p16^INK4a with its physiological targets, the cyclin‐dependent kinases (CDKs) CDK4 and CDK6. Assessing pathogenicity of nonsynonymous mutations is critical to evaluate melanoma risk in carriers. In the current study, we investigate 20 CDKN2A germline mutations whose effects on p16^INK4A structure and function have not been previously documented (Thr18_Ala19dup, Gly23Asp, Arg24Gln, Gly35Ala, Gly35Val, Ala57Val, Ala60Val, Ala60Arg, Leu65dup, Gly67Arg, Gly67_Asn71del, Glu69Gly, Asp74Tyr, Thr77Pro, Arg80Pro, Pro81Thr, Arg87Trp, Leu97Arg, Arg99Pro, and [Leu113Leu;Pro114Ser]). By considering genetic information, the predicted impact of each variant on the protein structure, its ability to interact with CDK4 and impede cell proliferation in experimental settings, we conclude that 18 of the 20 CDKN2A variants can be classed as loss of function mutations, whereas the results for two remain ambiguous. Discriminating between mutant and neutral variants of p16^INK4A not only adds to our understanding of the functionally critical residues in the protein but provides information that can be used for melanoma risk prediction. Hum Mutat 0, 1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Genotype‐specific progression of hereditary medullary thyroid cancer

Although already 25 years into the genomic era, age‐related progression of hereditary medullary thyroid cancer (MTC), the prevalence of which is estimated at one in 80,000 inhabitants, remains to be delineated for most unique RET (REarranged during Transfection) mutations. Included in this study were 567 RET carriers. The age‐related progression of MTC across histopathological groups (normal thyroid/C‐cell hyperplasia; node‐negative MTC; node‐positive MTC) was statistically significant for 13 unique RET mutations (p.Cys611Phe/c.1832G > T; p.Cys611Tyr; p.Cys618Ser/c.1852T > A; p.Cys620Arg; p.Cys634Arg; p.Cys634Phe; p.Cys634Ser; p.Cys634Tyr; p.Glu768Asp; p.Leu790Phe/c.2370G > T; p.Val804Met; p.Ser891Ala; p.Met918Thr), whereas two unique RET mutations (p.Cys618Phe; p.Cys634Gly) trended toward statistical significance. When grouped by mutational risk (highest; high; moderate – high; low – moderate; polymorphism), the age‐related progression of MTC was significant for all four categories of RET mutations, which differed significantly across and within the three histopathological groups. For high, for moderate–high, and for low–moderate risk RET mutations, the age‐related progression of MTC by mutated codon was broadly comparable across and within the three histopathological groups, and essentially unaffected by the amino acid substitutions examined. These data argue in favor of splitting the American Thyroid Association's moderate‐risk category into moderate–high and low–moderate risk categories, while emphasizing the need to contradistinguish the latter from rare nonpathogenic polymorphisms.     

Identification of seven novel SMPD1 mutations causing Niemann–Pick disease types A and B

Niemann–Pick disease (NPD) types A and B are autosomal, recessively inherited, lysosomal storage disorders caused by deficient activity of acid sphingomyelinase (E.C. 3.1.4.12) because of mutations in the sphingomyelin phosphodiesterase‐1 (SMPD1) gene. Here, we present the molecular analysis and clinical characteristics of 15 NPD type A and B patients. Sequencing the SMDP1 gene revealed eight previously described mutations and seven novel mutations including four missense [c.682T>C (p.Cys228Arg), c.1159T>C (p.Cys387Arg), c.1474G>A (p.Gly492Ser), and c.1795C>T (p.Leu599Phe)], one frameshift [c.169delG (p.Ala57Leufs*20)] and two splicing (c.316+1G>T and c.1341delG). The most frequent mutations were p.Arg610del (21%) and p.Gly247Ser (12%). Two patients homozygous for p.Arg610del and initially classified as phenotype B showed different clinical manifestations. Patients homozygous for p.Leu599Phe had phenotype B, and those homozygous for c.1341delG or c.316+1G>T presented phenotype A. The present results provide new insight into genotype/phenotype correlations in NPD and emphasize the difficulty of classifying patients into types A and B, supporting the idea of a continuum between these two classic phenotypes.     

