Purpose

Primary congenital glaucoma (PCG) is a genetically heterogeneous disorder caused by developmental defects in the anterior chamber and trabecular meshwork. This disease is an important cause of childhood blindness. In this study, we aim to identify the genetic determinants of PCG in three consanguineous families of Pakistani descent.

Methods

Affected members of all three families underwent detailed ophthalmological examination including slit-lamp biomicroscopy. Blood samples were collected from affected and healthy members of all three families, and genomic DNA was extracted. Linkage analysis was performed for the known or reported loci of PCG to localize the disease interval, and logarithm of odds (LOD) scores were calculated. All protein-coding exons of the candidate gene, latent transforming growth factor-beta binding protein 2 (LTBP2), were bidirectionally sequenced to identify the disease-causing mutation.

Results

Short tandem repeat (STR) marker-based linkage analysis localized the critical interval to chromosome 14q with a maximum two-point LOD score of 2.86 (PKGL076), 2.8 (PKGL015), and 2.92 (PKGL042). Bidirectional Sanger sequencing of LTBP2 revealed three novel pathogenic variants, i.e., c.3028G>A (p.Asp1010Asn), c.3427delC (p.Gln1143Argfs*35), and c.5270G>A (p.Cys1757Tyr) in PKGL076, PKGL015, and PKGL042, respectively. All three mutations segregated with the disease phenotype in their respective families and were absent in 200 ethnically matched normal chromosomes.

Conclusions

We identified three novel mutations, p.D1010N, p.Q1143Rfs*35, and p.C1757Y, in LTBP2 responsible for PCG.

Purpose

Transcriptome is the entire repertoire of transcripts present in a cell at any particular time. We undertook a next-generation whole transcriptome sequencing approach to gain insight into the transcriptional landscape of the developing mouse lens.

Methods

We ascertained mouse lenses at six developmental time points including two embryonic (E15 and E18) and four postnatal stages (P0, P3, P6, and P9). The ocular tissue at each time point was maintained as two distinct pools serving as biological replicates for each developmental stage. The mRNA and small RNA libraries were paired-end sequenced on Illumina HiSeq 2000 and subsequently analyzed using bioinformatics tools.

Results

Mapping of mRNA and small RNA libraries generated 187.56 and 154.22 million paired-end reads, respectively. We detected a total of 14,465 genes in the mouse ocular lens at the above-mentioned six developmental stages. Of these, 46 genes exhibited a 40-fold differential (higher or lower) expression at one the five developmental stages (E18, P0, P3, P6, and P9) compared with their expression level at E15. Likewise, small RNA profiling identified 379 microRNAs (miRNAs) expressed in mouse lens at six developmental time points. Of these, 49 miRNAs manifested an 8-fold differential (higher or lower) expression at one the five developmental stages, as mentioned above compared with their expression level at E15.

Conclusions

We report a comprehensive profile of developing murine lens transcriptome including both mRNA and miRNA through next-generation RNA sequencing. A complete repository of the lens transcriptome of six developmental time points will be monumental in elucidating processes essential for the development of the ocular lens and maintenance of its transparency.

We previously identified a homozygous G178R mutation in human ASRGL1 (hASRGL1) through whole-exome analysis responsible for early onset retinal degeneration (RD) in patients with cone-rod dystrophy. The mutant G178R ASRGL1 expressed in Cos-7 cells showed altered localization, while the mutant ASRGL1 in E. coli lacked the autocatalytic activity needed to generate the active protein. To evaluate the effect of impaired ASRGL1 function on the retina in vivo, we generated a mouse model with c.578_579insAGAAA (NM_001083926.2) mutation (Asrgl1^mut/mut) through the CRISPR/Cas9 methodology. The expression of ASGRL1 and its asparaginase activity were undetectable in the retina of Asrgl1^mut/mut mice. The ophthalmic evaluation of Asrgl1^mut/mut mice showed a significant and progressive decrease in scotopic electroretinographic (ERG) response observed at an early age of 3 months followed by a decrease in photopic response around 5 months compared with age-matched wildtype mice. Immunostaining and RT-PCR analyses with rod and cone cell markers revealed a loss of cone outer segments and a significant decrease in the expression of Rhodopsin, Opn1sw, and Opn1mw at 3 months in Asrgl1^mut/mut mice compared with age-matched wildtype mice. Importantly, the retinal phenotype of Asrgl1^mut/mut mice is consistent with the phenotype observed in patients harboring the G178R mutation in ASRGL1 confirming a critical role of ASRGL1 in the retina and the contribution of ASRGL1 mutations in retinal degeneration.

