RESUMO
Retinal dystrophies are a phenotypically and genetically complex group of conditions. Because of this complexity, it can be challenging in many families to determine the inheritance based on pedigree analysis alone. Clinical examinations were performed and blood samples were collected from a North American (M1186) and a consanguineous Pakistani (PKRD168) pedigree affected with two different retinal dystrophies (RD). Based on the structure of the pedigrees, inheritance patterns in the families were difficult to determine. In one family, linkage analysis was performed with markers on X-chromosome. In the second family, whole-exome sequencing (WES) was performed. Subsequent Sanger sequencing of genes of interest was performed. Linkage and haplotype analysis localized the disease interval to a 70 Mb region on the X chromosome that encompassed RP2 and RPGR in M1186 . The disease haplotype segregated with RD in all individuals except for an unaffected man (IV:3) and his affected son (V:1) in this pedigree. Subsequent analysis identified a novel RPGR mutation (p. Lys857Glu fs221X) in all affected members of M1186 except V:1. This information suggests that there is an unidentified second cause of retinitis pigmentosa (RP) within the family. A novel two-base-pair deletion (p. Tyr565Ter fsX) in CHM (choroideremia) was found to segregate with RD in PKRD168. This paper highlights the challenges of interpreting family history in families with RD and reports on the identification of novel mutations in two RD families.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Olho/genética , Degeneração Retiniana/genética , Deleção de Sequência , Códon sem Sentido , Consanguinidade , Feminino , Genes Ligados ao Cromossomo X , Ligação Genética , Haplótipos/genética , Humanos , Masculino , América do Norte , Paquistão , Linhagem , Sequenciamento do ExomaRESUMO
PURPOSE: To investigate the clinical characteristics and genetic basis of inherited retinal degeneration (IRD) in six unrelated pedigrees from Mexico. METHODS: A complete ophthalmic evaluation including measurement of visual acuities, Goldman kinetic or Humphrey dynamic perimetry, Amsler test, fundus photography, and color vision testing was performed. Family history and blood samples were collected from available family members. DNA from members of two pedigrees was examined for known mutations using the APEX ARRP genotyping microarray and one pedigree using the APEX LCA genotyping microarray. The remaining three pedigrees were analyzed using a custom-designed targeted capture array covering the exons of 233 known retinal degeneration genes. Sequencing was performed on Illumina HiSeq. Reads were mapped against hg19, and variants were annotated using GATK and filtered by exomeSuite. Segregation and ethnicity-matched control sample analyses were performed by dideoxy sequencing. RESULTS: Six pedigrees with IRD were analyzed. Nine rare or novel, potentially pathogenic variants segregating with the phenotype were detected in IMPDH1, USH2A, RPE65, ABCA4, and FAM161A genes. Among these, six were known mutations while the remaining three changes in USH2A, RPE65, and FAM161A genes have not been previously reported to be associated with IRD. Analysis of 100 ethnicity-matched controls did not detect the presence of these three novel variants indicating, these are rare variants in the Mexican population. CONCLUSIONS: Screening patients diagnosed with IRD from Mexico identified six known mutations and three rare or novel potentially damaging variants in IMPDH1, USH2A, RPE65, ABCA4, and FAM161A genes that segregated with disease.
Assuntos
Proteínas do Olho/genética , Mutação , Degeneração Retiniana/genética , Transportadores de Cassetes de Ligação de ATP/genética , Adolescente , Adulto , Idoso , Pré-Escolar , Análise Mutacional de DNA , Proteínas da Matriz Extracelular/genética , Feminino , Determinismo Genético , Técnicas de Genotipagem , Humanos , IMP Desidrogenase/genética , Masculino , México/epidemiologia , Pessoa de Meia-Idade , Linhagem , Fenótipo , Degeneração Retiniana/etnologia , Sequenciamento do Exoma , cis-trans-Isomerases/genéticaRESUMO
AIM: To test the utility of targeted sequencing as a method of clinical molecular testing in patients diagnosed with inherited retinal degeneration (IRD). METHODS: After genetic counseling, peripheral blood was drawn from 188 probands and 36 carriers of IRD. Single gene testing was performed on each patient in a Clinical Laboratory Improvement Amendment (CLIA) certified laboratory. DNA was isolated, and all exons in the gene of interest were analyzed along with 20 base pairs of flanking intronic sequence. Genetic testing was most often performed on ABCA4, CTRP5, ELOV4, BEST1, CRB1, and PRPH2. Pathogenicity of novel sequence changes was predicted by PolyPhen2 and sorting intolerant from tolerant (SIFT). RESULTS: Of the 225 genetic tests performed, 150 were for recessive IRD, and 75 were for dominant IRD. A positive molecular diagnosis was made in 70 (59%) of probands with recessive IRD and 19 (26%) probands with dominant IRD. Analysis confirmed 12 (34%) of individuals as carriers of familial mutations associated with IRD. Thirty-two novel variants were identified; among these, 17 sequence changes in four genes were predicted to be possibly or probably damaging including: ABCA4 (14), BEST1 (2), PRPH2 (1), and TIMP3 (1). CONCLUSIONS: Targeted analysis of clinically suspected genes in 225 subjects resulted in a positive molecular diagnosis in 26% of patients with dominant IRD and 59% of patients with recessive IRD. Novel damaging mutations were identified in four genes. Single gene screening is not an ideal method for diagnostic testing given the phenotypic and genetic heterogeneity among IRD cases. High-throughput sequencing of all genes associated with retinal degeneration may be more efficient for molecular diagnosis.
