Zubair M. Ahmed

Mutations of the protocadherin gene PCDH15 cause Usher syndrome type 1F.

Human chromosome 10q21-22 harbors USH1F in a region of conserved synteny to mouse chromosome 10. This region of mouse chromosome 10 contains Pcdh15, encoding a protocadherin gene that is mutated in ames waltzer and causes deafness and vestibular dysfunction. Here we report two mutations of protocadherin 15 (PCDH15) found in two families segregating Usher syndrome type 1F. A Northern blot probed with the PCDH15 cytoplasmic domain showed expression in the retina, consistent with its pathogenetic role in the retinitis pigmentosa associated with USH1F.

Targeted capture and next-generation sequencing identifies C9orf75, encoding taperin, as the mutated gene in nonsyndromic deafness DFNB79.

Targeted genome capture combined with next-generation sequencing was used to analyze 2.9 Mb of the DFNB79 interval on chromosome 9q34.3, which includes 108 candidate genes. Genomic DNA from an affected member of a consanguineous family segregating recessive, nonsyndromic hearing loss was used to make a library of fragments covering the DFNB79 linkage interval defined by genetic analyses of four pedigrees. Homozygosity for eight previously unreported variants in transcribed sequences was detected by evaluating a library of 402,554 sequencing reads and was later confirmed by Sanger sequencing. Of these variants, six were determined to be polymorphisms in the Pakistani population, and one was in a noncoding gene that was subsequently excluded genetically from the DFNB79 linkage interval. The remaining variant was a nonsense mutation in a predicted gene, C9orf75, renamed TPRN. Evaluation of the other three DFNB79-linked families identified three additional frameshift mutations, for a total of four truncating alleles of this gene. Although TPRN is expressed in many tissues, immunolocalization of the protein product in the mouse cochlea shows prominent expression in the taper region of hair cell stereocilia. Consequently, we named the protein taperin.

Mutations of MYO6 Are Associated with Recessive Deafness, DFNB37

Cosegregation of profound, congenital deafness with markers on chromosome 6q13 in three Pakistani families defines a new recessive deafness locus, DFNB37. Haplotype analyses reveal a 6-cM linkage region, flanked by markers D6S1282 and D6S1031, that includes the gene encoding unconventional myosin VI. In families with recessively inherited deafness, DFNB37, our sequence analyses of MYO6 reveal a frameshift mutation (36-37insT), a nonsense mutation (R1166X), and a missense mutation (E216V). These mutations, along with a previously published missense allele linked to autosomal dominant progressive hearing loss (DFNA22), provide an allelic spectrum that probes the relationship between myosin VI dysfunction and the resulting phenotype.

Novel association of hypertrophic cardiomyopathy, sensorineural deafness, and a mutation in unconventional myosin VI (MYO6)

Familial hypertrophic cardiomyopathy (FHC) is typically characterised by left ventricular hypertrophy, diastolic dysfunction, and hypercontractility, and is often associated with disabling symptoms, arrhythmias, and sudden death.1 FHC shows both non-allelic and allelic genetic heterogeneity, and results from any one of more than 100 mutations in genes encoding sarcomeric proteins.2 Identified genes include those encoding β myosin heavy chain, the myosin regulatory and essential light chains, myosin binding protein C, troponin I, troponin C, α cardiac actin, and titin.2,3 The FHC phenotype is characterised by hypertrophy, myocyte disarray and fibrosis, and results from the dominant negative expression of one of these (mainly missense) mutations. The resulting sarcomeric dysfunction leads ultimately, through mechanisms that remain obscure, to pathological left ventricular remodelling. However, as molecular defects are identified in only half the cases, it is likely that non-sarcomeric genes may also be responsible. Non-sarcomeric causes of FHC are largely uncharacterised, and may be associated with distinct or compound phenotypes. Similarly, hereditary sensorineural hearing loss shows a great degree of non-allelic and allelic genetic heterogeneity, and can be dominant, recessive, X linked, or mitochondrial.4–6 Hereditary sensorineural hearing loss is classified according to mode of inheritance and the presence of clinically detectable extra-auditory manifestations (syndromic deafness) or their absence (non-syndromic).6,7 The distributions of mutant gene expression are not necessarily restricted to clinically affected organ systems, and mutant genes associated with “non-syndromic” deafness may therefore have subtle extra-auditory manifestations. Genetic syndromes restricted to a cardio-auditory phenotype include the long QT syndrome (LQTS) caused by mutations in KvLQT1 ( KCNQ1 ) or in KCNE1 , where QT prolongation has autosomal dominant expression (Romano Ward syndrome), but congenital sensorineural hearing loss with LQTS is autosomal recessive (Jervell and Lange-Nielsen syndrome).8 In the LQTS, cardiac structure is normal. Recently, Schonberger et al …

