Purpose : To identify the molecular basis of disease in 4 pedigrees with IRD that remained unresolved after the initial analysis of WGS data.
Methods : Two pedigrees of Indian origin and one each of Hispanic and European American (EA) origin were analyzed. WGS was done on one affected individual and two unaffected siblings from each pedigree using Illumina HiSeq X Ten. Initial analysis by mapping reads to hg19 and variant calling using GATK did not identify disease causing variants. The data was further analyzed using two methods: (1) the reads were re-aligned to hg38 and variants were called using GATK; (2) read alignment and variant calling was done using the Illumina DRAGEN Bio-IT platform. Validation of a large inversion identified was performed by isolating high molecular weight DNA from the patient and unaffected family members using the Bionano DLS kit and analyzed on Bionano Saphyr. Segregation analysis was done by PCR followed by Sanger sequencing.
Results : Re-analysis using the hg38 reference genome identified 2 novel compound heterozygous variants p.[Pro3Ser;Thr1749Ala] in ALMS1 in one Indian pedigree. Analysis of remaining pedigrees using Illumina DRAGEN Bio-IT identified unique pathogenic variants in known IRD genes. In the second Indian pedigree, uniparental isodisomy (UPiD) involving a 39Mb homozygous region on chromosome-15 that included a novel pathogenic homozygous NR2E3 variant, p.Trp257Ser, was observed. In the EA pedigree, a heterozygous nonsense mutation p.Gln874* and a large inversion in the EYS gene were detected. This inversion (Chr6: g.65986120_67623958inv) was further characterized by analyzing the samples of the patient and unaffected family members using Bionano Saphyr. This analysis confirmed the presence of a 1.6Mb inversion on chromosome-6, which includes exons 1 to 12 of EYS. In the third Hispanic family with 1 affected female and 2 unaffected female siblings, a previously reported p.Glu746ArgfsTer23 mutation in RPGR was identified. All detected mutations segregated with IRD in all 4 families.
Conclusions : Analysis using the hg38 reference genome identified causative variants in one pedigree among four. Additional analysis using Illumina DRAGEN identified a 39Mb segmental UPiD, a mutation in RPGR and mutations in the EYS gene including a large inversion.
This is a 2021 ARVO Annual Meeting abstract.