Compound Heterozygosity for OTOA Truncating Variant and Genomic Rearrangement Cause Autosomal Recessive Sensorineural Hearing Loss in an Italian Family.

Hearing loss (HL) affects 1-3 newborns per 1000 and, in industrialized countries, recognizes a genetic etiology in more than 80% of the congenital cases. Excluding GJB2 and GJB6, OTOA is one of the leading genes associated with autosomal recessive non-syndromic HL. Allelic heterogeneity linked to OTOA also includes genomic rearrangements facilitated by non-allelic homologous recombination with the neighboring OTOAP1 pseudogene. We present a couple of Italian siblings affected by moderate to severe sensorineural hearing loss (SNHL) due to compound heterozygosity at the OTOA locus. Multigene panel next-generation sequencing identified the c.2223G>A, p.(Trp741*) variant transmitted from the unaffected mother. Assuming the existence of a second paternal deleterious variant which evaded detection at sequencing, genomic array analysis found a ~150 Kb microdeletion of paternal origin and spanning part of OTOA. Both deleterious alleles were identified for the first time. This study demonstrates the utility of an integrated approach to solve complex cases and allow appropriate management to affected individuals and at-risk relatives.

Could the interaction between LMX1B and PAX2 influence the severity of renal symptoms?

Nail Patella syndrome (NPS) is a rare autosomal dominant disease characterized by varying degrees of patella, nail, and elbows dysplasia and also ocular and renal congenital abnormalities. The renal involvement, ranging from hematuria and proteinuria to end-stage renal disease, is present in 22-60% of NPS cases. Heterozygous variants in LMX1B are known to be responsible of NPS and it has been hypothesized that the variable expressivity is due to the interaction of LMX1B with other developmental genes. We reported a case of co-presence of LMX1B and PAX2 variants in a child with extrarenal manifestation of NPS and end-stage renal disease but congenital bilateral renal hypodysplasia and vesicoureteral reflux. The LMX1B variant was de novo, whereas the PAX2 variant was inherited from the mother that had bilateral renal hypoplasia although in presence of only a mild chronic kidney disease. The molecular interaction between LMX1B and PAX2 has been already reported in vitro and this finding suggest that the worst renal NPS phenotype of our patient could be due to the defective expression of these two genes during nephrogenesis. In conclusion, our finding suggests that PAX2 may act as modifier gene in Nail Patella phenotype.

Putative TMPRSS3/GJB2 digenic inheritance of hearing loss detected by targeted resequencing.

The paper describes a putative digenic form of deafness in two siblings affected by non-syndromic hereditary hearing loss, detected by a Targeted resequencing approach. Given that a previous paper suggested TMPRSS3 and GJB2 genes as responsible for a digenic form of hearing loss, our data support and reinforce this hypothesis.

Expanding the mutation spectrum in 130 probands with ARPKD: identification of 62 novel PKHD1 mutations by sanger sequencing and MLPA analysis.

Autosomal recessive polycystic kidney disease (ARPKD) is a rare severe genetic disorder arising in the perinatal period, although a late-onset presentation of the disease has been described. Pulmonary hypoplasia is the major cause of morbidity and mortality in the newborn period. ARPKD is caused by mutations in the PKHD1 (polycystic kidney and hepatic disease 1) gene that is among the largest human genes. To achieve a molecular diagnosis of the disease, a large series of Italian affected subjects were recruited. Exhaustive mutation analysis of PKHD1 gene was carried out by Sanger sequencing and multiple ligation probe amplification (MLPA) technique in 110 individuals. A total of 173 mutations resulting in a detection rate of 78.6% were identified. Additional 20 unrelated patients, in whom it was not possible to analyze the whole coding sequence, have been included in this study. Taking into account the total number (n=130) of this cohort of patients, 107 different types of mutations have been detected in 193 mutated alleles. Out of 107 mutations, 62 were novel: 11 nonsense, 6 frameshift, 7 splice site mutations, 2 in-frame deletions and 2 multiexon deletion detected by MLPA. Thirty-four were missense variants. In conclusion, our report expands the spectrum of PKHD1 mutations and confirms the heterogeneity of this disorder. The population under study represents the largest Italian ARPKD cohort reported to date. The estimated costs and the time invested for molecular screening of genes with large size and allelic heterogeneity such as PKHD1 demand the use of next-generation sequencing (NGS) technologies for a faster and cheaper screening of the affected subjects.

EYA1-related disorders: two clinical cases and a literature review.