Insight into vitamin B6‐dependent epilepsy due to PLPBP (previously PROSC) missense mutations

Vitamin B₆‐dependent genetic epilepsy was recently associated to mutations in PLPBP (previously PROSC), the human version of the widespread COG0325 gene that encodes TIM‐barrel‐like pyridoxal phosphate (PLP)‐containing proteins of unclear function. We produced recombinantly, purified and characterized human PROSC (called now PLPHP) and its six missense mutants reported in epileptic patients. Normal PLPHP is largely a monomer with PLP bound through a Schiff‐base linkage. The PLP‐targeting antibiotic d ‐cycloserine decreased the PLP‐bound peak as expected for pseudo‐first‐order reaction. The p.Leu175Pro mutation grossly misfolded PLPHP. Mutations p.Arg241Gln and p.Pro87Leu decreased protein solubility and yield of pure PLPHP, but their pure forms were well folded, similarly to pure p.Pro40Leu, p.Tyr69Cys, and p.Arg205Gln mutants (judged from CD spectra). PLPHP stability was decreased in p.Arg241Gln, p.Pro40Leu, and p.Arg205Gln mutants (thermofluor assays). The p.Arg241Gln and p.Tyr69Cys mutants respectively lacked PLP or had a decreased amount of this cofactor. With p.Tyr69Cys there was extensive protein dimerization due to disulfide bridge formation, and PLP accessibility was decreased (judged from d ‐cycloserine reaction). A 3‐D model of human PLPHP allowed rationalizing the effects of most mutations. Overall, the six missense mutations caused ill effects and five of them impaired folding or decreased stability, suggesting the potential of pharmacochaperone‐based therapeutic approaches.     

Rare mutations in the autophagy‐regulating gene AMBRA1 contribute to human neural tube defects

Neural tube defects (NTDs) are severe congenital malformations caused by failed neural tube closure. Recently, autophagy is revealed to play a vital role in neuroepithelium development and neurulation. Autophagy and beclin 1 regulator 1 (Ambra1) is a crucial regulator of autophagy initiation, and its deficiency in mice leads to exencephaly and/or spina bifida. However, the genetic contribution of AMBRA1 to the etiology of human NTDs remains unknown. In this study, we identified five rare missense mutations of AMBRA1 in 352 NTDs cases, which were absent in 224 matched controls. Western blotting and fluorescence puncta counting for MAP1LC3A/LC3 in HEK293T cells suggested that four of the mutations (AMBRA1 p.Thr80Met, p.Leu274Phe, p.Ser743Phe, and p.Met884Val) affected autophagy initiation to various extents. Furthermore, these four mutations also displayed loss‐of‐function effects compared with wild‐type AMBRA1 when we injected messenger RNA (mRNA) to overexpress or rescue ambra1a‐morpholino oligos (MO) knockdown in zebrafish. It is intriguing that trehalose, a natural disaccharide, could rescue ambra1a‐MO knockdown in a dose‐dependent manner independently or together with AMBRA1 mRNA. Taken together, our findings suggest that rare mutations of the autophagy regulator gene AMBRA1 may contribute to the etiology of human neural tube defects, and trehalose is a promising treatment for a subset of NTDs caused by autophagy impairment.     

