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

Search for uniparental disomy (UPD) in patients with spinal muscular atrophy (SMA)

Objetivos: Padronizar a tecnica de pesquisa de DUP por marcadores microssatelites em gel de acrilamida e coloracao com a prata. Demonstrar a variacao do padrao de bandas dos marcadores microssatelites escolhidos em uma amostra controle. Realizar o diagnostico molecular da delecao do exon 7 no gene SMN1 em pacientes com AME. Verificar a presenca de DUP do cromossomo 5 em pacientes com AME sem recorrencia na irmandade e sem consanguinidade parental. Comparar os resultados obtidos atraves da tecnica de marcadores microssatelites e SNP array. Metodos: Foram utilizados 11 marcadores microssatelites para todos os pacientes. Em uma familia foi realizado o teste de portador para AME e cariotipo da probanda na qual foi encontrada regiao de DUP paterna. Para os experimentos com arrays foram utilizadas plataformas Genome-Wide Human SNP Array 6.0 (Affymetrix) segundo os protocolos do fabricante. Resultados: Na primeira etapa do trabalho os marcadores foram utilizados para padronizacao dos metodos. A segunda etapa foi composta por triades de pai, mae e filho com diagnostico clinico de AME. A analise com os marcadores microssatelites em 11 familias revelou padroes normais de heranca mendeliana biparental. A analise com os marcadores microssatelites em uma familia revelou ausencia de alelos maternos para dois marcadores, sugerindo DUP paterna do cromossomo 5. O estudo citogenetico realizado na probanda apresentou cariotipo normal. A analise quantitativa do numero de copias dos genes SMN1 e SMN2 no exon 7, revelou no pai uma copia do gene SMN1 e duas copias do gene SMN2, indicando que ele e portador assintomatico de AME e a analise realizada na mae revelou 2 copias do gene SMN1 e uma copia de SMN2, demonstrando que ela nao e portadora assintomatica de AME. A analise do resultado do SNP array verificou perda de heterozigosidade nas regioes 5p14.3, 5q23.1 e 5q31.2-q31.3. Alem da perda de heterozigosidade, o SNP array detectou uma delecao de 1.3Mb envolvendo o gene SMN1. Conclusoes: A pesquisa de DUP atraves do estudo de marcadores microssatelites foi padronizada de maneira rapida e eficiente. O padrao de bandas para os marcadores estudados nao variou na amostra estudada. O diagnostico molecular da delecao do exon 7 no gene SMN1, responsavel por 98% das mutacoes nesse gene, foi realizada em todos os pacientes. Foi possivel verificar regioes de DUP paterna em 1 dos 12 pacientes estudados. A tecnica de SNP array confirmou a presenca de regioes com perda de heterozigosidade. Adicionalmente foi encontrada uma delecao na regiao que contem o gene responsavel pela doenca em estudoBV 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

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

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