Exome and whole-genome analyses powered by next-generation sequencing (NGS) have become invaluable tools in identifying causal mutations responsible for Mendelian disorders. Given that individual exomes contain several thousand single nucleotide variants and insertions/deletions, it remains a challenge to analyze large numbers of variants from multiple exomes to identify causal alleles associated with inherited conditions. To this end, we have developed user-friendly software that analyzes variant calls from multiple individuals to facilitate identification of causal mutations. The software, termed exomeSuite, filters for putative causative variants of monogenic diseases inherited in one of three forms: dominant, recessive caused by a homozygous variant, or recessive caused by two compound heterozygous variants. In addition, exomeSuite can perform homozygosity mapping and analyze the variant data of multiple unrelated individuals. Here we demonstrate that filtering of variants with exomeSuite reduces datasets to a fraction of a percent of their original size. To the best of our knowledge this is the first freely available software developed to analyze variant data from multiple individuals that rapidly assimilates and filters large data sets based on pattern of inheritance.

We previously investigated the transcriptome and proteome profiles of the murine ocular lens at six developmental time points including two embryonic (E15 and E18) and four postnatal time points (P0, P3, P6, and P9). Here, we extend our analyses to identify novel transcripts and peptides in developing  mouse lens. We identified a total of 9,707 novel transcripts and 325 novel fusion genes in developing mouse lens. Additionally, we identified 13,281 novel alternative splicing (AS) events in mouse lens including 6,990 exon skipping (ES), 2,447 alternative 3' splice site (A3SS), 1,900 alternative 5' splice site (A5SS), 1,771 mutually exclusive exons (MXE), and 173 intron retention (IR). Finally, we integrated our OMIC (Transcriptome and Proteome) datasets identifying 20 novel peptides in mouse lens. All 20 peptides were validated through matching MS/MS spectra of synthetic peptides. To the best of our knowledge, this is the first report integrating OMIC datasets to identify novel peptides in developing murine lens.

Inherited retinal degenerations (IRDs) are a group of genetically heterogeneous conditions with a broad phenotypic heterogeneity. Here, we report detection and validation of the underlying cause of progressive retinal degeneration in a nuclear family of European descent with a single affected individual. Whole genome sequencing of the proband and her unaffected sibling identified a novel intron 8 donor splice site variant (c.1296 + 1G>A) and a novel 731 base pair deletion encompassing exon 9 (Chr2:g.112751488_112752218 del) resulting in c.1297_1451del; p.K433_G484fsTer3 in the Mer tyrosine kinase protooncogene (MERTK), which is highly expressed in the retinal pigment epithelium (RPE). The proband carried both variants in the heterozygous state, which segregated with disease in the pedigree. These MERTK variants are predicted to result in the defective splicing of exon 8 and loss of exon 9 respectively. To evaluate the impact of these novel variants, peripheral blood mononuclear cells of the proband and her parents were reprogrammed to humaninduced pluripotent stem cell (hiPSC) lines, which were subsequently differentiated to hiPSC-RPE. Analysis of the proband's hiPSC-RPE revealed the absence of both MERTK transcript and its respective protein as well as abnormal phagocytosis when compared with the parental hiPSC-RPE. In summary, whole genome sequencing identified novel compound heterozygous variants in MERTK as the underlying cause of progressive retinal degeneration in a simplex case. Further, analysis using an hiPSC-RPE model established the functional impact of novel MERTK mutations and revealed the potential mechanism underlying pathology in the proband.

This study was conducted to identify the genetic basis of retinal dystrophies in consanguineous Pakistani families. We recruited two families with retinitis pigmentosa (RP) displaying visual difficulties, including nyctalopia and constricted visual fields. Linkage analysis and Sanger sequencing resulted in the identification of a previously reported nonsense mutation, c.847C > T, in exon 5 of CERKL in one family and a novel four-base pair deletion in exon 4 of RP1, c.delAGAA4218_4221, leading to premature protein termination in the second family. Here, we report two RP-causing mutations extending the genetic heterogeneity of the disease.

