Combined CNV, haplotyping and whole exome sequencing enable identification of two distinct novel EYS mutations causing RP in a single inbred tribe

Abstract

Whole exome sequencing (WES) has become routine in clinical practice, especially in studies of recessive hereditary diseases in inbred consanguineous families, where homozygosity of a founder mutation is assumed. Multiple members of two consanguineous families of a single Bedouin tribe were diagnosed with apparently autosomal recessive/pseudo-dominant retinitis pigmentosa (RP). Affected individuals exhibited severe visual impairment with nyctalopia, marked constriction of visual fields, markedly reduced and delayed responses on electro-retinography (ERG) and eventual loss of central vision. Combined copy-number variant (CNV) analysis, haplotype reconstruction and WES of the kindred identified two distinct novel mutations in EYS (RP25): a p.(W1817*) nonsense mutation (identified through WES) and a large deletion encompassing 9 of the 43 exons, that was missed by WES and was identified through microarray CNV analysis. Segregation analysis of both mutations demonstrated that all affected individuals were either homozygous for one of the mutations, or compound heterozygous for both. The two mutations are predicted to cause loss of function of the encoded protein and were not present in screening of 200 ethnically-matched controls. Our findings of two distinct mutations in the same gene in a single inbred kindred, identified only through combined WES and microarray CNV analysis, highlight the limitations of either CNV or WES alone, as the heterozygous deletion had normal WES read-depth values. Moreover, they demonstrate pitfalls in homozygosity mapping for disease-causing variant identification in inbred communities.

De‐novo “germline second hit” loss‐of‐heterozygosity RBP3 deletion mutation causing recessive high myopia

“…As we showed previously, deletion mutations may be overlooked with WES 19 . Both WES and Sanger sequencing demonstrated apparent homozygosity of the RBP3 missense mutation.…”

“…As we showed previously, deletion mutations may be overlooked with WES. 19 Both WES and Sanger sequencing demonstrated apparent homozygosity of the RBP3 missense mutation. Notably, while small deletions are well-identified by WES, larger deletions are often missed.…”

“…Careful analysis of the WES data then unraveled low levels of reads of the patient compared to controls, corroborating the CMA results. Thus, in some cases it is important to combine sequencing with other tools to detect copy number variations 19 . Moreover, in WES data analysis, careful consideration should be given to identification of genomic regions with low level of reads, especially in loci of genes relevant to the disease phenotype.…”

Nationwide genetic analysis of more than 600 families with inherited eye diseases in Argentina

“…Certain variants reported herein were not only seen in European alleles (i.e., USH2A c.12575 G > A in Spain 49 , USH2A c.1751G > T in Italy 50 , PRPF31 c.371_375del in Germany 51 , TYR c.996 G > A in Denmark 52 , USH2A c.11864 G > A in UK) 53 , or other American countries ( COL2A1 c.3574 C > T in Brazil 54 , RPGR c.1345 C > T in North America 55 ), but also in populations from all around the world ( USH2A c.5329 C > T in Japan 56 , EYS c.5450 G > A in a Bedouin tribe in Israel 57 , FAM161A c.1003 C > T in Palestine 58 , CNGB3 c.1148del in Pingelapese islanders of Micronesia 59 , and USH2A c.5858 C > G in Tunisia, among others) 60 .…”