Matías Morín

Mutations in the seed region of human miR-96 are responsible for nonsyndromic progressive hearing loss

MicroRNAs (miRNAs) bind to complementary sites in their target mRNAs to mediate post-transcriptional repression, with the specificity of target recognition being crucially dependent on the miRNA seed region. Impaired miRNA target binding resulting from SNPs within mRNA target sites has been shown to lead to pathologies associated with dysregulated gene expression. However, no pathogenic mutations within the mature sequence of a miRNA have been reported so far. Here we show that point mutations in the seed region of miR-96, a miRNA expressed in hair cells of the inner ear, result in autosomal dominant, progressive hearing loss. This is the first study implicating a miRNA in a mendelian disorder. The identified mutations have a strong impact on miR-96 biogenesis and result in a significant reduction of mRNA targeting. We propose that these mutations alter the regulatory role of miR-96 in maintaining gene expression profiles in hair cells required for their normal function.

In vivo and in vitro effects of two novel gamma actin (ACTG1) mutations that cause DFNA20/26 hearing impairment

Here we report the functional assessment of two novel deafness-associated gamma-actin mutants, K118N and E241K, in a spectrum of different situations with increasing biological complexity by combining biochemical and cell biological analysis in yeast and mammalian cells. Our in vivo experiments showed that while the K118N had a very mild effect on yeast behaviour, the phenotype caused by the E241K mutation was very severe and characterized by a highly compromised ability to grow on glycerol as a carbon source, an aberrant multi-vacuolar pattern and the deposition of thick F-actin bundles randomly in the cell. The latter feature is consistent with the highly unusual spontaneous tendency of the E241K mutant to form bundles in vitro, although this propensity to bundle was neutralized by tropomyosin and the E241K filament bundles were hypersensitive to severing in the presence of cofilin. In transiently transfected NIH3T3 cells both mutant actins were normally incorporated into cytoskeleton structures, although cytoplasmic aggregates were also observed indicating an element of abnormality caused by the mutations in vivo. Interestingly, gene-gun mediated expression of these mutants in cochlear hair cells results in no gross alteration in cytoskeletal structures or the morphology of stereocilia. Our results provide a more complete picture of the biological consequences of deafness-associated gamma-actin mutants and support the hypothesis that the post-lingual and progressive nature of the DFNA20/26 hearing loss is the result of a progressive deterioration of the hair cell cytoskeleton over time.

DFNA8/12 caused by TECTA mutations is the most identified subtype of nonsyndromic autosomal dominant hearing loss.

The prevalence of DFNA8/DFNA12 (DFNA8/12), a type of autosomal dominant nonsyndromic hearing loss (ADNSHL), is unknown as comprehensive population‐based genetic screening has not been conducted. We therefore completed unbiased screening for TECTA mutations in a Spanish cohort of 372 probands from ADNSHL families. Three additional families (Spanish, Belgian, and English) known to be linked to DFNA8/12 were also included in the screening. In an additional cohort of 835 American ADNSHL families, we preselected 73 probands for TECTA screening based on audiometric data. In aggregate, we identified 23 TECTA mutations in this process. Remarkably, 20 of these mutations are novel, more than doubling the number of reported TECTA ADNSHL mutations from 13 to 33. Mutations lie in all domains of the α‐tectorin protein, including those for the first time identified in the entactin domain, as well as the vWFD1, vWFD2, and vWFD3 repeats, and the D1–D2 and TIL2 connectors. Although the majority are private mutations, four of them—p.Cys1036Tyr, p.Cys1837Gly, p.Thr1866Met, and p.Arg1890Cys—were observed in more than one unrelated family. For two of these mutations founder effects were also confirmed. Our data validate previously observed genotype–phenotype correlations in DFNA8/12 and introduce new correlations. Specifically, mutations in the N‐terminal region of α‐tectorin (entactin domain, vWFD1, and vWFD2) lead to mid‐frequency NSHL, a phenotype previously associated only with mutations in the ZP domain. Collectively, our results indicate that DFNA8/12 hearing loss is a frequent type of ADNSHL. Hum Mutat 32:1–10, 2011.

A de novo missense mutation in the gene encoding the SOX10 transcription factor in a Spanish sporadic case of Waardenburg syndrome type IV