To delineate the genetic determinants associated with retinitis pigmentosa (RP), a hereditary retinal disorder, we recruited four large families manifesting cardinal symptoms of RP. We localized these families to regions on the human genome harboring the α and β subunits of phosphodiesterase 6 and identified mutations that were absent in control chromosomes. Our data suggest that mutations in PDE6A and PDE6B are responsible for the retinal phenotype in these families.

Purpose

To localize and identify the gene and mutations causing autosomal recessive retinal dystrophy in two consanguineous Pakistani families.

Methods

Consanguineous families from Pakistan were ascertained to be affected with autosomal recessive retinal degeneration. All affected individuals underwent thorough ophthalmologic examinations. Blood samples were collected, and genomic DNA was extracted using a salting out procedure. Genotyping was performed using microsatellite markers spaced at approximately 10 cM intervals. Two-point linkage analysis was performed with the lod score method. Direct DNA sequencing of amplified genomic DNA was performed for mutation screening of candidate genes.

Results

Genome-wide linkage scans yielded a lod score of 3.05 at θ=0 for D17S1832 and 3.82 at θ=0 for D17S938, localizing the disease gene to a 12.22 cM (6.64 Mb) region flanked by D17S1828 and D17S1852 for family 61032 and family 61227, which contains aryl hydrocarbon receptor interacting protein-like 1 (AIPL1), a gene previously implicated in recessive Leber congenital amaurosis and autosomal dominant cone-rod dystrophy. Sequencing of AIPL1 showed a homozygous c.773G>C (p.Arg258Pro) sequence change in all affected individuals of family 61032 and a homozygous c.465G>T (p.(H93_Q155del)) change in all affected members of family 61227.

Conclusions

The results strongly suggest that the c.773G>C (p.R258P) and c.465G>T (p.(H93_Q155del)) mutations in AIPL1 cause autosomal recessive retinal degeneration in these consanguineous Pakistani families.

Purpose

This study was conducted to localize and identify causal mutations associated with autosomal recessive retinitis pigmentosa (RP) in consanguineous familial cases of Pakistani origin.

Methods

Ophthalmic examinations that included funduscopy and electroretinography (ERG) were performed to confirm the affectation status. Blood samples were collected from all participating individuals, and genomic DNA was extracted. A genome-wide scan was performed, and two-point logarithm of odds (LOD) scores were calculated. Sanger sequencing was performed to identify the causative variants. Subsequently, we performed whole exome sequencing to rule out the possibility of a second causal variant within the linkage interval. Sequence conservation was performed with alignment analyses of PDE6A orthologs, and in silico splicing analysis was completed with Human Splicing Finder version 2.4.1.

Results

A large multigenerational consanguineous family diagnosed with early-onset RP was ascertained. An ophthalmic clinical examination consisting of fundus photography and electroretinography confirmed the diagnosis of RP. A genome-wide scan was performed, and suggestive two-point LOD scores were observed with markers on chromosome 5q. Haplotype analyses identified the region; however, the region did not segregate with the disease phenotype in the family. Subsequently, we performed a second genome-wide scan that excluded the entire genome except the chromosome 5q region harboring PDE6A. Next-generation whole exome sequencing identified a splice acceptor site mutation in intron 16: c.2028-1G>A, which was completely conserved in PDE6A orthologs and was absent in ethnically matched 350 control chromosomes, the 1000 Genomes database, and the NHLBI Exome Sequencing Project. Subsequently, we investigated our entire cohort of RP familial cases and identified a second family who harbored a splice acceptor site mutation in intron 10: c.1408-2A>G. In silico analysis suggested that these mutations will result in the elimination of wild-type splice acceptor sites that would result in either skipping of the respective exon or the creation of a new cryptic splice acceptor site; both possibilities would result in retinal photoreceptor cells that lack PDE6A wild-type protein.

Conclusions

we report two splice acceptor site variations in PDE6A in consanguineous Pakistani families who manifested cardinal symptoms of RP. Taken together with our previously published work, our data suggest that mutations in PDE6A account for about 2% of the total genetic load of RP in our cohort and possibly in the Pakistani population as well.