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Non-syndromic sensorineural hearing loss (SNHL) is the most common sensory disorder, and it presents a high genetic heterogeneity. As part of our clinical genetic studies, we ascertained a novel mutation in CCDC50 (c.828_858del, p.(Asp276Glufs*40)) segregating with the hearing impairment in a Spanish family with autosomal dominant DFNA44 SNHL that is predicted to disrupt the protein function. To gain insight into the mechanism behind DFNA44 mutations, we analysed two Ccdc50 presumed loss-of-function mouse mutants which showed normal hearing thresholds up to 6 months old, thus indicating that haploinsufficiency is unlikely to be the pathogenic mechanism. We then carried out in vitro studies on a set of artificial mutants and on the p.(Asp276Glufs*40) and p.(Phe292Hisfs*37) human mutations, and determined that only the mutants containing the six amino acid sequence CLENGL as part of their aberrant protein tail showed an abnormal distribution consisting of perinuclear aggregates of the CCDC50-encoded protein Ymer. Therefore, we conclude that the CLENGL sequence is necessary to form the aggregates. Taken together the in vivo and in vitro results obtained in this study suggest that the two Spanish mutations in CCDC50 exert their effect through a dominant-negative or gain of function mechanism rather than by haploinsufficiency.

Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene. PKD-erythroid cells suffer from an energy imbalance caused by a reduction of erythroid pyruvate kinase (RPK) enzyme activity. PKD is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. More than 300 disease-causing mutations have been identified as causing PKD. Most mutations are missense mutations, commonly present as compound heterozygous. Therefore, specific correction of these point mutations might be a promising therapy for the treatment of PKD patients. We have explored the potential of precise gene editing for the correction of different PKD-causing mutations, using a combination of single-stranded oligodeoxynucleotides (ssODN) with the CRISPR/Cas9 system. We have designed guide RNAs (gRNAs) and single-strand donor templates to target four different PKD-causing mutations in immortalized patient-derived lymphoblastic cell lines, and we have detected the precise correction in three of these mutations. The frequency of the precise gene editing is variable, while the presence of additional insertions/deletions (InDels) has also been detected. Significantly, we have identified high mutation-specificity for two of the PKD-causing mutations. Our results demonstrate the feasibility of a highly personalized gene-editing therapy to treat point mutations in cells derived from PKD patients.

Hearing impairment not etiologically associated with clinical signs in other organs (non-syndromic) is genetically heterogeneous, so that over 120 genes are currently known to be involved. The frequency of mutations in each gene and the most frequent mutations vary throughout populations. Here we review the genetic etiology of non-syndromic hearing impairment (NSHI) in Europe. Over the years, epidemiological data were scarce because of the large number of involved genes, whose screening was not cost-effective until implementation of massively parallel DNA sequencing. In Europe, the most common form of autosomal recessive NSHI is DFNB1, which accounts for 11-57% of the cases. Mutations in STRC account for 16% of the recessive cases, and only a few more (MYO15A, MYO7A, LOXHD1, USH2A, TMPRSS3, CDH23, TMC1, OTOF, OTOA, SLC26A4, ADGRV1 and TECTA) have contributions higher than 2%. As regards autosomal-dominant NSHI, DFNA22 (MYO6) and DFNA8/12 (TECTA) represent the most common forms, accounting for 21% and 18% of elucidated cases, respectively. The contribution of ACTG1 and WFS1 drops to 9% in both cases, followed by POU4F3 (6.5%), MYO7A (5%), MYH14 and COL11A2 (4% each). Four additional genes contribute 2.5% each one (MITF, KCNQ4, EYA4, SOX10) and the remaining are residually represented. X-linked hearing loss and maternally-inherited NSHI have minor contributions in most countries. Further knowledge on the genetic epidemiology of NSHI in Europe needs a standardization of the experimental approaches and a stratification of the results according to clinical features, familial history and patterns of inheritance, to facilitate comparison between studies.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR–Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes. This work has been supported by grant nos. PID2019-109087RB-I00 (to R.P.-J.) and RTI2018-101223-B-I00 and PID2021-127644OB-I00 (to L.M.) from the Spanish Ministry of Science and Innovation. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 964764 (to R.P.-J.). The content presented in this document represents the views of the authors, and the European Commission has no liability in respect to the content. We acknowledge financial support from the Spanish Foundation for the Promotion of Research of Amyotrophic Lateral Sclerosis. A.F. acknowledges Spanish Center for Biomedical Network Research on Rare Diseases (CIBERE) intramural funds (no. ER19P5AC756/2021). F.J.M.M. acknowledges research support by Conselleria d’Educació, Investigació, Cultura i Esport from Generalitat Valenciana, research project nos. PROMETEO/2017/129 and PROMETEO/2021/057. M.M. acknowledges funding from CIBERER (grant no. ER19P5AC728/2021). The work has received funding from the Regional Government of Madrid (grant no. B2017/BMD3721 to M.A.M.-P.) and from Instituto de Salud Carlos III, cofounded with the European Regional Development Fund ‘A way to make Europe’ within the National Plans for Scientific and Technical Research and Innovation 2017–2020 and 2021–2024 (nos. PI17/1659, PI20/0429 and IMP/00009; to M.A.M.-P. B.P.K. was supported by an MGH ECOR Howard M. Goodman Award and NIH P01 HL142494.