Waardenburg syndrome type IV (WS4), also called Shah–Waardenburg syndrome and Waardenburg– Hirschsprung disease (OMIM 277580), is a rare congenital disorder comprising sensorineural deafness, pigmentary abnormalities of the skin, eyes and hair, and absence of ganglion cells in the myenteric and submucosal plexus of the gastrointestinal tract [Read and Newton, 1997; Brooks et al., 2004]. It is caused by mutations in any of three genes: EDN3 (MIM 131242), on 20q13, encoding endothelin-3 [Edery et al., 1996; Hofstra et al., 1996]; EDNRB (OMIM 131244), on 13q22, encoding the endothelinB receptor [Puffenberger et al., 1994]; and SOX10 (OMIM 602229), on 22q13, encoding the SOX10 transcription factor [Pingault et al., 1998] (Table I). The patterns of inheritance of WS4 are diverse, with incomplete penetrance and variable expressivity. There are autosomal recessive forms caused by mutations in the homozygous state in EDN3 or EDNRB [Puffenberger et al., 1994; Attié et al., 1995; Edery et al., 1996; Hofstra et al., 1996; Bidaud et al., 1997; Boardman et al., 2001; Verheij et al., 2002; Sangkhathat et al., 2005], and autosomal dominant forms caused by mutations in the heterozygous state in EDN3, EDNRB, or SOX10 [Pingault et al., 1998; Southard-Smith et al., 1999; Syrris et al., 1999; Pingault et al., 2001; Sham et al., 2001; Pingault et al., 2002]. In autosomal recessive WS4, heterozygous carriers sometimes show the colonic aganglionosis which is characteristic of isolated Hirschsprung disease. In addition, some patients with mutations in the SOX10 gene show a more severe WS4 phenotype, associating a Peripheral demyelinating neuropathy and a Central dysmyelinating leukodystrophy with Waardenburg–Hirschsprung manifestations (PCWH phenotype, OMIM 609136) [Inoue et al., 1999; Touraine et al., 2000; Inoue et al., 2002, 2004; Verheij et al., 2006]. Further complication is added by the existence of other disorders which are allelic with WS4. Firstly, the ABCD syndrome (OMIM 600501), an autosomal recessive condition of albinism, black locks, Hirschsprung disease and deafness, is caused by mutations in the EDNRB gene [Verheij et al., 2002]. Also, a missense mutation in SOX10 was shown to be responsible for the milder variant of the Yemenite deaf–blind hypopigmentation syndrome (OMIM 601706), a disorder whose clinical manifestations include cutaneous hypopigmented and hyperpigmented spots and patches, as well as severe sensorineural hearing impairment. This milder variant lacks two other clinical signs (microcornea and coloboma) that are characteristic of the severe form [Bondurand et al., 1999].

Three deaf mice: mouse models for TECTA-based human hereditary deafness reveal domain-specific structural phenotypes in the tectorial membrane

Tecta is a modular, non-collagenous protein of the tectorial membrane (TM), an extracellular matrix of the cochlea essential for normal hearing. Missense mutations in Tecta cause dominant forms of non-syndromic deafness and a genotype–phenotype correlation has been reported in humans, with mutations in different Tecta domains causing mid- or high-frequency hearing impairments that are either stable or progressive. Three mutant mice were created as models for human Tecta mutations; the TectaL1820F,G1824D/+ mouse for zona pellucida (ZP) domain mutations causing stable mid-frequency hearing loss in a Belgian family, the TectaC1837G/+ mouse for a ZP-domain mutation underlying progressive mid-frequency hearing loss in a Spanish family and the TectaC1619S/+ mouse for a zonadhesin-like (ZA) domain mutation responsible for progressive, high-frequency hearing loss in a French family. Mutations in the ZP and ZA domains generate distinctly different changes in the structure of the TM. Auditory brainstem response thresholds in the 8–40 kHz range are elevated by 30–40 dB in the ZP-domain mutants, whilst those in the ZA-domain mutant are elevated by 20–30 dB. The phenotypes are stable and no evidence has been found for a progressive deterioration in TM structure or auditory function. Despite elevated auditory thresholds, the Tecta mutant mice all exhibit an enhanced tendency to have audiogenic seizures in response to white noise stimuli at low sound pressure levels (≤84 dB SPL), revealing a previously unrecognised consequence of Tecta mutations. These results, together with those from previous studies, establish an allelic series for Tecta unequivocally demonstrating an association between genotype and phenotype.

Genetic and phenotypic heterogeneity in two novel cases of Waardenburg syndrome type IV

Genetic and Phenotypic Heterogeneity in Two Novel Cases of Waardenburg Syndrome Type IV Antonio Vi~nuela, Mat ıas Mor ın, Manuela Villamar, Constantino Morera, M. Jos e Lavilla, Laura Cavall e, Miguel A. Moreno-Pelayo, Felipe Moreno, and Ignacio del Castillo* Unidad de Gen etica Molecular, Hospital Universitario Ram on y Cajal, Madrid, Spain Centro de Investigaci on Biom edica en Red de Enfermedades Raras (CIBERER), Madrid, Spain Servicio de Otorrinolaringolog ıa, Hospital Universitario La Fe, Universidad de Valencia, Valencia, Spain Servicio de Otorrinolaringolog ıa, Hospital San Pedro de Alc antara, C aceres, Spain

Primary T-cell immunodeficiency with functional revertant somatic mosaicism in CD247

To the Editor: T lymphocytes detect antigens with the T-cell receptor (TCR) composed of a variable heterodimer (either ab or gd), 2 invariant heterodimers (CD3gε and CD3dε), and an invariant homodimer (CD247 or zz). Because of the crucial role of TCR signaling in thymic selection, mutations in TCR, CD3, or CD247 selectively impair T-cell development, albeit to different degrees: deficiency of CD3d or CD3ε, but not of CD3g or CD247, causes severe T-cell lymphopenia. Their clinical outcome is also disparate, because CD3g deficiency does not require urgent transplantation. Thus, TCR immunodeficiencies display a range of phenotypes and careful differential diagnosis is essential for appropriate therapy. We describe an infant born to consanguineous parents with early-onset chronic cytomegalovirus infection, severe immunodeficiency, and extremely low surface TCR levels. Her immunologic characterization at age 11 months is summarized in Table E1 in this article’s Online Repository at www.jacionline.

Parental Mosaicism in PAX6 Causes Intra-Familial Variability: Implications for Genetic Counseling of Congenital Aniridia and Microphthalmia

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.

Actin Mutations and Deafness

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.