Biallelic variant in DACH1, encoding Dachshund Homolog 1, defines a novel candidate locus for recessive postaxial polydactyly type A

Polydactyly or hexadactyly is characterized by an extra digit/toe with or without a bone. Currently, variants in ten genes have been implicated in the non-syndromic form of polydactyly.DNA from a single affected individual having bilateral postaxial polydactyly was subjected to whole exome sequencing (WES), followed by Sanger sequencing. Homology modeling was performed for the identified variant and advance microscopy imaging approaches were used to reveal the localization of the DACH1 protein at the base of primary cilia.A disease-causing biallelic missense variant (c.563G > A; p.Cys188Tyr; NM_080760.5) was identified in the DACH1 gene segregating perfectly within the family. Structural analysis using homology modeling of the DACH1 protein revealed secondary structure change that might result in loss of function or influence downstream interactions. Moreover, siRNA-mediated depletion of DACH1 showed a key role of DACH1 in ciliogenesis and cilia function.This study provides the first evidence of involvement of the DACH1 gene in digits development in humans and its role in primary cilia. This signifies the importance and yet unexplored role of DACH1.     

Morphogenesis of retinoic acid-induced postaxial polydactyly in mice

Previous experiments have demonstrated that oral administration of retinoic acid to ICR mice on day 10 of gestation results in an enhanced expression of postaxial polydactyly of the forelimbs. In the present investigations, the development of the limbs from the time of treatment until the appearance of the defect on day 14 were studied at 12- to 24-hr intervals with histological and vital staining technics. A diffuse cytotoxicity in the central proximal mesoderm was noted with a safranin-toluidine blue stain 12 h after treatment which then peaked 24 hr after treatment. Treated limbs stained with Nile blue sulfate showed a significant increase of necrotic mesodermal cells in the foyer preaxial primaire, foyer marginal I and foyer marginal V when compared to the respective control group. The treated postaxial ectoderm was more hyperplastic than was the control ectoderm on day 12. The treated group developed a squarish bulge on the postaxial boundary of the fifth digital ray on day 14 that was larger than those of the controls. Most of the controls had bulges that were completely necrotic. The treated group also had some completely necrotic bulges but a large number of the treated fetuses had bulges characterized as being less than 60% necrotic.     

Segregation distortion in the offspring of Afro-American fathers with postaxial polydactyly.

The unclear pattern of inheritance of postaxial polydactyly prompted this search for evidence of imprinting or change of expression in males and females using material of the Latin American Collaborative Study of Congenital Malformations. The frequency of affected offspring for 196 fathers with polydactyly was compared with that for 233 mothers with the same condition, stratified according to African and non-African ancestry. The postaxial polydactyly prevalence rate among the offspring of affected black fathers (44%) was larger than that in the group of affected black mothers (31%), with no difference between affected nonblack fathers (34%) and affected nonblack mothers (33%). The sex ratio (.51) observed in 631 black propositi and in 829 nonblack propositi with polydactyly (.58) could be a further indication of etiologic heterogeneity for polydactyly between these two ethnic groups. The segregation distortion in favor of affected among the offspring of affected black fathers could be interpreted as the effect of a sex-linked recessive modifier gene acting during gametogenesis on an autosomal dominant polydactyly gene, this modifier being more frequent in Africans.     

Characterization of a novel missense mutation in the prodomain of GDF5, which underlies brachydactyly type C and mild Grebe type chondrodysplasia in a large Pakistani family

All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T>C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.     

Identification of a novel missense mutation in Brazilian patient with a severe form of mucopolysaccharidosis type IVA

Mucopolysaccharidosis type IVA (MPS IVA) or Morquio syndrome type A is an autosomal recessive disease caused by deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfatase (GALNS). We report molecular characterization of a patient who presents the new missense mutation p.C165Y in homozygosis. Bioinformatics analysis predicted this mutation as being probably pathogenic. To evaluate the possibility that this alteration was a polymorphism we tested 100 alleles and all the results were negative. These findings together with the observation that this alteration is not present in controls, suggest that it is a disease-causing mutation, which was correlated with the severe phenotype observed in our patient. We conclude that molecular analysis of the GALNS gene, in addition to enzyme assays, is important for diagnosis and contributes to the better understanding of the relationship between genotype and phenotype, which is important as enzyme replacement therapy (ERT) will soon become available and treatment decisions will have to be take in such cases.     