12 páginas, 6 figuras, Coenzyme A (CoA) is an essential cofactor involved in a range of metabolic pathways including the activation of long-chain fatty acids for catabolism. Cells synthesize CoA de novo from vitamin B5 (pantothenate) via a pathway strongly conserved across prokaryotes and eukaryotes. In humans, it involves five enzymatic steps catalyzed by four enzymes: pantothenate kinase (PANK [isoforms 1-4]), 4'-phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC), and CoA synthase (COASY). To date, inborn errors of metabolism associated with all of these genes, except PPCDC, have been described, two related to neurodegeneration with brain iron accumulation (NBIA), and one associated with a cardiac phenotype. This paper reports another defect in this pathway (detected in two sisters), associated with a fatal cardiac phenotype, caused by biallelic variants (p.Thr53Pro and p.Ala95Val) of PPCDC. PPCDC enzyme (EC 4.1.1.36) catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine in CoA biosynthesis. The variants p.Thr53Pro and p.Ala95Val affect residues highly conserved across different species; p.Thr53Pro is involved in the binding of flavin mononucleotide, and p.Ala95Val is likely a destabilizing mutation. Patient-derived fibroblasts showed an absence of PPCDC protein, and nearly 50% reductions in CoA levels. The cells showed clear energy deficiency problems, with defects in mitochondrial respiration, and mostly glycolytic ATP synthesis. Functional studies performed in yeast suggest these mutations to be functionally relevant. In summary, this work describes a new, ultra-rare, severe inborn error of metabolism due to pathogenic variants of PPCDC., This work was funded by the Instituto de Salud Carlos (ISCIII), the European Regional Development Fund [PI19/01155], the Ministerio de Economía, Industria y Competitividad, Spain (BFU2017-82574-P), and the Consejería de Educacion, Juventud y Deporte, Comunidad de Madrid [B2017/BMD3721].

Collagen VI-related disorders are the second most common congenital muscular dystrophies for which no treatments are presently available. They are mostly caused by dominant-negative pathogenic variants in the genes encoding α chains of collagen VI, a heteromeric network forming collagen; for example, the c.877Ggt;A; p.Gly293Arg

Pathogenic variants in the PJVK gene cause the DFNB59 type of autosomal recessive non-syndromic hearing impairment (AR-NSHI). Phenotypes are not homogeneous, as a few subjects show auditory neuropathy spectrum disorder (ANSD), while others show cochlear hearing loss. The numbers of reported cases and pathogenic variants are still small to establish accurate genotype-phenotype correlations. We investigated a cohort of 77 Spanish familial cases of AR-NSHI, in whom DFNB1 had been excluded, and a cohort of 84 simplex cases with isolated ANSD in whom OTOF variants had been excluded. All seven exons and exon-intron boundaries of the PJVK gene were sequenced. We report three novel DFNB59 cases, one from the AR-NSHI cohort and two from the ANSD cohort, with stable, severe to profound NSHI. Two of the subjects received unilateral cochlear implantation, with apparent good outcomes. Our study expands the spectrum of PJVK mutations, as we report four novel pathogenic variants: p.Leu224Arg, p.His294Ilefs*43, p.His294Asp and p.Phe317Serfs*20. We review the reported cases of DFNB59, summarize the clinical features of this rare subtype of AR-NSHI and discuss the involvement of PJVK in ANSD.

