OSBPL2 encodes a protein of inner and outer hair cell stereocilia and is mutated in autosomal dominant hearing loss (DFNA67)

Background: Early-onset hearing loss is mostly of genetic origin. The complexity of the hearing process is reflected by its extensive genetic heterogeneity, with probably many causative genes remaining to be identified. Here, we aimed at identifying the genetic basis for autosomal dominant non-syndromic hearing loss (ADNSHL) in a large German family. Methods: A panel of 66 known deafness genes was analyzed for mutations by next-generation sequencing (NGS) in the index patient. We then conducted genome-wide linkage analysis, and whole-exome sequencing was carried out with samples of two patients. Expression of Osbpl2 in the mouse cochlea was determined by immunohistochemistry. Because Osbpl2 has been proposed as a target of miR-96, we investigated homozygous Mir96 mutant mice for its upregulation. Results: Onset of hearing loss in the investigated ADNSHL family is in childhood, initially affecting the high frequencies and progressing to profound deafness in adulthood. However, there is considerable intrafamilial variability. We mapped a novel ADNSHL locus, DFNA67, to chromosome 20q13.2-q13.33, and subsequently identified a co-segregating heterozygous frameshift mutation, c.141-142delTG (p.Arg50Alafs∗103), in OSBPL2, encoding a protein known to interact with the DFNA1 protein, DIAPH1. In mice, Osbpl2 was prominently expressed in stereocilia of cochlear outer and inner hair cells. We found no significant Osbpl2 upregulation at the mRNA level in homozygous Mir96 mutant mice. Conclusion: The function of OSBPL2 in the hearing process remains to be determined. Our study and the recent description of another frameshift mutation in a Chinese ADNSHL family identify OSBPL2 as a novel gene for progressive deafness.</p

Non-manifesting AHI1 truncations indicate localized loss-of-function tolerance in a severe Mendelian disease gene

Determination of variant pathogenicity represents a major challenge in the era of high-throughput sequencing. Erroneous categorization may result if variants affect genes that are in fact dispensable. We demonstrate that this also applies to rare, apparently unambiguous truncating mutations of an established disease gene. By whole-exome sequencing (WES) in a consanguineous family with congenital non-syndromic deafness, we unexpectedly identified a homozygous nonsense variant, p.Arg1066*, in AHI1, a gene associated with Joubert syndrome (JBTS), a severe recessive ciliopathy. None of four homozygotes expressed any signs of JBTS, and one of them had normal hearing, which also ruled out p.Arg1066* as the cause of deafness. Homozygosity mapping and WES in the only other reported JBTS family with a homozygous C-terminal truncation (p.Trp1088Leufs*16) confirmed AHI1 as disease gene, but based on a more N-terminal missense mutation impairing WD40-repeat formation. Morpholinos against N-terminal zebrafish Ahi1, orthologous to where human mutations cluster, produced a ciliopathy, but targeting near human p.Arg1066 and p.Trp1088 did not. Most AHI1 mutations in JBTS patients result in truncated protein lacking WD40-repeats and the SH3 domain; disease was hitherto attributed to loss of these protein interaction modules. Our findings indicate that normal development does not require the C-terminal SH3 domain. This has far-reaching implications, considering that variants like p.Glu984* identified by preconception screening (‘Kingsmore panel') do not necessarily indicate JBTS carriership. Genomes of individuals with consanguineous background are enriched for homozygous variants that may unmask dispensable regions of disease genes and unrecognized false positives in diagnostic large-scale sequencing and preconception carrier screenin

The supposed tumor suppressor gene WWOX is mutated in an early lethal microcephaly syndrome with epilepsy, growth retardation and retinal degeneration

Background: WWOX, encoding WW domain-containing oxidoreductase, spans FRA16D, the second most common chromosomal fragile site frequently altered in cancers. It is therefore considered a tumor suppressor gene, but its direct implication in cancerogenesis remains controversial. Methods and results: By whole-exome sequencing, we identified a homozygous WWOX nonsense mutation, p.Arg54*, in a girl from a consanguineous family with a severe syndrome of growth retardation, microcephaly, epileptic seizures, retinopathy and early death, a phenotype highly similar to the abormalities reported in lde/lde rats with a spontaneous functional null mutation of Wwox. As in rats, no tumors were observed in the patient or heterozygous mutation carriers. Conclusions: Our finding, a homozygous loss-of-function germline mutation in WWOX in a patient with a lethal autosomal recessive syndrome, supports an alternative role of WWOX and indicates its importance for human viability