OBJECTIVES: To delineate the diagnostic and rehabilitative aspects of syndromes that have overlapping features, we present the cases of two unrelated Caucasian males affected by hearing impairment, preauricular pits and cervical fistulae. Specific findings that are helpful in the diagnosis and management of EYA1-related disorders are highlighted. METHODS: Genetic, otologic, imaging, eye and renal evaluations were conducted to achieve a detailed and comprehensive assessment, leading to the most accurate diagnosis and appropriate treatment. A literature review was also carried out. RESULTS: Diagnostic criteria indicated that the two patients were affected by BOS1 (Branchio-Otic Syndrome 1). We also identified a novel sporadic missense mutation in the EYA1 gene: p.G533R (c.1597G>A, NM_000503.4), a highly conserved, heterozygotic amino acid substitution. In the other case, we identified the p.X593QextX6 (c.1777T>A, NM_000503.4) substitution. Both variants lead to isoform 1 (EYA1B and EYA1C) which is composed of 592 amino acids. Clinical and in silico evidence suggests a pathogenic role for the new mutations. Imaging evaluation revealed a complex pathology, characterized by external, inner and middle ear malformations, without renal anomalies. CONCLUSIONS: Our results demonstrate the importance of considering the imaging evaluation and the complete DNA sequencing of the EYA1 gene for the differential diagnosis of deafness and related branchio-oto-renal disorders.

Patient affected by neurofibromatosis type 1 and thyroid C-cell hyperplasia harboring pathogenic germ-line mutations in both NF1 and RET genes.

Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disease with an estimated incidence of 1 in 3000/3500 live births. NF1 is caused by a mutation in a gene which encodes a protein known as neurofibromin. In up to 5% of cases, NF1 is associated with pheochromocytomas. RET proto-oncogene encodes a member of the receptor tyrosine kinase family involved in the normal development or the neoplastic growth of neural crest cell lineages. Germ-line RET mutations account for cases of Multiple Endocrine Neoplasia type 2 (MEN2), an autosomal dominant genetic syndrome where medullary thyroid carcinoma (MTC) is the major and more clinically severe feature, with nearly complete penetrance. C-cell hyperplasia (CCH) is described in MEN2 patients, and it has been implicated as the precursor of in situ MTC. Patients with RET mutations develop pheochromocytomas in 50% of cases. Rarely, patients with NF1 have been found to present, in addition to the NF1 clinical picture, other lesions, such as parathyroid hyperplasia/adenoma and/or medullary thyroid carcinoma. In spite of the presence of these MEN2 lesions, in none of these patients mutations of gene RET have been found so far. In this report, we describe the first case of a patient affected by a germ-line mutation in both NF1 and RET genes.

Identification of a novel mutation in the SLC26A4 gene in an Italian with fluctuating sensorineural hearing loss.

Pendred syndrome is an autosomal recessive disorder characterized by congenital sensorineural deafness, goitre and defective iodide organification. Congenital and profound hearing loss is the hallmark of the syndrome, while goitre and thyroid dysfunction are highly variable even within the same family. Clinical features are due to altered formation of pendrin, a chloride/iodide transporter protein expressed in the inner ear, thyroid gland and kidney. A novel substitution was found in exon 7 of the pendrin encoding gene (SLC26A4) that leads to a stop codon, S314X. The new variation was found in compound heterozygosity with L445W mutation in a hearing impaired patient with bilateral Mondini's dysplasia and goitre.

Hearing loss features in GJB2 biallelic mutations and GJB2/GJB6 digenic inheritance in a large Italian cohort.

The aim of this study was to describe the clinical features of hearing loss due to mutations on connexin 26/30 coding genes (GJB2/GJB6). Mutations in the GJB2 gene are found to account for approximately 50% of cases of autosomal recessive non-syndromic deafness. Several European studies have estimated that the GJB2 healthy carrier condition involves about 2-4% of the population, with the 35delG mutations being the most common. A 342-kb deletion truncating the GJB6 gene (encoding connexin-30) has been associated with autosomal recessive non-syndromic deafness, mostly as digenic inheritance of the Cx30 deletion/Cx26 mutation. The following retrospective study describes audiological features and genotypes of a large cohort of 376 Italian hearing-impaired patients who underwent genetic screening for the GJB2/GJB6 genes and received follow-up care at our centre between January 2002 and October 2006. Sixteen different genotypes causing deafness in more than 27% of patients with either biallelic mutations or digenic inheritance GJB2/GJB6 were identified. The most frequent mutations were 35delG, M34T, L90P, and R184P.

A novel missense mutation in the Connexin 26 gene associated with autosomal recessive nonsyndromic sensorineural hearing loss in a consanguineous Tunisian family.