Pathogenic variants in the PJVK gene cause the DFNB59 type of autosomal recessive non-syndromic hearing impairment (AR-NSHI). Phenotypes are not homogeneous, as a few subjects show auditory neuropathy spectrum disorder (ANSD), while others show cochlear hearing loss. The numbers of reported cases and pathogenic variants are still small to establish accurate genotype-phenotype correlations. We investigated a cohort of 77 Spanish familial cases of AR-NSHI, in whom DFNB1 had been excluded, and a cohort of 84 simplex cases with isolated ANSD in whom OTOF variants had been excluded. All seven exons and exon-intron boundaries of the PJVK gene were sequenced. We report three novel DFNB59 cases, one from the AR-NSHI cohort and two from the ANSD cohort, with stable, severe to profound NSHI. Two of the subjects received unilateral cochlear implantation, with apparent good outcomes. Our study expands the spectrum of PJVK mutations, as we report four novel pathogenic variants: p.Leu224Arg, p.His294Ilefs*43, p.His294Asp and p.Phe317Serfs*20. We review the reported cases of DFNB59, summarize the clinical features of this rare subtype of AR-NSHI and discuss the involvement of PJVK in ANSD.

Simple Summary Ewing sarcoma is an aggressive tumor with still unacceptable survival rates, particularly in patients with metastatic disease and for which it is necessary to develop new and innovative therapies. These tumors are characterized by the presence of chromosomal translocations that give rise to chimeric transcription factors (i.e., EWSR1–FLI1) that govern the oncogenic process. In this article, we describe an efficient strategy to permanently inactivate the EWSR1–FLI1 oncogene characteristic of Ewing sarcoma using CRISPR/Cas9 gene editing technology. Although the application of gene therapy in cancer still has many limitations, for example, the strategy for delivery, studies like ours show that gene therapy can be a promising alternative, particularly for those tumors that are highly dependent on a particular oncogene as is the case in Ewing sarcoma. Abstract Ewing sarcoma is an aggressive bone cancer affecting children and young adults. The main molecular hallmark of Ewing sarcoma are chromosomal translocations that produce chimeric oncogenic transcription factors, the most frequent of which is the aberrant transcription factor EWSR1–FLI1. Because this is the principal oncogenic driver of Ewing sarcoma, its inactivation should be the best therapeutic strategy to block tumor growth. In this study, we genetically inactivated EWSR1–FLI1 using CRISPR-Cas9 technology in order to cause permanent gene inactivation. We found that gene editing at the exon 9 of FLI1 was able to block cell proliferation drastically and induce senescence massively in the well-studied Ewing sarcoma cell line A673. In comparison with an extensively used cellular model of EWSR1–FLI1 knockdown (A673/TR/shEF), genetic inactivation was more effective, particularly in its capability to block cell proliferation. In summary, genetic inactivation of EWSR1–FLI1 in A673 Ewing sarcoma cells blocks cell proliferation and induces a senescence phenotype that could be exploited therapeutically. Although efficient and specific in vivo CRISPR-Cas9 editing still presents many challenges today, our data suggest that complete inactivation of EWSR1–FLI1 at the cell level should be considered a therapeutic approach to develop in the future.

