Whole-exome sequencing reveals ZNF408 as a new gene associated with autosomal recessive retinitis pigmentosa with vitreal alterations

Retinitis pigmentosa (RP) is a group of progressive inherited retinal dystrophies that cause visual impairment as a result of photoreceptor cell death. RP is heterogeneous, both clinically and genetically making difficult to establish precise genotype–phenotype correlations. In a Spanish family with autosomal recessive RP (arRP), homozygosity mapping and whole-exome sequencing led to the identification of a homozygous mutation (c.358_359delGT; p.Ala122Leufs*2) in the ZNF408 gene. A screening performed in 217 additional unrelated families revealed another homozygous mutation (c.1621C>T; p.Arg541Cys) in an isolated RP case. ZNF408 encodes a transcription factor that harbors 10 predicted C2H2-type fingers thought to be implicated in DNA binding. To elucidate the ZNF408 role in the retina and the pathogenesis of these mutations we have performed different functional studies. By immunohistochemical analysis in healthy human retina, we identified that ZNF408 is expressed in both cone and rod photoreceptors, in a specific type of amacrine and ganglion cells, and in retinal blood vessels. ZNF408 revealed a cytoplasmic localization and a nuclear distribution in areas corresponding with the euchromatin fraction. Immunolocalization studies showed a partial mislocalization of the p.Arg541Cys mutant protein retaining part of the WT protein in the cytoplasm. Our study demonstrates that ZNF408, previously associated with Familial Exudative Vitreoretinopathy (FEVR), is a new gene causing arRP with vitreous condensations supporting the evidence that this protein plays additional functions into the human retina.This work is supported by CIBERER (06/07/0036), FIS (PI013/00226), Ministry of Economy and Competitiveness-FEDER (BFU2012-36845), RETICS (RD12/0034/0010), Fundación ONCE, Fundaluce and grants BIO2011-27069 from the Spanish Ministry of Economy and Competitiveness, and PROMETEOII/2014/025 from the Conselleria de Educacio of the Valencia Community. PC is supported by Fundación Conchita Rábago (FCR), MC by Miguel Servet ISCIII (CP/03256) and dS by CAPES Foundation, Ministry of Education of Brazil

New GJA8 variants and phenotypes highlight its critical role in a broad spectrum of eye anomalies

GJA8 encodes connexin 50 (Cx50), a transmembrane protein involved in the formation of lens gap junctions. GJA8 mutations have been linked to early onset cataracts in humans and animal models. In mice, missense mutations and homozygous Gja8 deletions lead to smaller lenses and microphthalmia in addition to cataract, suggesting that Gja8 may play a role in both lens development and ocular growth. Following screening of GJA8 in a cohort of 426 individuals with severe congenital eye anomalies, primarily anophthalmia, microphthalmia and coloboma, we identified four known [p.(Thr39Arg), p.(Trp45Leu), p.(Asp51Asn), and p.(Gly94Arg)] and two novel [p.(Phe70Leu) and p.(Val97Gly)] likely pathogenic variants in seven families. Five of these co-segregated with cataracts and microphthalmia, whereas the variant p.(Gly94Arg) was identified in an individual with congenital aphakia, sclerocornea, microphthalmia and coloboma. Four missense variants of unknown or unlikely clinical significance were also identified. Furthermore, the screening of GJA8 structural variants in a subgroup of 188 individuals identified heterozygous 1q21 microdeletions in five families with coloboma and other ocular and/or extraocular findings. However, the exact genotype–phenotype correlation of these structural variants remains to be established. Our data expand the spectrum of GJA8 variants and associated phenotypes, confirming the importance of this gene in early eye development

To analyse the polymorphism of gene in mothers of Down syndrome, measure the levels of serum homocysteine