Nonsyndromic sensorineural hearing impairment is inherited in a predominantly autosomal recessive manner in up to 70% of cases. The gene more often involved is GJB2, encoding the gap junction protein Connexin 26. We report here a novel missense mutation in the GJB2 gene found in a Tunisian family. A homozygous change C/G at nucleotide 263 was detected in the 4-year-old girl of this family, affected by congenital moderate hearing loss. This transversion leads to the replacement of a highly conserved alanine with glycine at codon 88 (A88G). The consanguineous parents of the child are healthy carriers of the mutation.

Are MYO1C and MYO1F associated with hearing loss?

The role of myosins in the pathogenesis of hearing loss is well established: five genes encoding unconventional myosins and two genes encoding nonmuscle conventional myosins have so far been described to be essential for normal auditory function and mutations in these genes associated with hearing impairment. To better understand the role of this gene family we performed a mutational screening on two candidate genes, MYO1C and MYO1F, analyzing hundreds of patients, affected by bilateral sensorineural hearing loss and coming from different European countries. This research activity led to the identification of 6 heterozygous missense mutations in MYO1C and additional 5 heterozygous missense mutations in MYO1F. Homology modelling suggests that some of these mutations could have a potential influence on the structure of the ATP binding site and could probably affect the ATPase activity or the actin binding process of both myosins. This study suggests a role of the above mentioned myosin genes in the pathogenesis of hearing loss.

Pathogenetic role of the deafness-related M34T mutation of Cx26.

Mutations in the GJB2 gene, which encodes the gap junction protein connexin26 (Cx26), are the major cause of genetic non-syndromic hearing loss. The role of the allelic variant M34T in causing hereditary deafness remains controversial. By combining genetic, clinical, biochemical, electrophysiological and structural modeling studies, we have re-assessed the pathogenetic role of the M34T mutation. Genetic and audiological data indicate that the majority of heterozygous carriers and all five compound heterozygotes exhibited an impaired auditory function. Functional expression in transiently transfected HeLa cells showed that, although M34T was correctly synthesized and targeted to the plasma membrane, it inefficiently formed intercellular channels that displayed an abnormal electrical behavior and retained only 11% of the unitary conductance of the wild-type protein (HCx26wt). Moreover, M34T channels failed to support the intercellular diffusion of Lucifer Yellow and the spreading of mechanically induced intercellular Ca2+ waves. When co-expressed together with HCx26wt, M34T exerted dominant-negative effects on cell-cell coupling. Our findings are consistent with a structural model, predicting that the mutation leads to a constriction of the channel pore. These data support the view that M34T is a pathological variant of Cx26 associated with hearing impairment.

Functional characterization of a novel Cx26 (T55N) mutation associated to non-syndromic hearing loss.

Mutations of the GJB2 gene, encoding connexin 26, are the most common cause of hereditary congenital hearing loss in many countries and account for up to 50% of cases of autosomal-recessive non-syndromic deafness. By contrast, only a few GJB2 mutations have been reported to cause an autosomal-dominant form of non-syndromic deafness. Here, we report a family from Southern Italy affected by non-syndromic autosomal dominant post-lingual hearing loss, due to a novel missense mutation in the GJB2 gene, a threonine to asparagine amino acid substitution at codon 55 (T55N). Functional studies indicated that the mutation T55N produces a protein that, although expressed to levels similar to those of the wt counterpart, is deeply impaired in its intracellular trafficking and fails to reach the plasma membrane. The mutation T55N is located at the apex of the first extracellular loop of the protein, a region suggested to play a role in protein targeting and a site for other two mutations, G59A and D66H, causing dominant forms of deafness.

Mitochondrial DNA mutations in patients with postlingual, nonsyndromic hearing impairment.

Mitochondrial mutations have previously been reported anecdotally in families with maternally inherited, nonsyndromic hearing impairment. To ascertain the contribution of mitochondrial mutations to postlingual but early-onset, nonsyndromic hearing impairment, we screened patients collected from within two different populations (southern Italy and UK) for previously reported mtDNA mutations associated with hearing disorders. Primer extension (SNP analysis) was used to screen for specific mutations, revealing cases of heteroplasmy and its extent. The most frequently implicated tRNA genes, Leu(UUR) and Ser(UCN), were also sequenced in all Italian patients. All tRNA genes were sequenced in those UK patients showing the clearest likelihood of maternal inheritance. Causative mtDNA mutations were found in approximately 5% of patients in both populations, representing almost 10% of cases that were clearly familial. Age of onset, where known, was generally before adulthood, and hearing loss was typically progressive. Haplogroup analysis revealed a possible excess of haplogroup cluster HV in the patients, compared with population controls, but of borderline statistical significance. In contrast, we did not find any of the previously reported mtDNA mutations, nor a significant deviation from haplogroup cluster frequencies typical of the control population, in patients with late adult-onset hearing loss (age-related hearing impairment) from the UK or Finland.

