Hearing impairment (HI) affects 1 in 650 newborns, which makes it the most common congenital sensory impairment. Despite extraordinary genetic heterogeneity, mutations in one gene, GJB2, which encodes the connexin 26 protein and is involved in inner ear homeostasis, are found in up to 50% of patients with autosomal recessive nonsyndromic hearing loss. Because of the high frequency of GJB2 mutations, mutation analysis of this gene is widely available as a diagnostic test. In this study, we assessed the association between genotype and degree of hearing loss in persons with HI and biallelic GJB2 mutations. We performed cross-sectional analyses of GJB2 genotype and audiometric data from 1,531 persons, from 16 different countries, with autosomal recessive, mildto-profound nonsyndromic HI. The median age of all participants was 8 years; 90% of persons were within the age range of 0-26 years. Of the 83 different mutations identified, 47 were classified as nontruncating, and 36 as truncating. A total of 153 different genotypes were found, of which 56 were homozygous truncating (T/T), 30 were homozygous nontruncating (NT/NT), and 67 were compound heterozygous truncating/nontruncating (T/ NT). The degree of HI associated with biallelic truncating mutations was significantly more severe than the HI associated with biallelic nontruncating mutations (). The HI of 48 different genotypes was less severe P ! .0001 than that of 35delG homozygotes. Several common mutations (M34T, V37I, and L90P) were associated with mildto-moderate HI (median 25-40 dB). Two genotypes-35delG/R143W (median 105 dB) and 35delG/dela(GJB6-D13S1830) (median 108 dB)-had significantly more-severe HI than that of 35delG homozygotes.

Autosomal recessive nonsyndromic hearing impairment (NSHI) is a heterogeneous condition, for which 53 genetic loci have been reported, and 29 genes have been identified to date. One of these, OTOF, encodes otoferlin, a membrane-anchored calcium-binding protein that plays a role in the exocytosis of synaptic vesicles at the auditory inner hair cell ribbon synapse. We have investigated the prevalence and spectrum of deafness-causing mutations in the OTOF gene. Cohorts of 708 Spanish, 83 Colombian, and 30 Argentinean unrelated subjects with autosomal recessive NSHI were screened for the common p.Gln829X mutation. In compound heterozygotes, the second mutant allele was identified by DNA sequencing. In total, 23 Spanish, two Colombian and two Argentinean subjects were shown to carry two mutant alleles of OTOF. Of these, one Colombian and 13 Spanish subjects presented with auditory neuropathy. In addition, a cohort of 20 unrelated subjects with a diagnosis of auditory neuropathy, from several countries, was screened for mutations in OTOF by DNA sequencing. A total of 11 of these subjects were shown to carry two mutant alleles of OTOF. In total, 18 pathogenic and four neutral novel alleles of the OTOF gene were identified. Haplotype analysis for markers close to OTOF suggests a common founder for the novel c.2905_2923delinsCTCCGAGCGCA mutation, frequently found in Argentina. Our results confirm that mutation of the OTOF gene correlates with a phenotype of prelingual, profound NSHI, and indicate that OTOF mutations are a major cause of inherited auditory neuropathy.

The mode of inheritance of Alport syndrome (ATS) has long been controversial. In 1927, the disease was hypothesized as a dominant condition in which males were more severely affected than females. In 1990, it was considered an X-linked (XL) semidominant condition, due to COL4A5 mutations. Later on, a rare autosomal recessive (AR) form due to COL4A3/COL4A4 mutations was identified. An autosomal dominant (AD) form was testified more recently by the description of some large pedigrees but the real existence of this form is still questioned by many and its exact prevalence is unknown. The introduction of next generation sequencing (NGS) allowed us to perform an unbiased simultaneous COL4A3-COL4A4-COL4A5 analysis in 87 Italian families (273 individuals) with clinical suspicion of ATS. In 48 of them (55%), a mutation in one of the three genes was identified: the inheritance was XL semidominant in 65%, recessive in 4% and most interestingly AD in 31% (15 families). The AD form must therefore be seriously taken into account in all pedigrees with affected individuals in each generation. Furthermore, a high frequency of mutations (>50%) was shown in patients with only 1 or 2 clinical criteria, suggesting NGS as first-level analysis in cases with a clinical suspicion of ATS.

Human ciliopathies are hereditary conditions caused by defects of proteins expressed at the primary cilium. Among ciliopathies, Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS) and nephronophthisis (NPH) present clinical and genetic overlap, being allelic at several loci. One of the most interesting gene is TMEM67, encoding the transmembrane protein meckelin. We performed mutation analysis of TMEM67 in 341 probands, including 265 JSRD representative of all clinical subgroups and 76 MKS fetuses. We identified 33 distinct mutations, of which 20 were novel, in 8/10 (80%) JS with liver involvement (COACH phenotype) and 12/76 (16%) MKS fetuses. No mutations were found in other JSRD subtypes, confirming the strong association between TMEM67 mutations and liver involvement. Literature review of all published TMEM67 mutated cases was performed to delineate genotype-phenotype correlates. In particular, comparison of the types of mutations and their distribution along the gene in lethal versus non lethal phenotypes showed in MKS patients a significant enrichment of missense mutations falling in TMEM67 exons 8 to 15, especially when in combination with a truncating mutation. These exons encode for a region of unknown function in the extracellular domain of meckelin. ©2010Wiley-Liss, Inc.

