Identification of a novel biallelic missense variant in the KIAA0825 underlies postaxial polydactyly type A

Postaxial polydactyly (PAP) is characterized by development of extra digits, which mostly segregates in autosomal recessive pattern. The underlying genetic cause of recessive non-syndromic PAP type A has been associated with sequence variants in five different genes (ZNF141, IQCE, GLI1, FAM92A, KIAA0825).The present study was aimed to investigate clinical and genetic causes of PAPA in a consanguineous family of Pakistani origin. Microsatellite-based linkage analysis was used to search for the disease-causing gene. Linkage in the family was established at chromosome 5q15 harbouring a candidate gene KIAA0825. Subsequently, Sanger sequencing revealed a novel homozygous missense variant [c.50T>C; p. (Leu17Ser)] in the gene, which co-segregated with the disease within the family. Protein structural analysis predicted a substantial change in the secondary structure of the mutant protein affecting its function. This is the third disease causing variant identified in the KIAA0825. This has not only expanded spectrum of the mutations in the gene but also further substantiated its role in the limb development in human.     

A null mutation in TNIK defines a novel locus for intellectual disability

Intellectual disability (ID) is one of the most common disabilities and, although many genes have been implicated in its etiology, the genetic heterogeneity of ID continues to expand. The purpose of the study was to describe a novel autosomal recessive non-syndromic ID locus. Autozygome and linkage analysis, and exome sequencing followed by RNA and protein analysis of the candidate disease gene were performed. We describe two multiplex consanguineous families with non-syndromic ID phenotype, which maps to a critical linkage locus on 3q26. Exome sequencing of the index in each family revealed the same homozygous truncating mutation in TNIK that results in complete loss of the protein. TNIK is a kinase with a well-established role in dendrite development and synaptic transmission. The phenotype we observe in human patients who lack TNIK is consistent with the previously published Tnik ^?/? phenotype in the murine model. Our data strongly implicate TNIK deficiency in the causation of ID in humans.     

Non-Syndromic Hearing Impairment in India: High Allelic Heterogeneity among Mutations in TMPRSS3, TMC1, USHIC,CDH23 and TMIE

Mutations in the autosomal genes TMPRSS3, TMC1, USHIC, CDH23 and TMIE are known to cause hereditary hearing loss. To study the contribution of these genes to autosomal recessive, non-syndromic hearing loss (ARNSHL) in India, we examined 374 families with the disorder to identify potential mutations. We found four mutations in TMPRSS3, eight in TMC1, ten in USHIC, eight in CDH23 and three in TMIE. Of the 33 potentially pathogenic variants identified in these genes, 23 were new and the remaining have been previously reported. Collectively, mutations in these five genes contribute to about one-tenth of ARNSHL among the families examined. New mutations detected in this study extend the allelic heterogeneity of the genes and provide several additional variants for structure-function correlation studies. These findings have implications for early DNA-based detection of deafness and genetic counseling of affected families in the Indian subcontinent.     

The Role of a Novel TRMT1 Gene Mutation and Rare GRM1 Gene Defect in Intellectual Disability in Two Azeri Families

Cognitive impairment or intellectual disability (ID) is a widespread neurodevelopmental disorder characterized by low IQ (below 70). ID is genetically heterogeneous and is estimated to affect 1–3% of the world’s population. In affected children from consanguineous families, autosomal recessive inheritance is common, and identifying the underlying genetic cause is an important issue in clinical genetics. In the framework of a larger project, aimed at identifying candidate genes for autosomal recessive intellectual disorder (ARID), we recently carried out single nucleotide polymorphism-based genome-wide linkage analysis in several families from Ardabil province in Iran. The identification of homozygosity-by-descent loci in these families, in combination with whole exome sequencing, led us to identify possible causative homozygous changes in two families. In the first family, a missense variant was found in GRM1 gene, while in the second family, a frameshift alteration was identified in TRMT1, both of which were found to co-segregate with the disease. GRM1, a known causal gene for autosomal recessive spinocerebellar ataxia (SCAR13, MIM#614831), encodes the metabotropic glutamate receptor1 (mGluR1). This gene plays an important role in synaptic plasticity and cerebellar development. Conversely, the TRMT1 gene encodes a tRNA methyltransferase that dimethylates a single guanine residue at position 26 of most tRNAs using S-adenosyl methionine as the methyl group donor. We recently presented TRMT1 as a candidate gene for ARID in a consanguineous Iranian family (Najmabadi et al., 2011). We believe that this second Iranian family with a biallelic loss-of-function mutation in TRMT1 gene supports the idea that this gene likely has function in development of the disorder.     