Nonmuscle myosin heavy-chain gene MYH14 is expressed in cochlea and mutated in patients affected by autosomal dominant hearing impairment (DFNA4).

Myosins have been implicated in various motile processes, including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Different members of the myosin superfamily are responsible for syndromic and nonsyndromic hearing impairment in both humans and mice. MYH14 encodes one of the heavy chains of the class II nonmuscle myosins, and it is localized within the autosomal dominant hearing impairment (DFNA4) critical region. After demonstrating that MYH14 is highly expressed in mouse cochlea, we performed a mutational screening in a large series of 300 hearing-impaired patients from Italy, Spain, and Belgium and in a German kindred linked to DFNA4. This study allowed us to identify a nonsense and two missense mutations in large pedigrees, linked to DFNA4, as well as a de novo allele in a sporadic case. Absence of these mutations in healthy individuals was tested in 200 control individuals. These findings clearly demonstrate the role of MYH14 in causing autosomal dominant hearing loss and further confirm the crucial role of the myosin superfamily in auditive functions.

Multiple mutations of MYO1A, a cochlear-expressed gene, in sensorineural hearing loss.

Myosin I isozymes have been implicated in various motile processes, including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Unconventional myosins were among the first family of proteins found to be associated with hearing loss in both humans and mice. Here, we report the identification of a nonsense mutation, of a trinucleotide insertion leading to an addition of an amino acid, and of six missense mutations in MYO1A cDNA sequence in a group of hearing-impaired patients from Italy. MYO1A, which is located within the DFNA48 locus, is the first myosin I family member found to be involved in causing deafness and may be a major contributor to autosomal dominant-hearing loss.

A new locus (DFNA47) for autosomal dominant non-syndromic inherited hearing loss maps to 9p21-22 in a large Italian family.

Hearing loss is the most common sensory disorder in humans, and genetic factors are a major cause. Approximately 15-20% of genetic cases exhibit an autosomal dominant pattern of transmission. So far, 41 autosomal dominant loci have been mapped and 17 genes have been identified. Here we report the mapping of a novel locus for autosomal dominant non-syndromic hearing loss, DFNA47, to chromosome 9p21-22 in a large multigenerational Italian family with progressive hearing impairment. Most affected individuals noticed hearing impairment after their teens with subsequent gradual progression to a moderate-severe loss. There were no obvious vestibular dysfunction and other associated abnormalities. A maximum lod score of 3.14 was obtained with marker D9S157 (at theta=0) after a genome wide search. The study of additional markers allowed us to confirm this region with positive lod scores of 3.58 (at theta=0 from D9S285) and of 3.67 (at theta=0 from D9S162). Recombinants define a region of approximately 9 cM flanked by markers D9S268 and D9S942. Multipoint linkage analysis showed a Lod score of 4.26. Few known genes map to the region, and those possibly related by function to hearing are being screened for disease-causing mutations.

Hearing loss: frequency and functional studies of the most common connexin26 alleles.

Mutations in the GJB2 gene, encoding the gap-junction channel protein connexin 26, account for the majority of recessive forms and some of the dominant cases of deafness. Here, we report the frequency of GJB2 alleles in the Italian population affected by hearing loss and the functional analysis of six missense mutations. Genetic studies indicate that, apart from the common 35delG, only few additional mutations can be detected with a significant frequency in our population. Transfection of communication-incompetent HeLa cells with Cx26 missense mutations revealed three distinct classes of functional deficits in terms of protein expression, subcellular localisation and/or functional activity. Moreover, the M34T mutant acted as a dominant inhibitor of wild-type Cx26 channel activity when the two proteins were co-expressed in a manner mimicking a heterozygous genotype. These data support the hypothesis of a functional role for M34T as a dominant allele and represent a further step towards a complete understanding of the role of GJB2 in causing hearing loss.

A common frameshift mutation and other variants in GJB4 (connexin 30.3): Analysis of hearing impairment families.

Mutations in GJB1, GJB2, GJB3 and GJB6 are involved in hearing impairment. GJB2, GJB3 and GJB6 are also mutated in patients with hyperproliferative skin disorders. The human GJB4 gene has been deduced in silico and a mutation in a family with erythrokeratodermia variabilis has been reported. We describe here the analysis of the GJB4 gene in hearing impairment patients and control subjects. We have identified a common (4%) frameshift mutation (154del4) in GJB4 in both affected and hearing subjects, one patient being homozygous for the mutation. We have also detected five amino acid variants (R103C, R124Q, R160C, C169W and E204A) in individuals that have not skin disorders. While mutation 154del4 is not associated with hearing impairment the involvement of some of the amino acid variants detected here is uncertain. These GJB4 variants should help to define the putative role of connexin 30.3 in both skin disorders and hearing impairment.