Introdução: A síndrome de Down resulta da presença de três cópias do cromossomo 21. Na maioria dos casos, o cromossomo extra origina-se da segregação anormal dos cromossomos durante a meiose (origem materna em 95 por cento dos casos). Estudos demonstram que a hipometilação do DNA genômico está associada à instabilidade cromossômica, podendo levar ao aumento da taxa de quebras e erros na segregação dos cromossomos, embora o mecanismo molecular responsável não seja conhecido. A S-adenosilmetionina (SAM) age como um fator crítico na regulação dos processos de metilação celular, pois é doadora de radicais metil para muitos substratos, incluindo o DNA. A SAM é formada durante o metabolismo da metionina/homocisteína e sua síntese depende do bom funcionamento das enzimas envolvidas nesse processo, como a metilenotetraidrofolato redutase (MTHFR), a metionina sintase (MTR), a metionina sintase redutase (MTRR), a cistationina j3-sintase (CBS) e a diidrofolato redutase (DHFR). Objetivos: Analisar os polimorfismos dos genes MTHFR (C677T e A1298C), MTR (A2756G), MTRR (A66G), CBS (844ins68) e DHFR (deI19pb) em 154 mães de crianças com síndrome de Down e 158 mães-controles, dosar os níveis de homocisteína plasmática. Resultados: Os níveis de Hcy encontrados no grupo-caso foram maiores do que aqueles obtidos para o grupo-controle (p=O,OO2). Somente o polimorfismo C677T pôde ser associado com níveis aumentados de Hcy no grupo-caso (p=O,OO6), especialmente quando a mutação era encontrada em homozigose. No grupo-controle, a associação do genótipo TT com níveis aumentados de Hcy apresentou resultados estatísticos com margem de significância (p=O,OO54). Todos os polimorfismos apresentaram distribuição genotípica similar nos grupos-caso e controle (p=O,05), embora uma maior freqüência do alelo 677T tenha sido encontrada nas mães-caso (p=O,049). Conclusões: O aumento dos níveis de Hcy no grupo-caso pode estar refletindo um prejuízo nos processos de metilação celular e pode estar relacionado com um risco maior de não-disjunção cromossômica. Embora o alelo 677T pareça contribuir com o aumento dos níveis de Hcy, sua presença deve exercer uma influência sutil como fator de risco para a síndrome de Down. Os outros polimorfismos estudados não mostraram relação com o aumento de risco para a doença quando avaliados isoladamente (p=O,05). No entanto, quando os seis genes foram avaliados em conjunto, observamos que a presença de cada alelo mutado mostrou contribuir com um aumento de 20,3 por cento na chance de associação com a condição-caso, corroborando com a teoria da etiologia multifatorial da não-disjunção cromossômica.BV UNIFESP: Teses e dissertaçõe

New GJA8 variants and phenotypes highlight its critical role in a broad spectrum of eye anomalies

GJA8 encodes connexin 50 (Cx50), a transmembrane protein involved in the formation of lens gap junctions. GJA8 mutations have been linked to early onset cataracts in humans and animal models. In mice, missense mutations and homozygous Gja8 deletions lead to smaller lenses and microphthalmia in addition to cataract, suggesting that Gja8 may play a role in both lens development and ocular growth. Following screening of GJA8 in a cohort of 426 individuals with severe congenital eye anomalies, primarily anophthalmia, microphthalmia and coloboma, we identified four known [p.(Thr39Arg), p.(Trp45Leu), p.(Asp51Asn), and p.(Gly94Arg)] and two novel [p.(Phe70Leu) and p.(Val97Gly)] likely pathogenic variants in seven families. Five of these co-segregated with cataracts and microphthalmia, whereas the variant p.(Gly94Arg) was identified in an individual with congenital aphakia, sclerocornea, microphthalmia and coloboma. Four missense variants of unknown or unlikely clinical significance were also identified. Furthermore, the screening of GJA8 structural variants in a subgroup of 188 individuals identified heterozygous 1q21 microdeletions in five families with coloboma and other ocular and/or extraocular findings. However, the exact genotype–phenotype correlation of these structural variants remains to be established. Our data expand the spectrum of GJA8 variants and associated phenotypes, confirming the importance of this gene in early eye development.</p

Mutational spectrum of CHM characterized families.

<p>Mutational spectrum of CHM characterized families.</p

Molecular strategy followed up for the diagnosis of CHM families.

<p>Molecular strategy followed up for the diagnosis of CHM families.</p

Haplotypes from families presenting the recurrent <i>CHM</i> mutations.

<p>Identified pedigrees carrying the exon 9 deletion <b>(A)</b>, the p.Arg293* <b>(B)</b> and the p.Lys178Argfs*5 <b>(C)</b> mutations are shown. For exon 9 deletion, haplotypes analysis demonstrated identity by descent in the Spanish families RP-1310, RP-1560 and RP-2128 but independent origin for the Portuguese family RP-0779, defined by the alleles located along the black bar. For the p.Arg293* and p.Lys178Argfs*5 mutations, haplotypes indicates an independent origin for both variants defined by the alleles located along the black bar.</p

Clinical findings identified in affected individuals carrying mutations in the <i>CHM</i> gene.

<p>Clinical findings identified in affected individuals carrying mutations in the <i>CHM</i> gene.</p