Haplotype analysis of DFNB8/10 locus reveals contribution of TMPRSS3 mutations in Pakistani deaf population

TMPRSS3 mutations are associated with non-syndromic recessive deafness (DFNB8/10). To evaluate the frequency of TMPRSS3 mutation in Pakistani population, highly consanguineous families were enrolled. A group of five consanguineous families without any history of associated environmental causes were found to be linked to DFNB8/10 locus. To correlate haplotypes and to evaluate founder affect 17 other families linked to DFNB8/10 were provided by NCEMB DNA bank. Haploytpe analysis revealed that out of 22 families, haplotypes of 8 families (42 %) were found similar to PKDF003 and PKDF311 having 207delC mutation, 5 (26 %) families had haplotypes similar to PKDF040 and 4 families (15.7 %) shared halpotypes similar to PKDF064, having C407R (1219T>C) and C194F (518G>T) mutations, respectively. Interestingly, PKDF321 and PKDF337 (10.5 %) showed different haplotypes and might harbor novel mutation. Taken together, these data imply that Punjab region is more affected by TMPRSS3 mutation, and the founder-effect mutation might be traced back to Punjab region.     

Analysis of two Arab families reveals additional support for a DFNB2 nonsyndromic phenotype of MYO7A

Variants in the head and tail domains of the MYO7A gene, encoding myosin VIIA, cause Usher syndrome type 1B (USH1B) and nonsyndromic deafness (DFNB2, DFNA11). In order to identify the genetic defect(s) underling profound deafness in two consanguineous Arab families living in UAE, we have sequenced a panel of 19 genes involved in Usher syndrome and nonsyndromic deafness in the index cases of the two families. This analysis revealed a novel homozygous insertion of AG (c.1952_1953insAG/p.C652fsX11) in exon 17 of the MYO7A gene in an Iraqi family, and a homozygous point mutation (c.5660C>T/p.P1887L) in exon 41 affecting the same gene in a large Palestinian family. Moreover, some individuals from the Palestinian family also harbored a novel heterozygous truncating variant (c.1267C>T/p.R423X) in the DFNB31 gene, which is involved in autosomal recessive nonsyndromic deafness type DFNB31 and Usher syndrome type II. Assuming an autosomal recessive mode of inheritance in the two inbred families, we conclude that the homozygous variants in the MYO7A gene are the disease-causing mutations in these families. Furthermore, given the absence of retinal disease in all affected patients examined, particularly a 28 year old patient, suggests that at least one family may segregate a DFNB2 presentation rather than USH1B. This finding further supports the premise that the MYO7A gene is responsible for two distinct diseases and gives evidence that the p.P1887L mutation in a homozygous state may be responsible for nonsyndromic hearing loss.     

Comparative genomics and gene expression analysis identifies BBS9, a new Bardet-Biedl syndrome gene

Bardet-Biedl syndrome (BBS) is an autosomal recessive, genetically heterogeneous, pleiotropic human disorder characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, and hypogenitalism. Eight BBS genes representing all known mapped loci have been identified. Mutation analysis of the known BBS genes in BBS patients indicate that additional BBS genes exist and/or that unidentified mutations exist in the known genes. To identify new BBS genes, we performed homozygosity mapping of small, consanguineous BBS pedigrees, using moderately dense SNP arrays. A bioinformatics approach combining comparative genomic analysis and gene expression studies of a BBS-knockout mouse model was used to prioritize BBS candidate genes within the newly identified loci for mutation screening. By use of this strategy, parathyroid hormone-responsive gene B1 (B1) was found to be a novel BBS gene (BBS9), supported by the identification of homozygous mutations in BBS patients. The identification of BBS9 illustrates the power of using a combination of comparative genomic analysis, gene expression studies, and homozygosity mapping with SNP arrays in small, consanguineous families for the identification of rare autosomal recessive disorders. We also demonstrate that small, consanguineous families are useful in identifying intragenic deletions. This type of mutation is likely to be underreported because of the difficulty of deletion detection in the heterozygous state by the mutation screening methods that are used in many studies.     