The miR-96, miR-182 and miR-183 microRNA (miRNA) family is essential for differentiation and function of the vertebrate inner ear. Recently, point mutations within the seed region of miR-96 were reported in two Spanish families with autosomal dominant non-syndromic sensorineural hearing loss (NSHL) and in a mouse model of NSHL. We screened 882 NSHL patients and 836 normal-hearing Italian controls and identified one putative novel mutation within the miR-96 gene in a family with autosomal dominant NSHL. Although located outside the mature miR-96 sequence, the detected variant replaces a highly conserved nucleotide within the companion miR-96*, and is predicted to reduce the stability of the pre-miRNA hairpin. To evaluate the effect of the detected mutation on miR-96/mir-96* biogenesis, we investigated the maturation of miR-96 by transient expression in mammalian cells, followed by real-time reverse-transcription polymerase chain reaction (PCR). We found that both miR-96 and miR-96* levels were significantly reduced in the mutant, whereas the precursor levels were unaffected. Moreover, miR-96 and miR-96* expression levels could be restored by a compensatory mutation that reconstitutes the secondary structure of the pre-miR-96 hairpin, demonstrating that the mutation hinders precursor processing, probably interfering with Dicer cleavage. Finally, even though the mature miR-96 sequence is not altered, we demonstrated that the identified mutation significantly impacts on miR-96 regulation of selected targets. In conclusion, we provide further evidence of the involvement of miR-96 mutations in human deafness and demonstrate that a quantitative defect of this miRNA may contribute to NSHL.

BACKGROUND: Hemolytic uremic syndrome associated with Shiga toxin-producing Escherichia coli (STEC-HUS) is a severe acute illness without specific treatment except supportive care; fluid management is concentrated on preventing fluid overload for patients, who are often oligoanuric. Hemoconcentration at onset is associated with more severe disease, but the benefits of volume expansion after hemolytic uremic syndrome (HUS) onset have not been explored.

Two familial and seven sporadic patients with neurofibromatosis 1-who showed dysmorphism, learning disabilities/mental retardation, and additional signs and carried deletions of the NF1 gene-were investigated by use of a two-step FISH approach to characterize the deletions. With FISH of YAC clones belonging to a 7-Mb 17q11.2 contig, we estimated the extension of all of the deletions and identified the genomic regions harboring the breakpoints. Mosaicism accounted for the mild phenotype in two patients. In subsequent FISH experiments, performed with locus-specific probes generated from the same YACs by means of a novel procedure, we identified the smallest region of overlapping (SRO), mapped the deletion breakpoints, and identified the genes that map to each deletion interval. From centromere to telomere, the approximately 0.8-Mb SRO includes sequence-tagged site 64381, the SUPT6H gene (encoding a transcription factor involved in chromatin structure), and NF1. Extending telomerically from the SRO, two additional genes-BLMH, encoding a hydrolase involved in bleomycin resistance, and ACCN1, encoding an amiloride-sensitive cation channel expressed in the CNS-were located in the deleted intervals of seven and three patients, respectively. An apparently common centromeric deletion breakpoint was shared by all of the patients, whereas a different telomeric breakpoint defined a deletion interval of 0.8-3 Mb. There was no apparent correlation between the extent of the deletion and the phenotype. This characterization of gross NF1 deletions provides the premise for addressing correctly any genotype-phenotype correlation in the subset of patients with NF1 deletions.

The imprinted expression of the IGF2 and H19 genes is controlled by the Imprinting Centre 1 (IC1) at chromosome 11p15.5. This is a methylation-sensitive chromatin insulator that works by binding the zinc-finger protein CTCF in a parent-specific manner. Microdeletions abolishing some of the CTCF target sites (CTSs) of IC1 have been associated with the Beckwith-Wiedemann syndrome (BWS). However, the link between these mutations and the molecular and clinical phenotypes was debated. We have identified two novel families with IC1 deletions, in which individuals with the clinical features of the BWS are present in multiple generations. By analysing the methylation pattern at the IGF2-H19 locus together with the clinical phenotypes in the individuals with maternal and those with paternal transmission of five different deletions, we demonstrate that maternal transmission of 1.4-1.8 kb deletions in the IC1 region co-segregates with the hypermethylation of the residual CTSs and BWS phenotype with complete penetrance, whereas normal phenotype is observed upon paternal transmission. Although gene expression could not be assayed in all cases, the methylation detected at the IGF2 DMR2 and H19 promoter suggests that IC1 hypermethylation is consistently associated with biallelic activation of IGF2 and biallelic silencing of H19. Comparison of these deletions with a 2.2 kb one previously reported by another group indicates that the spacing of the CTSs on the deleted allele is critical for the gain of the abnormal methylation and penetrance of the clinical phenotype. Furthermore, we observe that the hypermethylation resulting from the deletions is always mosaic, suggesting that the epigenetic defect at the IGF2-H19 locus is established post-zygotically and may cause body asymmetry and heterogeneity of the clinical phenotype. Finally, the IC1 microdeletions are associated with a high incidence of Wilms' tumour, making their molecular diagnosis particularly important for genetic counselling and tumour surveillance at follow-up.