Whole-exome sequencing identifies novel compound heterozygous mutations in USH2A in Spanish patients with autosomal recessive retinitis pigmentosa.
OBJECTIVE
Retinitis pigmentosa (RP) is an inherited retinal dystrophy characterized by extreme genetic and clinical heterogeneity. Thus, the diagnosis is not always easily performed due to phenotypic and genetic overlap. Current clinical practices have focused on the systematic evaluation of a set of known genes for each phenotype, but this approach may fail in patients with inaccurate diagnosis or infrequent genetic cause. In the present study, we investigated the genetic cause of autosomal recessive RP (arRP) in a Spanish family in which the causal mutation has not yet been identified with primer extension technology and resequencing.


METHODS
We designed a whole-exome sequencing (WES)-based approach using NimbleGen SeqCap EZ Exome V3 sample preparation kit and the SOLiD 5500×l next-generation sequencing platform. We sequenced the exomes of both unaffected parents and two affected siblings. Exome analysis resulted in the identification of 43,204 variants in the index patient. All variants passing filter criteria were validated with Sanger sequencing to confirm familial segregation and absence in the control population. In silico prediction tools were used to determine mutational impact on protein function and the structure of the identified variants.


RESULTS
Novel Usher syndrome type 2A (USH2A) compound heterozygous mutations, c.4325T>C (p.F1442S) and c.15188T>G (p.L5063R), located in exons 20 and 70, respectively, were identified as probable causative mutations for RP in this family. Family segregation of the variants showed the presence of both mutations in all affected members and in two siblings who were apparently asymptomatic at the time of family ascertainment. Clinical reassessment confirmed the diagnosis of RP in these patients.


CONCLUSIONS
Using WES, we identified two heterozygous novel mutations in USH2A as the most likely disease-causing variants in a Spanish family diagnosed with arRP in which the cause of the disease had not yet been identified with commonly used techniques. Our data reinforce the clinical role of WES in the molecular diagnosis of highly heterogeneous genetic diseases where conventional genetic approaches have previously failed in achieving a proper diagnosis.
Department of Genetics, Reproduction and Fetal Medicine, Institute of Biomedicine of Seville, University Hospital Virgen del Rocío/CSIC/University of Seville, Seville, Spain ; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Seville, Spain.
Cristina
C
Méndez-Vidal
María
M
González-Del Pozo
Alicia
A
Vela-Boza
Javier
J
Santoyo-López
Francisco J
FJ
López-Domingo
Carmen
C
Vázquez-Marouschek
Joaquin
J
Dopazo
Salud
S
Borrego
Guillermo
G
Antiñolo
D000328:Adult; D001483:Base Sequence; D020090:Chromosome Segregation / Q000235:genetics; D004252:DNA Mutational Analysis; D059472:Exome / Q000235:genetics*; D005091:Exons / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000235:genetics*; D005260:Female; D005808:Genes, Recessive / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D010375:Pedigree; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D015203:Reproducibility of Results; D012174:Retinitis Pigmentosa / Q000150:complications* / Q000235:genetics*; D035781:Siblings; D013030:Spain; D052245:Usher Syndromes / Q000150:complications* / Q000235:genetics*
D016326:Extracellular Matrix Proteins; C112735:USH2A protein, human