DFNB68, a novel autosomal recessive non-syndromic hearing impairment locus at chromosomal region 19p13.2

From a large collection of families with autosomal recessive non-syndromic hearing impairment (NSHI) from Pakistan, linkage has been established for two unrelated consanguineous families to 19p13.2. This new locus was assigned the name DFNB68. A 10 cM genome scan and additional fine mapping were carried out using microsatellite marker loci. Linkage was established for both families to DFNB68 with maximum multipoint LOD scores of 4.8 and 4.6. The overlap of the homozygous regions between the two families was bounded by D19S586 and D19S584, which limits the locus interval to 1.9 cM and contains 1.4 Mb. The genes CTL2, KEAP1 and CDKN2D were screened but were negative for functional sequence variants.Regie Lyn P. Santos and Muhammad Jawad Hassan contributed equally to this work.     

<Emphasis Type="Italic">CHX10</Emphasis> mutations cause non-syndromic microphthalmia/anophthalmia in Arab and Jewish kindreds

Microphthalmia/anophthalmia is a clinically heterogeneous disorder of eye formation, ranging from small size of a single eye to complete bilateral absence of ocular tissues. The genetic defect underlying isolated autosomal recessive microphthalmia/anophthalmia is yet unclear. We studied four families (two of Arab origin, one of Bedouin origin, and one of Persian-Jewish origin) with autosomal recessive microphthalmia/anophthalmia and no associated eye anomalies, and one Syrian–Jewish family with associated colobomas. Assuming a founder effect in each of the families, we performed homozygosity mapping using polymorphic markers adjacent to human homologues of genes known to be associated with eye absence in various species, namely EYA1, EYA2, EYA3, SIX4, SIX6, PAX6 and CHX10. No association was found with EYA1, EYA2, EYA3, SIX6 or PAX6. In two families, linkage analysis was consistent with possible association with SIX4, but no mutations were found in the coding region of the gene or its flanking intron sequences. In three of the five families, linkage analysis followed by sequencing demonstrated that affected individuals in each family were homozygous for a different CHX10 aberration: a mutation in the CVC domain and a deletion of the homeobox domain were found in two Arab families, and a mutation in the donor-acceptor site in the first intron in the Syrian-Jewish family. There was phenotypic variation between families having different mutations, but no significant phenotypic variation within each family. It has been previously shown that mutations in a particular nucleotide in CHX10 are associated with an autosomal recessive syndrome of microphthalmia/anophthalmia with iris colobomas and cataracts in two families. We now show that different mutations in other domains of the same gene underlie isolated microphthalmia/anophthalmia.     

A Systematic Approach to Mapping Recessive Disease Genes in Individuals from Outbred Populations

The identification of recessive disease-causing genes by homozygosity mapping is often restricted by lack of suitable consanguineous families. To overcome these limitations, we apply homozygosity mapping to single affected individuals from outbred populations. In 72 individuals of 54 kindred ascertained worldwide with known homozygous mutations in 13 different recessive disease genes, we performed total genome homozygosity mapping using 250,000 SNP arrays. Likelihood ratio Z-scores (ZLR) were plotted across the genome to detect ZLR peaks that reflect segments of homozygosity by descent, which may harbor the mutated gene. In 93% of cases, the causative gene was positioned within a consistent ZLR peak of homozygosity. The number of peaks reflected the degree of inbreeding. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations within a single ZLR peak of homozygosity as short as 2 Mb, containing an average of only 16 candidate genes. As many specialty clinics have access to cohorts of individuals from outbred populations, and as our approach will result in smaller genetic candidate regions, the new strategy of homozygosity mapping in single outbred individuals will strongly accelerate the discovery of novel recessive disease genes.     

Genome-wide association and linkage analyses localize a progressive retinal atrophy locus in Persian cats

Hereditary eye diseases of animals serve as excellent models of human ocular disorders and assist in the development of gene and drug therapies for inherited forms of blindness. Several primary hereditary eye conditions affecting various ocular tissues and having different rates of progression have been documented in domestic cats. Gene therapy for canine retinopathies has been successful, thus the cat could be a gene therapy candidate for other forms of retinal degenerations. The current study investigates a hereditary, autosomal recessive, retinal degeneration specific to Persian cats. A multi-generational pedigree segregating for this progressive retinal atrophy was genotyped using a 63 K SNP array and analyzed via genome-wide linkage and association methods. A multi-point parametric linkage analysis localized the blindness phenotype to a ~1.75 Mb region with significant LOD scores (Z ≈ 14, θ = 0.00) on cat chromosome E1. Genome-wide TDT, sib-TDT, and case–control analyses also consistently supported significant association within the same region on chromosome E1, which is homologous to human chromosome 17. Using haplotype analysis, a ~1.3 Mb region was identified as highly associated for progressive retinal atrophy in Persian cats. Several candidate genes within the region are reasonable candidates as a potential causative gene and should be considered for molecular analyses.     

