John Suk

Investigating the Molecular Basis of Retinal Degeneration in a Familial Cohort of Pakistani Decent by Exome Sequencing.

Purpose To define the molecular basis of retinal degeneration in consanguineous Pakistani pedigrees with early onset retinal degeneration. Methods A cohort of 277 individuals representing 26 pedigrees from the Punjab province of Pakistan was analyzed. Exomes were captured with commercial kits and sequenced on an Illumina HiSeq 2500. Candidate variants were identified using standard tools and analyzed using exomeSuite to detect all potentially pathogenic changes in genes implicated in retinal degeneration. Segregation analysis was performed by dideoxy sequencing and novel variants were additionally investigated for their presence in ethnicity-matched controls. Results We identified a total of nine causal mutations, including six novel variants in RPE65, LCA5, USH2A, CNGB1, FAM161A, CERKL and GUCY2D as the underlying cause of inherited retinal degenerations in 13 of 26 pedigrees. In addition to the causal variants, a total of 200 variants each observed in five or more unrelated pedigrees investigated in this study that were absent from the dbSNP, HapMap, 1000 Genomes, NHLBI ESP6500, and ExAC databases were identified, suggesting that they are common in, and unique to the Pakistani population. Conclusions We identified causal mutations associated with retinal degeneration in nearly half of the pedigrees investigated in this study through next generation whole exome sequencing. All novel variants detected in this study through exome sequencing have been cataloged providing a reference database of variants common in, and unique to the Pakistani population.

Whole Genome Sequencing Revealed Mutations in Two Independent Genes as the Underlying Cause of Retinal Degeneration in an Ashkenazi Jewish Pedigree

Retinitis pigmentosa (RP) causes progressive photoreceptor loss resulting from mutations in over 80 genes. This study identified the genetic cause of RP in three members of a non-consanguineous pedigree. Detailed ophthalmic evaluation was performed in the three affected family members. Whole exome sequencing (WES) and whole genome sequencing (WGS) were performed in the three affected and the two unaffected family members and variants were filtered to detect rare, potentially deleterious variants segregating with disease. WES and WGS did not identify potentially pathogenic variants shared by all three affected members. However, WES identified a previously reported homozygous nonsense mutation in KIZ (c.226C>T, p.Arg76*) in two affected sisters, but not in their affected second cousin. WGS revealed a novel 1.135 kb homozygous deletion in a retina transcript of C21orf2 and a novel 30.651 kb heterozygous deletion in CACNA2D4 in the affected second cousin. The sisters with the KIZ mutation carried no copies of the C21orf2 or CACNA2D4 deletions, while the second cousin with the C21orf2 and CACNA2D4 deletions carried no copies of the KIZ mutation. This study identified two independent, homozygous mutations in genes previously reported in autosomal recessive RP in a non-consanguineous family, and demonstrated the value of WGS when WES fails to identify likely disease-causing mutations.

Molecular Diagnostic Testing by eyeGENE: Analysis of Patients With Hereditary Retinal Dystrophy Phenotypes Involving Central Vision Loss

PURPOSE To analyze the genetic test results of probands referred to eyeGENE with a diagnosis of hereditary maculopathy. METHODS Patients with Best macular dystrophy (BMD), Doyne honeycomb retinal dystrophy (DHRD), Sorsby fundus dystrophy (SFD), or late-onset retinal degeneration (LORD) were screened for mutations in BEST1, EFEMP1, TIMP3, and CTRP5, respectively. Patients with pattern dystrophy (PD) were screened for mutations in PRPH2, BEST1, ELOVL4, CTRP5, and ABCA4; patients with cone-rod dystrophy (CRD) were screened for mutations in CRX, ABCA4, PRPH2, ELOVL4, and the c.2513G>A p.Arg838His variant in GUCY2D. Mutation analysis was performed by dideoxy sequencing. Impact of novel variants was evaluated using the computational tool PolyPhen. RESULTS Among the 213 unrelated patients, 38 had BMD, 26 DHRD, 74 PD, 8 SFD, 6 LORD, and 54 CRD; six had both PD and BMD, and one had no specific clinical diagnosis. BEST1 variants were identified in 25 BMD patients, five with novel variants of unknown significance (VUS). Among the five patients with VUS, one was diagnosed with both BMD and PD. A novel EFEMP1 variant was identified in one DHRD patient. TIMP3 novel variants were found in two SFD patients, PRPH2 variants in 14 PD patients, ABCA4 variants in four PD patients, and p.Arg838His GUCY2D mutation in six patients diagnosed with dominant CRD; one patient additionally had a CRX VUS. ABCA4 mutations were identified in 15 patients with recessive CRD. CONCLUSIONS Of the 213 samples, 55 patients (26%) had known causative mutations, and 13 (6%) patients had a VUS that was possibly pathogenic. Overall, selective screening for mutations in BEST1, PRPH2, and ABCA4 would likely yield the highest success rate in identifying the genetic basis for macular dystrophy phenotypes. Because of the overlap in phenotypes between BMD and PD, it would be beneficial to screen genes associated with both diseases.