PURPOSE Pathogenic variants in GJB2 are the most common cause of autosomal recessive sensorineural hearing loss. The classification of c.101T>C/p.Met34Thr and c.109G>A/p.Val37Ile in GJB2 are controversial. Therefore, an expert consensus is required for the interpretation of these two variants. METHODS The ClinGen Hearing Loss Expert Panel collected published data and shared unpublished information from contributing laboratories and clinics regarding the two variants. Functional, computational, allelic, and segregation data were also obtained. Case-control statistical analyses were performed. RESULTS The panel reviewed the synthesized information, and classified the p.Met34Thr and p.Val37Ile variants utilizing professional variant interpretation guidelines and professional judgment. We found that p.Met34Thr and p.Val37Ile are significantly overrepresented in hearing loss patients, compared to population controls. Individuals homozygous or compound heterozygous for p.Met34Thr or p.Val37Ile typically manifest mild to moderate hearing loss. Several other types of evidence also support pathogenic roles for these two variants. CONCLUSION Resolving controversies in variant classification requires coordinated effort among a panel of international multi-institutional experts to share data, standardize classification guidelines, review evidence, and reach a consensus. We concluded that p.Met34Thr and p.Val37Ile variants in GJB2 are pathogenic for autosomal recessive nonsyndromic hearing loss with variable expressivity and incomplete penetrance.

HIV remains incurable because of viral persistence in latent reservoirs that are inaccessible to antiretroviral therapy. A potential curative strategy is to reactivate viral gene expression in latently infected cells. However, no drug so far has proven to be successful in vivo in reducing the reservoir, and therefore new anti-latency compounds are needed. We explored the role of microRNAs (miRNAs) in latency maintenance and their modulation as a potential anti-latency strategy. Latency models based on treating resting CD4 T cells with chemokine (C-C motif) ligand 19 (CCL19) or interleukin-7 (IL7) before HIV infection and next-generation sequencing were used to identify the miRNAs involved in HIV latency. We detected four upregulated miRNAs (miRNA-98, miRNA-4516, miRNA-4488, and miRNA-7974). Individual or combined inhibition of these miRNAs was performed by transfection into cells latently infected with HIV. Viral replication, assessed 72 h after transfection, did not increase after miRNA modulation, despite miRNA inhibition and lack of toxicity. Furthermore, the combined modulation of five miRNAs previously associated with HIV latency was not effective in these models. Our results do not support the modulation of miRNAs as a useful strategy for the reversal of HIV latency. As shown with other drugs, the potential of miRNA modulation as an HIV reactivation strategy could be dependent on the latency model used, This work wasfunded by the Spanish Ministry of Economy and Competitiveness(PIE 13/00040) and the Spanish AIDS Research Network (RIS)(RD16/0025/0001) as part of the Plan Estatal I+D+I and co-financedby Instituto de Salud Carlos III (ISCIII)-Subdirección General deEvaluación y Fomento de la Investigación and Fondo Europeo deDesarrollo Regional (FEDER) (European Regional DevelopmentFund). M.R.L-H. was supported by the Spanish Ministry of Economyand Competitiveness with ISCIII-FEDER funding (PIE 13/00040 and RD12/0017/0017). N.M.E. and C.G. were supported bythe Spanish AIDS Research Network (RD12/0017/0017 and RD16/0025/0017

PurposeProper interpretation of genomic variants is critical to successful medical decision making based on genetic testing results. A fundamental prerequisite to accurate variant interpretation is the clear understanding of the clinical validity of gene-disease relationships. The Clinical Genome Resource (ClinGen) has developed a semi-quantitative framework to assign clinical validity to gene-disease relationships.MethodsThe ClinGen Hearing Loss Gene Curation Expert Panel (HL GCEP) uses this framework to perform evidence-based curations of genes present on testing panels from 17 clinical laboratories in the Genetic Testing Registry. The HL GCEP curated and reviewed 142 genes and 164 gene-disease pairs, including 105 nonsyndromic and 59 syndromic forms of hearing loss.ResultsThe final outcome included 82 Definitive (50%), 12 Strong (7%), 25 Moderate (15%), 32 Limited (20%), 10 Disputed (6%), and 3 Refuted (2%) classifications. The summary of each curation is date stamped with the HL GCEP approval, is live, and will be kept up-to-date on the ClinGen website (https://search.clinicalgenome.org/kb/gene-validity).ConclusionThis gene curation approach serves to optimize the clinical sensitivity of genetic testing while reducing the rate of uncertain or ambiguous test results caused by the interrogation of genes with insufficient evidence of a disease link.