Mutations of 60 known causative genes in 157 families with retinitis pigmentosa based on exome sequencing

Retinitis pigmentosa (RP) is the most common and highly heterogeneous form of hereditary retinal degeneration. This study was to identify mutations in the 60 genes that were known to be associated with RP in 157 unrelated Chinese families with RP. Genomic DNA from probands was initially analyzed by whole exome sequencing. Sanger sequencing was used to confirm potential candidate variants affecting the encoded residues in the 60 genes, including heterozygous variants from genes that are related to autosomal dominant RP, homozygous or compound heterozygous variants from genes that are related to autosomal recessive RP, and hemizygous variants from genes that are related to X-linked RP. Synonymous and intronic variants were also examined to confirm whether they could affect splicing. A total of 244 candidate variants were detected by exome sequencing. Sanger sequencing confirmed 240 variants out of the 244 candidates. Informatics and segregation analyses suggested 110 potential pathogenic mutations in 28 out of the 60 genes involving 79 of the 157 (50 %) families, including 31 (39 %, 31/79) families with heterozygous mutations in autosomal dominant genes, 37 (47 %, 37/79) families with homozygous (9) or compound heterozygous (28) mutations in autosomal recessive genes, and 11 (14 %, 11/79) families with hemizygous mutations in X-linked genes. Of the 110 identified variants, 74 (67 %) were novel. The genetic defects in approximately half of the 157 studies families were detected by exome sequencing. A comprehensive analysis of the 60 known genes not only expanded the mutation spectrum and frequency of the 60 genes in Chinese patients with RP, but also provided an overview of the molecular etiology of RP in Chinese patients. The analysis of the known genes also supplied the foundation and clues for discovering novel causative RP genes.     

Novel mutation G324C in <Emphasis Type="Italic">WNT1</Emphasis> mapped in a large Pakistani family with severe recessively inherited <Emphasis Type="Italic">Osteogenesis Imperfecta</Emphasis>

Introduction

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disease with skeletal fragility and variable extra-skeletal manifestations. To date several point mutations in 18 different genes causing different types of OI have been identified. Mutations in WNT1 compromise activity of the osteoblasts leading to disturbed bone mass accrual, fragility fractures and progressive skeletal abnormalities. The present study was conducted to determine the underlying genetic cause of an autosomal recessive skeletal dysplasia in a large consanguineous family from Chinute, Pakistan.

Materials and methods

Blood was collected from 24 individuals of affected family along with clinical data. Homozygosity mapping was performed to confirm consanguinity. SNPs were identified, followed by whole exome and Sanger sequencing. In silico characterization of WNT1 mutation was performed using multiple platforms.

Results

Nine affected family members exhibited severe bone deformities, recurrent fractures, short stature and low bone mineral density. SNP array data revealed homozygous segments >?1 Mb in length accounting for 2.1–12.7% of the genome in affected individuals and their siblings and a single 6,344,821 bp homozygous region in all affected individuals on chromosome 12q12-q13. This region includes two potential OI candidate genes WNT1 and VDR. We did whole-exome sequencing for both genes in two patients and identified a novel damaging missense mutation in exon 4 of WNT1: c.1168G?>?T (NM_005430) resulting in p.G324C. Sanger sequencing confirmed segregation of mutation with the disease in family.

Conclusion

We report a novel mutation responsible for OI and our investigation expands the spectrum of disease-causing WNT1 mutations and the resulting OI phenotypes.

     

Exome Sequencing Identifies Three Novel Candidate Genes Implicated in Intellectual Disability

Intellectual disability (ID) is a major health problem mostly with an unknown etiology. Recently exome sequencing of individuals with ID identified novel genes implicated in the disease. Therefore the purpose of the present study was to identify the genetic cause of ID in one syndromic and two non-syndromic Pakistani families. Whole exome of three ID probands was sequenced. Missense variations in two plausible novel genes implicated in autosomal recessive ID were identified: lysine (K)-specific methyltransferase 2B (KMT2B), zinc finger protein 589 (ZNF589), as well as hedgehog acyltransferase (HHAT) with a de novo mutation with autosomal dominant mode of inheritance. The KMT2B recessive variant is the first report of recessive Kleefstra syndrome-like phenotype. Identification of plausible causative mutations for two recessive and a dominant type of ID, in genes not previously implicated in disease, underscores the large genetic heterogeneity of ID. These results also support the viewpoint that large number of ID genes converge on limited number of common networks i.e. ZNF589 belongs to KRAB-domain zinc-finger proteins previously implicated in ID, HHAT is predicted to affect sonic hedgehog, which is involved in several disorders with ID, KMT2B associated with syndromic ID fits the epigenetic module underlying the Kleefstra syndromic spectrum. The association of these novel genes in three different Pakistani ID families highlights the importance of screening these genes in more families with similar phenotypes from different populations to confirm the involvement of these genes in pathogenesis of ID.     