Increasing the complexity: new genes and new types of albinism

Albinism is a rare genetic condition globally characterized by a number of specific deficits in the visual system, resulting in poor vision, in association with a variable hypopigmentation phenotype. This lack or reduction in pigment might affect the eyes, skin, and hair (oculocutaneous albinism, OCA), or only the eyes (ocular albinism, OA). In addition, there are several syndromic forms of albinism (e.g. Hermansky–Pudlak and Chediak–Higashi syndromes, HPS and CHS, respectively) in which the described hypopigmented and visual phenotypes coexist with more severe pathological alterations. Recently, a locus has been mapped to the 4q24 human chromosomal region and thus represents an additional genetic cause of OCA, termed OCA5, while the gene is eventually identified. In addition, two new genes have been identified as causing OCA when mutated: SLC24A5 and C10orf11, and hence designated as OCA6 and OCA7, respectively. This consensus review, involving all laboratories that have reported these new genes, aims to update and agree upon the current gene nomenclature and types of albinism, while providing additional insights from the function of these new genes in pigment cells.

Identities, frequencies and origins of TMC1 mutations causing DFNB7/B11 deafness in Pakistan

Non‐syndromic deafness is genetically heterogeneous. We previously reported that mutations of transmembrane channel‐like gene 1 (TMC1) cause non‐syndromic recessive deafness at the DFNB7/B11 locus on chromosome 9q13–q21 in nine Pakistani families. The goal of this study was to define the identities, origins and frequencies of TMC1 mutations in an expanded cohort of 557 large Pakistani families segregating recessive deafness. We screened affected family members for homozygosity at short‐tandem repeats flanking known autosomal recessive (DFNB) deafness loci, followed by TMC1 sequence analysis in families segregating deafness linked to DFNB7/B11. We identified 10 new families segregating DFNB7/B11 deafness and TMC1 mutations, including three novel alleles. Overall, 9 different TMC1 mutations account for deafness in 19 (3.4%) of the 557 Pakistani families. A single mutation, p.R34X, causes deafness in 10 (1.8%) of the families. Genotype analysis of p.R34X‐linked markers indicates that it arose from a common founder. We also detected p.R34X among normal control samples of African‐American and northern European origins, raising the possibility that p.R34X and other mutations of TMC1 are prevalent contributors to the genetic load of deafness across a variety of populations and continents.

Mutation of ATF6 causes autosomal recessive achromatopsia

Achromatopsia (ACHM) is an early-onset retinal dystrophy characterized by photophobia, nystagmus, color blindness and severely reduced visual acuity. Currently mutations in five genes CNGA3, CNGB3, GNAT2, PDE6C and PDE6H have been implicated in ACHM. We performed homozygosity mapping and linkage analysis in a consanguineous Pakistani ACHM family and mapped the locus to a 15.12-Mb region on chromosome 1q23.1–q24.3 with a maximum LOD score of 3.6. A DNA sample from an affected family member underwent exome sequencing. Within the ATF6 gene, a single-base insertion variant c.355_356dupG (p.Glu119Glyfs*8) was identified, which completely segregates with the ACHM phenotype within the family. The frameshift variant was absent in public variant databases, in 130 exomes from unrelated Pakistani individuals, and in 235 ethnically matched controls. The variant is predicted to result in a truncated protein that lacks the DNA binding and transmembrane domains and therefore affects the function of ATF6 as a transcription factor that initiates the unfolded protein response during endoplasmic reticulum (ER) stress. Immunolabeling with anti-ATF6 antibodies showed localization throughout the mouse neuronal retina, including retinal pigment epithelium, photoreceptor cells, inner nuclear layer, inner and outer plexiform layers, with a more prominent signal in retinal ganglion cells. In contrast to cytoplasmic expression of wild-type protein, in heterologous cells ATF6 protein with the p.Glu119Glyfs*8 variant is mainly confined to the nucleus. Our results imply that response to ER stress as mediated by the ATF6 pathway is essential for color vision in humans.