Mutations in PAX6 are involved in several developmental eye disorders. These disorders have considerable phenotypic variability, ranging from panocular forms of congenital aniridia and microphthalmia to isolated anomalies of the anterior or posterior segment. Here, we describe 3 families with variable inter-generational ocular expression of aniridia, iris coloboma, or microphthalmia, and an unusual transmission of PAX6 mutations from an unaffected or mildly affected parent; all of which raised suspicion of gonosomal mosaicism. We first identified two previously known nonsense mutations and one novel likely pathogenic missense variant in PAX6 in probands by means of targeted NGS. The subsequent segregation analysis by Sanger sequencing evidenced the presence of highly probable mosaic events in paternal blood samples. Mosaicism was further confirmed by droplet digital PCR analysis in several somatic tissues of mosaic fathers. Quantification of the mutant allele fraction in parental samples showed a marked deviation from 50%, with a range between 12 and 29% depending on cell type. Gonosomal mosaicsm was definitively confirmed in one of the families thanks to the availability of a sperm sample from the mosaic father. Thus, the recurrence risk in this family was estimated to be about one-third. This is the first report confirming parental PAX6 mosaicism as a cause of disease recurrence in aniridia and other related phenotypes. In addition, we demonstrated that post-zygotic mosaicism is a frequent and underestimated pathogenic mechanism in aniridia, explaining intra-familial phenotypic variability in many cases. Our findings may have substantial implications for genetic counseling in congenital aniridia. Thus, we also highlight the importance of comprehensive genetic screening of parents for new sporadic cases with aniridia or related developmental eye disease to more accurately assess recurrence risk. In conclusion, somatic and/or gonosomal mosaicism should be taken into consideration as a genetic factor to explain not only families with unaffected parents despite multiple affected children but also variable expressivity, apparent de novo cases, and even uncharacterized cases of aniridia and related developmental eye disorders, apparently lacking PAX6 mutations.

Mutations in PAX6 are involved in several developmental eye disorders. These disorders have considerable phenotypic variability, ranging from panocular forms of congenital aniridia and microphthalmia to isolated anomalies of the anterior or posterior segment. Here, we describe 3 families with variable inter-generational ocular expression of aniridia, iris coloboma, or microphthalmia, and an unusual transmission of PAX6 mutations from an unaffected or mildly affected parent; all of which raised suspicion of gonosomal mosaicism. We first identified two previously known nonsense mutations and one novel likely pathogenic missense variant in PAX6 in probands by means of targeted NGS. The subsequent segregation analysis by Sanger sequencing evidenced the presence of highly probable mosaic events in paternal blood samples. Mosaicism was further confirmed by droplet digital PCR analysis in several somatic tissues of mosaic fathers. Quantification of the mutant allele fraction in parental samples showed a marked deviation from 50%, with a range between 12 and 29% depending on cell type. Gonosomal mosaicsm was definitively confirmed in one of the families thanks to the availability of a sperm sample from the mosaic father. Thus, the recurrence risk in this family was estimated to be about one-third. This is the first report confirming parental PAX6 mosaicism as a cause of disease recurrence in aniridia and other related phenotypes. In addition, we demonstrated that post-zygotic mosaicism is a frequent and underestimated pathogenic mechanism in aniridia, explaining intra-familial phenotypic variability in many cases. Our findings may have substantial implications for genetic counseling in congenital aniridia. Thus, we also highlight the importance of comprehensive genetic screening of parents for new sporadic cases with aniridia or related developmental eye disease to more accurately assess recurrence risk. In conclusion, somatic and/or gonosomal mosaicism should be taken into consideration as a genetic factor to explain not only families with unaffected parents despite multiple affected children but also variable expressivity, apparent de novo cases, and even uncharacterized cases of aniridia and related developmental eye disorders, apparently lacking PAX6 mutations.