Establishing the involvement of the novel gene AGBL5 in retinitis pigmentosa by whole genome sequencing

While more than 250 genes are known to cause inherited retinal degenerations (IRD), nearly 40-50% of families have the genetic basis for their disease unknown. In this study we sought to identify the underlying cause of IRD in a family by whole genome sequence (WGS) analysis. Clinical characterization including standard ophthalmic examination, fundus photography, visual field testing, electroretinography, and review of medical and family history was performed. WGS was performed on affected and unaffected family members using Illumina HiSeq X10. Sequence reads were aligned to hg19 using BWA-MEM and variant calling was performed with Genome Analysis Toolkit. The called variants were annotated with SnpEff v4.11, PolyPhen v2.2.2, and CADD v1.3. Copy number variations were called using Genome STRiP (svtoolkit 2.00.1611) and SpeedSeq software. Variants were filtered to detect rare potentially deleterious variants segregating with disease. Candidate variants were validated by dideoxy sequencing. Clinical evaluation revealed typical adolescent-onset recessive retinitis pigmentosa (arRP) in affected members. WGS identified about 4 million variants in each individual. Two rare and potentially deleterious compound heterozygous variants p.Arg281Cys and p.Arg487* were identified in the gene ATP/GTP binding protein like 5 (AGBL5) as likely causal variants. No additional variants in IRD genes that segregated with disease were identified. Mutation analysis confirmed the segregation of these variants with the IRD in the pedigree. Homology models indicated destabilization of AGBL5 due to the p.Arg281Cys change. Our findings establish the involvement of mutations in AGBL5 in RP and validate the WGS variant filtering pipeline we designed.

Genetic analysis of 10 pedigrees with inherited retinal degeneration by exome sequencing and phenotype-genotype association

Our purpose was to identify causative mutations and characterize the phenotype associated with the genotype in 10 unrelated families with autosomal recessive retinal degeneration. Ophthalmic evaluation and DNA isolation were carried out in 10 pedigrees with inherited retinal degenerations (IRD). Exomes of probands from eight pedigrees were captured using Nimblegen V2/V3 or Agilent V5+UTR kits, and sequencing was performed on Illumina HiSeq. The DHDDS gene was screened for mutations in the remaining two pedigrees with Ashkenazi Jewish ancestry. Exome variants were filtered to detect candidate causal variants using exomeSuite software. Segregation and ethnicity-matched control sample analysis were performed by dideoxy sequencing. Retinal histology of a patient with DHDDS mutation was studied by microscopy. Genetic analysis identified six known mutations in ABCA4 (p.Gly1961Glu, p.Ala1773Val, c.5461-10T>C), RPE65 (p.Tyr249Cys, p.Gly484Asp), PDE6B (p.Lys706Ter) and DHDDS (p.Lys42Glu) and ten novel potentially pathogenic variants in CERKL (p.Met323Val fsX20), RPE65 (p.Phe252Ser, Thr454Leu fsX31), ARL6 (p.Arg121His), USH2A (p.Gly3142Ter, p.Cys3294Trp), PDE6B (p.Gln652Ter), and DHDDS (p.Thr206Ala) genes. Among these, variants/mutations in two separate genes were observed to segregate with IRD in two pedigrees. Retinal histopathology of a patient with a DHDDS mutation showed severe degeneration of retinal layers with relative preservation of the retinal pigment epithelium. Analysis of exome variants in ten pedigrees revealed nine novel potential disease-causing variants and nine previously reported homozygous or compound heterozygous mutations in the CERKL, ABCA4, RPE65, ARL6, USH2A, PDE6B, and DHDDS genes. Mutations that could be sufficient to cause pathology were observed in more than one gene in one pedigree.

Identification of the genetic determinants responsible for retinal degeneration in families of Mexican descent

ABSTRACT 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.

Correction: Gustafson et al., Whole Genome Sequencing Revealed Mutations in Two Independent Genes as the Underlying Cause of Retinal Degeneration in an Ashkenazi Jewish Pedigree. Genes 2017, 8, 210

Following publication of our article [1], we identified discrepancies between the pedigree shown in Figure 1 and the rest of the text.[...].