Neuropathy target esterase impairments cause Oliver–McFarlane and Laurence–Moon syndromes

Background Oliver–McFarlane syndrome is characterised by trichomegaly, congenital hypopituitarism and retinal degeneration with choroidal atrophy. Laurence–Moon syndrome presents similarly, though with progressive spinocerebellar ataxia and spastic paraplegia and without trichomegaly. Both recessively inherited disorders have no known genetic cause. Methods Whole-exome sequencing was performed to identify the genetic causes of these disorders. Mutations were functionally validated in zebrafish pnpla6 morphants. Embryonic expression was evaluated via in situ hybridisation in human embryonic sections. Human neurohistopathology was performed to characterise cerebellar degeneration. Enzymatic activities were measured in patient-derived fibroblast cell lines. Results Eight mutations in six families with Oliver–McFarlane or Laurence–Moon syndrome were identified in the PNPLA6 gene, which encodes neuropathy target esterase (NTE). PNPLA6 expression was found in the developing human eye, pituitary and brain. In zebrafish, the pnpla6 curly-tailed morphant phenotype was fully rescued by wild-type human PNPLA6 mRNA and not by mutation-harbouring mRNAs. NTE enzymatic activity was significantly reduced in fibroblast cells derived from individuals with Oliver–McFarlane syndrome. Intriguingly, adult brain histology from a patient with highly overlapping features of Oliver–McFarlane and Laurence–Moon syndromes revealed extensive cerebellar degeneration and atrophy. Conclusions Previously, PNPLA6 mutations have been associated with spastic paraplegia type 39, Gordon–Holmes syndrome and Boucher–Neuhäuser syndromes. Discovery of these additional PNPLA6-opathies further elucidates a spectrum of neurodevelopmental and neurodegenerative disorders associated with NTE impairment and suggests a unifying mechanism with diagnostic and prognostic importance.

CD44 is a marker for the outer pillar cells in the early postnatal mouse inner ear.

Cluster of differentiation antigens (CD proteins) are classically used as immune cell markers. However, their expression within the inner ear is still largely undefined. In this study, we explored the possibility that specific CD proteins might be useful for defining inner ear cell populations. mRNA expression profiling of microdissected auditory and vestibular sensory epithelia revealed 107 CD genes as expressed in the early postnatal mouse inner ear. The expression of 68 CD genes was validated with real-time RT-PCR using RNA extracted from microdissected sensory epithelia of cochleae, utricles, saccules, and cristae of newborn mice. Specifically, CD44 was identified as preferentially expressed in the auditory sensory epithelium. Immunohistochemistry revealed that within the early postnatal organ of Corti, the expression of CD44 is restricted to outer pillar cells. In order to confirm and expand this finding, we characterized the expression of CD44 in two different strains of mice with loss- and gain-of-function mutations in Fgfr3 which encodes a receptor for FGF8 that is essential for pillar cell development. We found that the expression of CD44 is abolished from the immature pillar cells in homozygous Fgfr3 knockout mice. In contrast, both the outer pillar cells and the aberrant Deiters’ cells in the Fgfr3P244R/+ mice express CD44. The deafness phenotype segregating in DFNB51 families maps to a linkage interval that includes CD44. To study the potential role of CD44 in hearing, we characterized the auditory system of CD44 knockout mice and sequenced the entire open reading frame of CD44 of affected members of DFNB51 families. Our results suggest that CD44 does not underlie the deafness phenotype of the DFNB51 families. Finally, our study reveals multiple potential new cell type-specific markers in the mouse inner ear and identifies a new marker for outer pillar cells.

Genetic Analysis through OtoSeq of Pakistani Families Segregating Prelingual Hearing Loss

Objective To identify the genetic cause of prelingual sensorineural hearing loss in Pakistani families using a next-generation sequencing (NGS)–based mutation screening test named OtoSeq. Study Design Prospective study. Setting Research laboratory. Subjects and Methods We used 3 fluorescently labeled short tandem repeat (STR) markers for each of the known autosomal recessive nonsyndromic (DFNB) and Usher syndrome (USH) locus to perform a linkage analysis of 243 multigenerational Pakistani families segregating prelingual hearing loss. After genotyping, we focused on 34 families with potential linkage to MYO7A, CDH23, and SLC26A4. We screened affected individuals from a subset of these families using the OtoSeq platform to identify underlying genetic variants. Sanger sequencing was performed to confirm and study the segregation of mutations in other family members. For novel mutations, normal hearing individuals from ethnically matched backgrounds were also tested. Results Hearing loss was found to co-segregate with locus-specific STR markers for MYO7A in 32 families, CDH23 in 1 family, and SLC26A4 in 1 family. Using the OtoSeq platform, a microdroplet PCR-based enrichment followed by NGS, we identified mutations in 28 of the 34 families including 11 novel mutations. Sanger sequencing of these mutations showed 100% concordance with NGS data and co-segregation of the mutant alleles with the hearing loss phenotype in the respective families. Conclusion Using NGS-based platforms like OtoSeq in families segregating hearing loss will contribute to the identification of common and population-specific mutations, early diagnosis, genetic counseling, and molecular epidemiology.