Mutations in GJB2, the gene encoding connexin-26 at the DFNB1 locus on 13q12, are found in as many as 50% of subjects with autosomal recessive, nonsyndromic prelingual hearing impairment. However, genetic diagnosis is complicated by the fact that 10%-50% of affected subjects with GJB2 mutations carry only one mutant allele. Recently, a deletion truncating the GJB6 gene (encoding connexin-30), near GJB2 on 13q12, was shown to be the accompanying mutation in approximately 50% of these deaf GJB2 heterozygotes in a cohort of Spanish patients, thus becoming second only to 35delG at GJB2 as the most frequent mutation causing prelingual hearing impairment in Spain. Here, we present data from a multicenter study in nine countries that shows that the deletion is present in most of the screened populations, with higher frequencies in France, Spain, and Israel, where the percentages of unexplained GJB2 heterozygotes fell to 16.0%-20.9% after screening for the del(GJB6-D13S1830) mutation. Our results also suggest that additional mutations remain to be identified, either in DFNB1 or in other unlinked genes involved in epistatic interactions with GJB2. Analysis of haplotypes associated with the deletion revealed a founder effect in Ashkenazi Jews and also suggested a common founder for countries in Western Europe. These results have important implications for the diagnosis and counseling of families with DFNB1 deafness.

Inherited hearing impairment affects one in 2,000 newborns. Nonsyndromic prelingual forms are inherited mainly as autosomal recessive traits, for which 16 genes are currently known. Mutations in the genes encoding connexins 26 and 30 account for up to 50% of these cases. However, the individual contribution of the remaining genes to the whole remains undetermined. In addition, for most of the genes there is a need for studies on genotype–phenotype correlations, to identify distinctive clinical features which may direct the molecular diagnosis to specific genes. Here we present a mutation analysis and a genotype–phenotype correlation study on the gene encoding otoferlin (OTOF), responsible for the DFNB9 subtype of prelingual hearing impairment. Four novel mutations were identified: c.2122C>T (p.Arg708Ter), c.4275G>A (p.Trp1425Ter), c.4362+2T>G, and c.5860_5862delATC (p.Ile1954del). A total of 37 subjects with muta-tions in OTOF were studied clinically. They were phenotypically homogeneous, having profound hearing impairment with very early onset, as shown by pure-tone audiometry and auditory brainstem responses. Magnetic resonance imaging and computed tomography did not reveal any inner ear malformation. Unexpectedly, transient evoked otoacoustic emissions (TEOAEs) were present, either bilaterally or unilaterally in 11 subjects. Altogether, clinical data of these subjects met the diagnostic criteria of auditory neuropathy. A total of 10 subjects had been successfully provided with cochlear implants. The results of our study indicate that genetic diagnosis of subjects with auditory neuropathy and profound hearing impairment should be directed to the otoferlin gene. Our data are of concern to universal screening programs which use TEOAEs as the first detection test for hearing impairment in newborns, since this technique may overlook a nonnegligible proportion of cases. Hum Mutat 22:451–456, 2003. © 2003 Wiley-Liss, Inc.

Hereditary non-syndromic sensorineural hearing loss (NSSHL) is a genetically highly heterogeneous group of disorders. Autosomal dominant forms account for up to 20% of cases. To date, 39 loci have been identified by linkage analysis of affected families that segregate NSSHL forms in the autosomal dominant mode (DFNA). Investigation of a large Spanish pedigree with autosomal dominant inheritance of bilateral and progressive NSSHL of postlingual onset excluded linkage to known DFNA loci and, in a subsequent genome-wide scan, the disorder locus was mapped to 3q28-29. A maximum two-point LOD score of 4.36 at theta=0 was obtained for marker D3S1601. Haplotype analysis placed the novel locus, DFNA44, within a 3-cM genetic interval defined by markers D3S1314 and D3S2418. Heteroduplex analysis and DNA sequencing of coding regions and exon/intron boundaries of two genes (CLDN16 and FGF12) in this interval did not reveal disease-causing mutations.