Mutations in the planar cell polarity gene, Fuzzy, are associated with neural tube defects in humans

Neural tube defects (NTDs) are a heterogeneous group of common severe congenital anomalies which affect 1-2 infants per 1000 births. Most genetic and/or environmental factors that contribute to the pathogenesis of human NTDs are unknown. Recently, however, pathogenic mutations of VANGL1 and VANGL2 genes have been associated with some cases of human NTDs. Vangl genes encode proteins of the planar cell polarity (PCP) pathway that regulates cell behavior during early stages of neural tube formation. Homozygous disruption of PCP genes in mice results in a spectrum of NTDs, including defects that affect the entire neural axis (craniorachischisis), cranial NTDs (exencephaly) and spina bifida. In this paper, we report the dynamic expression of another PCP gene, Fuzzy, during neural tube formation in mice. We also identify non-synonymous Fuzzy amino acid substitutions in some patients with NTDs and demonstrate that several of these Fuzzy mutations affect formation of primary cilia and ciliary length or affect directional cell movement. Since Fuzzy knockout mice exhibit both NTDs and defective primary cilia and Fuzzy is expressed in the emerging neural tube, we propose that mutations in Fuzzy may account for a subset of NTDs in humans.     

Significant association of rare variant p.Gly8Ser in cardiac sodium channel β4‐subunit SCN4B with atrial fibrillation

Atrial fibrillation (AF) affects 33.5 million individuals worldwide. It accounts for 15% of strokes and increases risk of heart failure and sudden death. The voltage‐gated cardiac sodium channel complex is responsible for the generation and conduction of the cardiac action potential, and composed of the main pore‐forming α‐subunit Na_v1.5 (encoded by the SCN5A gene) and one or more auxiliary β‐subunits, including Na_vβ1 to Na_vβ4 encoded by SCN1B to SCN4B, respectively. We and others identified loss‐of‐function mutations in SCN1B and SCN2B and dominant‐negative mutations in SCN3B in patients with AF. Three missense variants in SCN4B were identified in sporadic AF patients and small nuclear families; however, the association between SCN4B variants and AF remains to be further defined. In this study, we performed mutational analysis in SCN4B using a panel of 477 AF patients, and identified one nonsynonymous genomic variant p.Gly8Ser in four patients. To assess the association between the p.Gly8Ser variant and AF, we carried out case‐control association studies with two independent populations (944 AF patients vs. 9,81 non‐AF controls in the first discovery population and 732 cases and 1,291 controls in the second replication population). Significant association was identified in the two independent populations and in the combined population (p = 4.16 × 10^?4, odds ratio [OR] = 3.14) between p.Gly8Ser and common AF as well as lone AF (p = 0.018, OR = 2.85). These data suggest that rare variant p.Gly8Ser of SCN4B confers a significant risk of AF, and SCN4B is a candidate susceptibility gene for AF.     

Functional Studies of Tyrosine Hydroxylase Missense Variants Reveal Distinct Patterns of Molecular Defects in Dopa‐Responsive Dystonia

Congenital tyrosine hydroxylase deficiency (THD) is found in autosomal‐recessive Dopa‐responsive dystonia and related neurological syndromes. The clinical manifestations of THD are variable, ranging from early‐onset lethal disease to mild Parkinson disease‐like symptoms appearing in adolescence. Until 2014, approximately 70 THD patients with a total of 40 different disease‐related missense mutations, five nonsense mutations, and three mutations in the promoter region of the tyrosine hydroxylase (TH) gene have been reported. We collected clinical and biochemical data in the literature for all variants, and also generated mutant forms of TH variants previously not studied (N = 23). We compared the in vitro solubility, thermal stability, and kinetic properties of the TH variants to determine the cause(s) of their impaired enzyme activity, and found great heterogeneity in all these properties among the mutated forms. Some TH variants had specific kinetic anomalies and phenylalanine hydroxylase, and Dopa oxidase activities were measured for variants that showed signs of altered substrate binding. p.Arg233His, p.Gly247Ser, and p.Phe375Leu had shifted substrate specificity from tyrosine to phenylalanine and Dopa, whereas p.Cys359Phe had an impaired activity toward these substrates. The new data about pathogenic mechanisms presented are expected to contribute to develop individualized therapy for THD patients.     