Identification of recurrent c.742G>T nonsense mutation in ECM1 in Pakistani families suffering from lipoid proteinosis

Lipoid proteinosis (LP) is one of the rare, recessive autosomal disorders clinically characterized by widespread deposition of hyaline-like material in the skin, mucosa and viscera. Classical features include beaded eyelid papules, laryngeal infiltration and hoarseness of voice caused by pathogenic mutations in the ECM1gene located on 1q21.2. In present study ethnically different, three consanguineous Pakistani families with typical cutaneous features of LP were analysed to investigate the underlying molecular basis. PCR based linkage analysis using microsatellite markers localized the families to locus 1q21.2, harboring ECM1 gene. To identify the mutation in the candidate gene (ECM1), Sanger sequencing was carried out. All the families were found to carry c.742 G>T nonsense mutation in exon 7 of the ECM1 gene that resulted in a truncated ECM1 protein containing 247 amino acids instead of 540 (p.E248X). To further investigate the impact and importance of mutation in LP pathogenesis we applied different bioinformatics tools. In silico studies has predicted lack of functional domains and 65 % shorter ECM1 mutant protein. It is the first report of recurrence mutation from Pakistan as c.742G>T nonsense mutation was found in three ethnically different Pakistani families with LP. Study strengthens the conclusion that c.742G>T mutation is the pathological cause of LP. Furthermore, data also support the fact that exon 7 is one of the most common hot spots of pathological mutations in ECM1. The absence of functional domains and truncated sequence most likely contribute to the lack of ECM1 function and thereby influence several aspects of dermal homeostasis that leads to LP pathogenesis.     

Genome-wide SNP-based linkage analysis for ADNSHL families identifies novel susceptibility loci with positive evidence for linkage

The linkage search for susceptibility loci using SNP markers in hereditary hearing loss has proven challenging due to genetic heterogeneity. We conducted a genome-wide linkage analysis using high-density SNP markers in two Korean families (families coded SD-J and SR-167) with autosomal dominant non-syndromic hearing loss (ADNSHL). Evidence was found of linkage at 8q24.13~q24.3 and 10p11.21~q22.2 (LOD 3.01) in the SD-J family. In the case of family SR-167, which had the most affected members, the parametric LOD score was low owing to the lack of power for linkage analysis. However, using non-parametric linkage analysis, it was possible to obtain significant evidence for linkage at 10q22.1~q23.31 (LOD 1.79; NPL 6.47, P<0.00001). There is an overlapping region with a significant LOD score between the SD-J and SR-167 families, which encompasses 4 cM at 10q22.1~22.2. Interestingly, the characteristics of hearing loss in both families were similar, and the haplotype within overlapping region was shared in the affected individuals of the two families. We performed direct sequencing of the candidate genes that are thought to be causing the condition, but no disease-causing mutations were identified.     

Comprehensive Analysis of Deafness Genes in Families with Autosomal Recessive Nonsyndromic Hearing Loss

Comprehensive genetic testing has the potential to become the standard of care for individuals with hearing loss. In this study, we investigated the genetic etiology of autosomal recessive nonsyndromic hearing loss (ARNSHL) in a Turkish cohort including individuals with cochlear implant, who had a pedigree suggestive of an autosomal recessive inheritance. A workflow including prescreening of GJB2 and a targeted next generation sequencing panel (Illumına TruSight^TM Exome) covering 2761 genes that we briefly called as mendelian exome sequencing was used. This panel includes 102 deafness genes and a number of genes causing Mendelian disorders. Using this approach, we identified causative variants in 21 of 29 families. Three different GJB2 variants were present in seven families. Remaining 14 families had 15 different variants in other known NSHL genes (MYO7A, MYO15A, MARVELD2, TMIE, DFNB31, LOXHD1, GPSM2, TMC1, USH1G, CDH23). Of these variants, eight are novel. Mutation detection rate of our workflow is 72.4%, confirming the usefulness of targeted sequencing approach in NSHL.