Inherited hearing impairment affects about 1 in 2000 newborns. Up to 50 percent of all patients with autosomal recessive nonsyndromic prelingual deafness in different populations have mutations in the gene encoding the gap-junction protein connexin 26 (GJB2) at locus DFNB1 on chromosome 13q12. However, a large fraction (10 to 42 percent) of patients with GJB2 mutations have only one mutant allele; the accompanying mutation has not been identified. DFNB1-linked familial cases with no mutation in GJB2 have also been reported.We evaluated 33 unrelated probands with nonsyndromic prelingual deafness who had only one GJB2 mutant allele. Nine subjects had evidence of linkage to DFNB1. We used haplotype analysis for markers on 13q12 to search for mutations other than the one involving GJB2.We identified a 342-kb deletion in the gene encoding connexin 30 (GJB6), a protein that is reported to be expressed with connexin 26 in the inner ear. The deletion extended distally to GJB2, which remained intact. The break-point junction of the deletion was isolated and sequenced, and a specific diagnostic test was developed for this common mutation. Twenty-two of the 33 subjects were heterozygous for both the GJB6 and GJB2 mutations, including all 9 with evidence of linkage to DFNB1. Two subjects were homozygous for the GJB6 mutation.A 342-kb deletion in GJB6 is the second most frequent mutation causing prelingual deafness in the Spanish population. Our data suggest that mutations in the complex locus DFNB1, which contains two genes (GJB2 and GJB6), can result in a monogenic or a digenic pattern of inheritance of prelingual deafness.

Mohr-Tranebjaerg syndrome is a rare X-linked condition characterized by the association of dystonia and progressive postlingual sensorineural hearing impairment. Here we report the clinical and genetic findings in a Spanish patient with MTS carrying a novel mutation in the DDP1 (deafness-dystonia peptide 1) gene, which encodes TIMM8a, a component of the mitochondrial protein translocation system. The phenotypic variability observed in patients with Mohr-Tranebjaerg syndrome suggests the involvement of modifier factors which may modulate the clinical manifestations of the syndrome.

Hearing loss comprises a group of disorders with great social implications. During the past decade, tremendous progress has been made in the investigation of hereditary hearing impairment, especially in the nonsyndromic forms, in which the hearing deficit is not accompanied by other clinical signs. These isolated conditions are the most frequent traits and are characterized by a vast genetic and clinical heterogeneity. Among them, those inherited following an autosomal dominant pattern (ADNSHL) represents around 10–20% of hereditary cases for which more than 50 loci (DFNA) have been mapped so far. One of these subtypes, DFNA20/A26, has been reported to be associated with ACTG1, the gene encoding γ-actin.

We previously mapped a novel autosomal dominant deafness locus, DFNA44, by studying a family with postlingual, progressive, nonsyndromic hearing loss. We report here on the identification of a mutation in CCDC50 as the cause of hearing loss in the family. CCDC50 encodes Ymer, an effector of epidermal growth factor (EGF)–mediated cell signaling that is ubiquitously expressed in different organs and has been suggested to inhibit down-regulation of the EGF receptor. We have examined its expression pattern in mouse inner ear. Western blotting and cell transfection results indicate that Ymer is a soluble, cytoplasmic protein, and immunostaining shows that Ymer is expressed in a complex spatiotemporal pattern during inner ear development. In adult inner ear, the expression of Ymer is restricted to the pillar cells of the cochlea, the stria vascularis, and the vestibular sensory epithelia, where it shows spatial overlap with the microtubule-based cytoskeleton. In dividing cells, Ymer colocalizes with microtubules of the mitotic apparatus. We suggest that DFNA44 hearing loss may result from a time-dependent disorganization of the microtubule-based cytoskeleton in the pillar cells and stria vascularis of the adult auditory system.

Molecular testing for mutations in the gene encoding connexin-26 (GJB2) at the DFNB1 locus has become the standard of care for genetic diagnosis and counseling of autosomal recessive non-syndromic hearing impairment (ARNSHI). The spectrum of mutations in GJB2 varies considerably among the populations, different alleles predominating in different ethnic groups. A cohort of 34 families of Spanish Romani (gypsies) with ARNSHI was screened for mutations in GJB2. We found that DFNB1 deafness accounts for 50% of all ARNSHI in Spanish gypsies. The predominating allele is W24X (79% of the DFNB1 alleles), and 35delG is the second most common allele (17%). An allele-specific PCR test was developed for the detection of the W24X mutation. By using this test, carrier frequencies were determined in two sample groups of gypsies from different Spanish regions (Andalusia and Catalonia), being 4% and 0%, respectively. Haplotype analysis for microsatellite markers closely flanking the GJB2 gene revealed five different haplotypes associated with the W24X mutation, all sharing the same allele from marker D13S141, suggesting that a founder effect for this mutation is responsible for its high prevalence among Spanish gypsies.