Recurrent and Prolonged Infections in a Child with a Homozygous IFIH1 Nonsense Mutation

In an Egyptian girl born to consanguineous parents, whole-exome sequencing (WES) identified a homozygous mutation in PHGDH, c. 1273G>A (p.Val425Met), indicating 3-phosphoglycerate dehydrogenase deficiency. This diagnosis was compatible with the patient's microcephaly, severe psychomotor retardation, seizures and cataracts. However, she additionally suffered from recurrent (at least monthly) episodes of prolonged and severe chest infections requiring hospitalization, suggesting a secondary, predisposing and potentially Mendelian, condition. A local reactivation of an EBV infection in the respiratory tract was detected after a recent chest infection, likely representing an opportunistic infection based on a compromised immune system. Further inspection of WES data revealed a homozygous nonsense mutation, c.2665A>T (p.Lys889*), in IFIH1, encoding MDA5. MDA5 detects long viral double-stranded RNA that is generated during replication of picorna viruses, and thereby activates the type I interferon signaling pathway. The results of Western blot analysis of protein from cultured fibroblasts of the patient indicates absence of wild type MDA5/ IFIH1, compatible with NMD. We propose that, analogous to the severe course of primary influenza infection due to biallelic deficiency of a downstream effector, IRF7, homozygous loss of IFIH1 defines a novel Mendelian immunodeficiency disorder that increases susceptibility to severe viral infections. This is contrasted to heterozygous gain-offunction IFIH1 mutations in autoimmune diseases. Our findings highlight the potential of comprehensive genomic investigations in patients from consanguineous families to identify monogenic predispositions to severe infections

A TUBB6 mutation is associated with autosomal dominant non-progressive congenital facial palsy, bilateral ptosis and velopharyngeal dysfunction

Congenital cranial dysinnervation disorders (CCDDs) comprise a heterogeneous spectrum of diseases characterized by congenital, non-progressive impairment of eye, eyelid and/or facial movements including Mobius syndrome, Duane retraction syndrome, congenital ptosis, and congenital fibrosis of the extraocular muscles. Over the last 20 years, several CCDDs have been identified as neurodevelopmental disorders that are caused by mutations of genes involved in brain and cranial nerve development, e.g. KIF21A and TUBB3 that each plays a pivotal role for microtubule function. In a five-generation pedigree, we identified a heterozygous mutation of TUBB6, a gene encoding a class V tubulin which has not been linked to a human hereditary disease so far. The missense mutation (p.Phe394Ser) affects an amino acid residue highly conserved in evolution, and co-segregates with a phenotype characterized by congenital non-progressive bilateral facial palsy and congenital velopharyngeal dysfunction presenting with varying degrees of hypomimia, rhinophonia, impaired gag reflex and bilateral ptosis. Expression of the mutated protein in yeast led to an impaired viability compared to wildtype cells when exposed to the microtubule-poison benomyl. Our findings enlarge the spectrum of tubulinopathies and emphasize that mutations of TUBB6 should be considered in patients with congenital non-progressive facial palsy. Further studies are needed to verify whether this phenotype is indeed part of the CCDD spectrum

PEX6 is Expressed in Photoreceptor Cilia and Mutated in Deafblindness with Enamel Dysplasia and Microcephaly

Deafblindness is part of several genetic disorders. We investigated a consanguineous Egyptian family with two siblings affected by congenital hearing loss and retinal degeneration, initially diagnosed as Usher syndrome type 1. At teenage, severe enamel dysplasia, developmental delay, and microcephaly became apparent. Genome-wide homozygosity mapping and whole-exome sequencing detected a homozygous missense mutation, c.1238G>T (p.Gly413Val), affecting a highly conserved residue of peroxisomal biogenesis factor 6, PEX6. Biochemical profiling of the siblings revealed abnormal and borderline plasma phytanic acid concentration, and cerebral imaging revealed white matter disease in both. We show that Pex6 localizes to the apical extensions of secretory ameloblasts and differentiated odontoblasts at early stages of dentin synthesis in mice, and to cilia of retinal photoreceptor cells. We propose PEX6, and possibly other peroxisomal genes, as candidate for the rare cooccurrence of deafblindness and enamel dysplasia. Our study for the first time links peroxisome biogenesis disorders to retinal ciliopathies. (C) 2015 Wiley Periodicals, Inc