Mapping one form of autosomal dominant postaxial polydactyly type A to chromosome 7p15-q11.23 by linkage analysis.

Postaxial polydactyly type-A (PAP-A) in humans is an autosomal dominant trait characterized by an extra digit in the ulnar and/or fibular side of the upper and/or lower extremities. The extra digit is well formed and articulates with the fifth, or extra, metacarpal/metatarsal, and thus it is usually functional. In order to map the gene responsible for PAP-A, we studied a five-generation Indian family of 37 individuals (15 of whom were affected). A genomewide search with highly informative polymorphic markers on part of the pedigree showed linkage between the PAP-A phenotype and markers on chromosome 7p15-q11.23 (no crossovers were found with D7S526, D7S795, D7S528, D7S521, D7S691, D7S667, D7S478, D7S1830, D7S803, D7S801, or ELN). The highest LOD score was obtained with marker D7S801 (zeta max = 4.21; theta = 0). Haplotype analysis enabled the mapping of the PAP-A phenotype in this family between markers D7S2848 and D7S669. Analysis of additional families with PAP-A will narrow down the critical genomic region, facilitate positional cloning of the PAP-A gene, and/or uncover potential genetic heterogeneity.     

Scalp defect associated with postaxial polydactyly: Confirmation of a distinct entity with autosomal dominant inheritance

Summary The association of postaxial polydactyly type A and congenital scalp defect in a 15-year-old severely mentally retarded male confirms the previously reported suggestion that the combination of both anomalies represents a new distinct entity with autosomal dominant inheritance and variable expression.     

Identification of a novel variant in the phosphoenolpyruvate carboxykinase gene promoter in Japanese patients with type 2 diabetes.

Phospho enolpyruvate carboxykinase (PEPCK) plays an important role in gluconeogenesis and hepatic glucose production. To test the hypothesis that mutations of the PEPCK gene promoter contribute to the increased hepatic glucose production that leads to diabetes, we screened for polymorphisms of the PEPCK promoter region in 252 Japanese type 2 diabetic patients and 188 non-diabetic control subjects. A novel variant at position - 232 (C to G) was found at a similar frequency in type 2 diabetes patients (32 %) and control subjects (35 %) (p = 0.26). However, patients with the - 232 G/G genotype had an earlier age of onset than those with the - 232 C/C or - 232 C/G genotypes (p = 0.028). As the variant might well otherwise influence hormonal action, we transfected PEPCK-luciferase fusion gene constructs with the variant into human hepatoma cells and examined the response to dexamethasone, insulin, and cAMP. The reporter assay showed no significant difference in hormonal responses with the fusion gene containing the variant. Accordingly, the single-base variant at position - 232 of the PEPCK gene promoter is most probably not a major contributor to the pathogenesis of type 2 diabetes. However, this variation may be useful as a genetic marker for other metabolic disorders, especially in Japanese.     

Identification and characterization of a novel splice variant of MuSK

MuSK is a receptor tyrosine kinase that initiates the formation of neuromuscular junctions in response to agrin. Little is known about the ligand-induced activation and kinase-dependent signalling that leads to the clustering of acetylcholine receptors. The ectodomain of these molecule is composed of four Ig-like domains. We describe here the isolation of a novel MuSK splice variant that lacks the third Ig-like domain in its ectodomain. The corresponding RNA is the result of alternative splicing which eliminates two exons. There is 10 times less mRNA for this shorter form than for the long form of MuSK and both forms are regulated coordinately. They decrease strongly after birth and are elevated in denervated muscle. Gene transfer by muscle injection of MuSK DNA into individual muscle fibers demonstrates that kinase-induced acetylcholine receptor clustering caused by overexpression of the two kinases does not depend on the presence of the third Ig-like domain.     