Mutation analysis of 73 southern Chinese Wilson's disease patients: identification of 10 novel mutations and its clinical correlation

This study was designed to investigate the molecular basis and the correlation between genotype and phenotype in the southern Chinese patients with Wilson's disease (WD). Genotypes of the ATP7B gene in 73 WD patients were examined by denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing. A total of 38 different disease-causing mutations were identified, including 10 novel mutations: missense mutations (p.Gln707Arg, p.Cys1079Phe, p.Gly1149Glu, p.Ser855Tyr, p.Ala874Pro and p.Ser921Arg), nonsense mutation (p.Arg1228Stop), splice-site mutations (2121+3A>T and 3244-2A>G) and frameshift mutation (1875_1876insAATT). We found that a pair of siblings carried the same genotype but different clinical type, and two patients were found to have three mutations. In addition, we compared the clinical data for p.Arg778Leu homozygotes and compound heterozygotes. Our research has enriched the mutation spectrum of the ATP7B gene in the Chinese population and can serve as the basis for genetic counseling and clinical/prenatal diagnosis to prevent WD in China.     

Vaccinia topoisomerase mutants illuminate roles for Phe59, Gly73, Gln69 and Phe215

Vaccinia topoisomerase provides a model system for structure-function analysis of the type IB topoisomerase family. Here we performed an alanine scan of eight positions in the beta4 and beta5 strands of the N-terminal domain (Leu57, Ile58, Phe59, Val60, Gly61, Ser62, Gln69 and Gly73) and eight positions in the alpha8-alpha9 loop of the C-terminal catalytic domain (Ser241, Ile242, Ser243, Pro244, Leu245, Pro246, Ser247, and Pro248). Mutants F59A, G73A, and Q69A displayed rate defects in relaxing supercoiled DNA that were attributed to effects on DNA binding rather than transesterification chemistry. Replacing Gln69 conservatively with Asn, Glu or Lys failed to restore relaxation activity. Gln69 is located along a concave DNA-binding surface of the N-terminal domain and it makes direct contact with the +2A base of the 5'-CCCTT/3-GGGAA target site for DNA cleavage. Gly73 is located at the junction between the N-terminal domain and catalytic domain and it is likely to act as a swivel for the large domain movements that coordinate DNA ingress and closure of the topoisomerase clamp around the duplex. Previous alanine scanning had identified Phe215 in helix alpha7 of the catalytic domain as contributing to DNA relaxation activity. Here we find that F215L resembles F215A in its diminished relaxation activity and its sensitivity to inhibition by salt. The Phe215 side chain makes van der Waals contacts to Ile98, Met121 and Phe101, which we propose stabilize a three helix bundle and promote clamp closure.     

Functional Characterization of Novel Mutations Affecting Survivin (BIRC5)‐Mediated Therapy Resistance in Head and Neck Cancer Patients

Survivin (BIRC5) is an acknowledged cancer therapy‐resistance factor and overexpressed in head and neck squamous cell carcinomas (HNSCC). Driven by its nuclear export signal (NES), Survivin shuttles between the nucleus and the cytoplasm, and is detectable in both cellular compartments in tumor biopsies. Although predominantly nuclear Survivin is considered a favorable prognostic disease marker for HNSCC patients, the underlying molecular mechanisms are not resolved. Hence, we performed immunohistochemical and mutational analyses using laser capture microdissection on HNSCC biopsies from patients displaying high levels of nuclear Survivin. We found somatic BIRC5 mutations, c.278T>C (p.Phe93Ser), c.292C>T (p.Leu98Phe), and c.288A>G (silent), in tumor cells, but not in corresponding normal tissues. Comprehensive functional characterization of the Survivin mutants by ectopic expression and microinjection experiments revealed that p.Phe93Ser, but not p.Leu98Phe inactivated Survivin's NES, resulted in a predominantly nuclear protein, and attenuated Survivin's dual cytoprotective activity against chemoradiation‐induced apoptosis. Notably, in xenotransplantation studies, HNSCC cells containing the p.Phe93Ser mutation responded significantly better to cisplatin‐based chemotherapy. Collectively, our results underline the disease relevance of Survivin's nucleocytoplasmic transport, and provide first evidence that genetic inactivation of Survivin's NES may account for predominantly nuclear Survivin and increased therapy response in cancer patients.     