Generation of a set of isogenic iPSC lines carrying all APOE genetic variants (epsilon 2/epsilon 3/epsilon 4) and knock-out for the study of APOE biology in health and disease

APOE genotype is the strongest genetic risk factor for Alzheimer's Disease (AD). The low degree of homology between mouse and human APOE is a concerning issue in preclinical models currently used to study the role of this gene in AD pathophysiology. A key objective of ADAPTED (Alzheimer's Disease Apolipoprotein Pathology for Treatment Elucidation and Development) project was to generate in vitro models that better recapitulate human APOE biology. We describe a new set of induced pluripotent stem cells (iPSC) lines carrying common APOE variants (epsilon 2,epsilon 3, and epsilon 3/epsilon 4) and a knock-out isogenic to the parental APOE epsilon 4/epsilon 4 line (UKBi011-A)

Autosomal dominant SCA5 and autosomal recessive infantile SCA are allelic conditions resulting from SPTBN2 mutations

Although many genes have been identified for the autosomal recessive cerebellar ataxias (ARCAs), several patients are unlinked to the respective loci, suggesting further genetic heterogeneity. We combined homozygosity mapping and exome sequencing in a consanguineous Egyptian family with congenital ARCA, mental retardation and pyramidal signs. A homozygous 5-bp deletion in SPTBN2, the gene whose in-frame mutations cause autosomal dominant spinocerebellar ataxia type 5, was shown to segregate with ataxia in the family. Our findings are compatible with the concept of truncating SPTBN2 mutations acting recessively, which is supported by disease expression in homozygous, but not heterozygous, knockout mice, ataxia in Beagle dogs with a homozygous frameshift mutation and, very recently, a homozygous SPTBN2 nonsense mutation underlying infantile ataxia and psychomotor delay in a human family. As there was no evidence for mutations in 23 additional consanguineous families, SPTBN2-related ARCA is probably rare

Non-manifesting AHI1 truncations indicate localized loss-of-function tolerance in a severe Mendelian disease gene

Determination of variant pathogenicity represents a major challenge in the era of high-throughput sequencing. Erroneous categorization may result if variants affect genes that are in fact dispensable. We demonstrate that this also applies to rare, apparently unambiguous truncating mutations of an established disease gene. By whole-exome sequencing (WES) in a consanguineous family with congenital non-syndromic deafness, we unexpectedly identified a homozygous nonsense variant, p.Arg1066*, in AHI1, a gene associated with Joubert syndrome (JBTS), a severe recessive ciliopathy. None of four homozygotes expressed any signs of JBTS, and one of them had normal hearing, which also ruled out p.Arg1066* as the cause of deafness. Homozygosity mapping and WES in the only other reported JBTS family with a homozygous C-terminal truncation (p.Trp1088Leufs*16) confirmed AHI1 as disease gene, but based on a more N-terminal missense mutation impairing WD40-repeat formation. Morpholinos against N-terminal zebrafish Ahi1, orthologous to where human mutations cluster, produced a ciliopathy, but targeting near human p.Arg1066 and p.Trp1088 did not. Most AHI1 mutations in JBTS patients result in truncated protein lacking WD40-repeats and the SH3 domain; disease was hitherto attributed to loss of these protein interaction modules. Our findings indicate that normal development does not require the C-terminal SH3 domain. This has far-reaching implications, considering that variants like p.Glu984* identified by preconception screening ('Kingsmore panel') do not necessarily indicate JBTS carriership. Genomes of individuals with consanguineous background are enriched for homozygous variants that may unmask dispensable regions of disease genes and unrecognized false positives in diagnostic large-scale sequencing and preconception carrier screening