Next-Generation Exome Sequencing of Primary Congenital Glaucoma (PCG) Families

Abstract

Purpose: To screen a group of familial and sporadic PCG cases by next-generation exome sequencing (NGES), aiming to identify new PCG-causing genes.

Methods: Our screening panel consisted of 78 subjects (30 PCG) from 24 families. We used 39 subjects (17 PCG siblings and 22 parents) from 11 multiply affected families (6 with consanguinity) and another 39 unrelated trios (13 sporadic PCG and 26 parents) for exome sequencing. PCG subjects were negative for both CYP1B1 and LTBP2 mutations. Exome capture was performed using the NimbleGen SeqCap EZ V2 followed by sequencing on the Illumina HiSeq 2000. Reads were mapped to human genome (hg19) with ELAND aligner and variants were called with SAMtools.

Results: Initially, exome sequencing revealed 2,806,964 variants, including 252,607 novel ones. Considering a Quality Score of ≥60, 88% showed coverage of at least 8-times. After removing duplicates, 540,710 unique variants remained (208,948 Missense; 12,776 Nonsense; 104,428 Synonymous; 214,558 Non-Coding) with a mean value of 6,932 changes per individual. Further filtrations against 1,000 Genome, NHLBI Exomes, dbSNP, Simple Repeats and Genome Segmental Duplications revealed 247 novel amino acid changes in 235 genes. Homozygote changes were observed in only 22 of these novel genes. While heterozygote de novo variations were common, only 2 homozygote amino acid alterations were detected in our PCG cases. We are currently evaluating the causative nature of these candidate genes by Bioinformatic analysis and direct-Sanger sequencing of our original exome-sequenced subjects and their immediate family members. We further observed another 26,656 insertions or deletions (InDels) in these 78 subjects including, 55 novel variants, of which 6 were present in 2 or more families. The exact contribution of these InDels and their roles in the etiology of PCG phenotype is also being actively evaluated.

Conclusions: To date, exome sequencing of PCG trios has not been previously reported. Our NGES study of 24 families with 30 PCG subjects revealed that many of these do not have a co-segregating homozygous variation, as anticipated for an autosomal recessive condition. Our PCG cases may have resulted from compound-heterozygote mutations in two or more genes or caused by other de novo mutations. We are currently prioritizing potential genes of interest and further analyzing each variant as identified by our NGES data.

Keywords: 539 genetics • 440 candidate gene analysis