Inherited hearing impairment affects one in 2,000 newborns. Nonsyndromic prelingual forms are inherited mainly as autosomal recessive traits, for which 16 genes are currently known. Mutations in the genes encoding connexins 26 and 30 account for up to 50% of these cases. However, the individual contribution of the remaining genes to the whole remains undetermined. In addition, for most of the genes there is a need for studies on genotype-phenotype correlations, to identify distinctive clinical features which may direct the molecular diagnosis to specific genes. Here we present a mutation analysis and a genotype-phenotype correlation study on the gene encoding otoferlin (OTOF), responsible for the DFNB9 subtype of prelingual hearing impairment. Four novel mutations were identified: c.2122CT (p.Arg708Ter), c.4275GA (p.Trp1425Ter), c.4362+2TG, and c.5860_5862delATC (p.Ile1954del). A total of 37 subjects with mutations in OTOF were studied clinically. They were phenotypically homogeneous, having profound hearing impairment with very early onset, as shown by pure-tone audiometry and auditory brainstem responses. Magnetic resonance imaging and computed tomography did not reveal any inner ear malformation. Unexpectedly, transient evoked otoacoustic emissions (TEOAEs) were present, either bilaterally or unilaterally in 11 subjects. Altogether, clinical data of these subjects met the diagnostic criteria of auditory neuropathy. A total of 10 subjects had been successfully provided with cochlear implants. The results of our study indicate that genetic diagnosis of subjects with auditory neuropathy and profound hearing impairment should be directed to the otoferlin gene. Our data are of concern to universal screening programs which use TEOAEs as the first detection test for hearing impairment in newborns, since this technique may overlook a nonnegligible proportion of cases.

The A1555G mutation in the mitochondrial genome causes sensorineural hearing loss and familial aminoglycoside ototoxicity.Screening for the A1555G mutation was performed on 72 patients with nonsyndromic sensorineural hearing loss.The A1555G mutation was identified in 15 patients (20.8%). All of them presented maternal relatives with deafness. Individuals with the A1555G mutation that had been treated with aminoglycosides developed more severe hearing loss.The A1555G mutation should be screened in individuals with maternal relatives with hearing loss before administering aminoglycosides.

So far, 37 different loci for autosomal dominant, sensorineural, non-syndromic hearing loss (ADSNSHL), have been mapped and 11 genes have been cloned.1 Among them, the TECTA gene, mapped to chromosome 11q22-24 (locus DFNA8/A12 ), encodes α-tectorin,2 a non-collagenous component of the cochlear tectorial membrane. This membrane is an extracellular matrix that covers the apical surface of the sensory epithelium in the cochlea and plays an important role in transmitting the mechanical energy of sound to the mechanosensitive stereociliary bundles of the hair cells, where the sound is transduced into neural potentials. In previous studies, five different missense mutations resulting in ADSNSHL have been described in the TECTA gene in four DFNA8/A12 families, affecting different domains of the protein and showing different phenotypes (table 1): a prelingual, non-progressive hearing loss affecting mid frequencies or a postlingual, progressive, high frequency hearing impairment. View this table: Table 1 TECTA mutations described in the DFNA8/A12 families In this study, we report a Spanish family with a novel phenotype of postlingual, progressive, mid frequency hearing loss resulting from a new mutation in the TECTA gene. The family consists of 47 members including nine affected subjects with ADSNSHL (fig 1A). Appropriate informed consent was obtained from all those studied. Clinical examination was performed and blood samples were obtained from 33 family members. Environmental factors were eliminated as the cause of deafness in all affected family members. Features suggestive of syndromic anomalies were not present. Otoscopic examination and use of the tuning fork test ruled out conductive hearing loss. Pure tone audiometry was performed to test for air conduction (frequencies of 125-8000 Hz) and bone conduction (frequencies of 250-8000 Hz). Affected subjects showed bilateral sensorineural hearing impairment. In the beginning, the hearing loss in this family is mild, mainly affecting mid frequencies (500, 1000, and …