Single‐molecule detection of cancer mutations using a novel PCR‐LDR‐qPCR assay

Detection of low‐abundance mutations in cell‐free DNA is being used to identify early cancer and early cancer recurrence. Here, we report a new PCR‐LDR‐qPCR assay capable of detecting point mutations at a single‐molecule resolution in the presence of an excess of wild‐type DNA. Major features of the assay include selective amplification and detection of mutant DNA employing multiple nested primer‐binding regions as well as wild‐type sequence blocking oligonucleotides, prevention of carryover contamination, spatial sample dilution, and detection of multiple mutations in the same position. Our method was tested to interrogate the following common cancer somatic mutations: BRAF:c.1799T>A (p.Val600Glu), TP53:c.743G>A (p.Arg248Gln), KRAS:c.35G>C (p.Gly12Ala), KRAS:c.35G>T (p.Gly12Val), KRAS:c.35G>A (p.Gly12Asp), KRAS:c.34G>T (p.Gly12Cys), and KRAS:c.34G>A (p.Gly12Ser). The single‐well version of the assay detected 2–5 copies of these mutations, when diluted with 10,000 genome equivalents (GE) of wild‐type human genomic DNA (hgDNA) from buffy coat. A 12‐well (pixel) version of the assay was capable of single‐molecule detection of the aforementioned mutations at TP53, BRAF, and KRAS (specifically p.Gly12Val and p.Gly12Cys), mixed with 1,000–2,250 GE of wild‐type hgDNA from plasma or buffy coat. The assay described herein is highly sensitive, specific, and robust, and potentially useful in liquid biopsies.     

Vacuolating megalencephalic leukoencephalopathy with subcortical cysts: functional studies of novel variants in MLC1

Nine new unrelated patients presenting vacuolating myelinopathy with subcortical cysts were identified and analyzed for variations in the MLC1 gene. We detected 12 mutations (p.Leu37fs, p.Met80Val, p.Leu83Phe, p.Pro92Ser, p.Ser93Leu, p.Ile108fs, p.Gly130Arg, p.Cys171fs, p.Glu202Lys, p.Ser269Tyr, p.Ala275Asn, and p.Leu310_311insLeu) of which nine were novel. In one patient we did not detect mutations. Using a heterologous system, three new missense variants (p.Glu202Lys, p.Ser269Tyr, and p.Ala275Asn) and a single leucine insertion (p.Leu310insLeu)--lying in a stretch of seven leucines--were functionally assayed by determining total protein levels and mutant protein expression at the plasma membrane. No correlation was observed between mutation, clinical features, and plasma membrane expression of mutant protein.     

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. Hum Mutat 33:1450–1455, 2012. © 2012 Wiley Periodicals, Inc.     

Identification of eight novel glucokinase mutations in Italian children with maturity-onset diabetes of the young

Maturity-onset diabetes of the young (MODY) is a clinically heterogeneous group of disorders characterized by early onset non-insulin-dependent diabetes mellitus, autosomal dominant inheritance, and primary defect in the function of the beta cells of the pancreas. Mutations in the glucokinase (GCK) gene account for 8%-56% of MODY, with the highest prevalences being found in the southern Europe. While screening for GCK mutations in 28 MODY families of Italian origin, we identified 17 different mutations (corresponding to 61% prevalence), including eight previously undescribed ones. The novel sequence variants included five missense mutations (p.Lys161Asn c.483G>C in exon 4, p.Phe171Leu c.511T>C in exon 5 and p.Thr228Ala c.682A>G, p.Thr228Arg c.683C>G, p.Gly258Cys c.772G>T in exon 7), one nonsense mutation (p.Ser383Ter c.1148C>A in exon 9), the splice site variant c.1253+1G>T in intron 9, and the deletion of 12 nucleotides in exon 10 (p.Ser433_Ile436del c.1298_1309del12). Our study indicates that mutations in the GCK/MODY2 gene are a very common cause of MODY in the Italian population and broadens our knowledge of the naturally occurring GCK mutation repertoire.