Identification of a candidate missense mutation in a family with von Willebrand disease type IIC

A screening project to identify candidate molecular defects causing von Willebrand disease type IIC (VWD IIC) in a German family was carried out using polymerase chain reaction (PCR) amplification of all 52 exons of the von Willebrand factor (VWF) gene, subsequent electrophoresis of single and double stranded DNA and direct sequencing of PCR products with aberrant electrophoretic patterns. Only one candidate mutation, G550R, caused by a GA transition, was detected in exon 14 of the pro-VWF gene sequence. This mutation was not found on 200 chromosomes of normal individuals. The propositus was homozygous for the mutation and for an extended intragenic haplotype, composed of eight polymorphic markers. Further family members were heterozygous for the mutation and were phenotypically normal or only mildly affected, in accordance with the recessive pattern of inheritance for VWD type IIC. The mutation could influence one of the presumed active centers for the suspected multimerizing enzymatic activity of pro-VWF localized in the D1 and D2 domain, which corresponds to exon 5 and exon 14 of the VWF gene.     

Identification of three novel missense PKU mutations among Chinese.

Three novel missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese individuals afflicted with various degrees of phenylketonuria (PKU). A T-to-C transition was observed in exon 5 of the gene, resulting in the substitution of Phe161 by Ser161. Two substitutions, G-to-T and T-to-G, were observed in exon 7, resulting in the substitution of Gly247 by Val247 and Leu255 by Val255, respectively. Expression analysis demonstrated that these mutant proteins produced between 0 and 15% of normal PAH enzyme activity. Population screening of a Chinese sample population indicates that these mutations are quite rare, together accounting for only about 4% of all PKU alleles among the Chinese. The P161S and G247V mutations were each present on a single PAH RFLP haplotype 4 chromosome in patients form Northern China, while the L255V mutation was present on chromosomes of both haplotypes 18 and 21 in patients from Southern China. These results suggest that the remaining 30% of uncharacterized PKU alleles in the Chinese population may bear a large number of relatively rare PAH mutations.     

Identification of a novel prostate cancer susceptibility variant in the KLK3 gene transcript

Genome-wide association studies (GWAS) have identified more than 30 prostate cancer (PrCa) susceptibility loci. One of these (rs2735839) is located close to a plausible candidate susceptibility gene, KLK3, which encodes prostate-specific antigen (PSA). PSA is widely used as a biomarker for PrCa detection and disease monitoring. To refine the association between PrCa and variants in this region, we used genotyping data from a two-stage GWAS using samples from the UK and Australia, and the Cancer Genetic Markers of Susceptibility (CGEMS) study. Genotypes were imputed for 197 and 312 single nucleotide polymorphisms (SNPs) from HapMap2 and the 1000 Genome Project, respectively. The most significant association with PrCa was with a previously unidentified SNP, rs17632542 (combined P?=?3.9?×?10(-22)). This association was confirmed by direct genotyping in three stages of the UK/Australian GWAS, involving 10,405 cases and 10,681 controls (combined P?=?1.9?×?10(-34)). rs17632542 is also shown to be associated with PSA levels and it is a non-synonymous coding SNP (Ile179Thr) in KLK3. Using molecular dynamic simulation, we showed evidence that this variant has the potential to introduce alterations in the protein or affect RNA splicing. We propose that rs17632542 may directly influence PrCa risk.     

Phenotypic features of pure 9p deletion in a male infant include cryptorchidism, congenital heart defects and postaxial polydactyly

We report a 2 1/2-year-old male infant with a karyotype of 46,XY,del(9)(p22) and the phenotypic features of craniofacial dysmorphisms, hypotonia, psychomotor developmental delay, mental retardation, ventricular septal defect, atrial septal defect, cryptorchidism and postaxial polydactyly of the fingers. A rudimentary poorly developed extra digit in the ulnar side of the fifth finger was observed in each hand. The present case adds to the literature of postaxial hexadactyly of the fingers in chromosome 9p deletion syndrome. We suggest that 9pter-p22 may contain genetic loci associated with human postaxial polydactyly.