Identification of Genes in Pakistani Families with Inherited Eye Diseases

Abstract

The development and function of eye in humans is coordinated by well-organized set of events. Several genes play important role in these processes and mutations in these genes can lead to different types of ocular disorders including structural and functional disorders. Anophthalmia, microphthalmia (A/M) and glaucoma are among the defects which arise mostly due to structural abnormalities in the anterior segment of the eye. Inherited retinal dystrophies (IRDs) are among the functional eye disorders which affects retina, the innermost layer of the eye. Genetic and phenotypic overlap makes the diagnosis of these ocular disorders very challenging and frequently require help of comprehensive genomic applications for the accurate diagnosis. Though there are prior studies on ocular disorders from Pakistan, but underlying genes and mutations are still unknown for a large majority of families/patients. Therefore, there is a dire need to expand the genetic spectrum of inherited eye disorders in Pakistan. In this study, 28 families from different regions of Pakistan were recruited and were divided into four groups based on clinical presentation. Out of 28, 6 families were placed in the glaucoma group (A1-A6) and were subjected to either targeted Sanger sequencing (TSS) or exome sequencing (ES). However, these analyses identified mutation in all six families in CYP1B1. The mutation p.(Arg390His) was identified in three families, while in other families one recurrent p.(Pro442Glnfs*15) and two novel mutations p.(Pro118Leu) and p.(Met1?) were identified. Similarly, TSS of FOXE3 in eight (FamilyB1-B8) families with A/M phenotype identified p.(Cys240*) variant in three families while a missense p.(Ile97Val) variant was identified in one family. Four families (B3, B4, B5, B8) unsolved with TSS were subjected to genome sequencing (GS) which identified an already known missense variant p.(Asp183His) in SIX6 gene, a novel 13-bp deletion in VSX2 and a known 1-bp deletion p.(Cys857Alafs*5) and a novel deep-intronic mutation (c.3609-1307G>A) in PXDN. The probable effect p.(Arg1203Serfs*76) and severity of deep-intronic variant was confirmed through minigene splice assay performed in HEK293T cells which showed the activation of a pseudoexon. Single molecule molecular inversion probes (smMIPs) based panel sequencing was used for the remaining 14 families (Family C1-C11 and Family D1-D3) with different types of IRDs. Two types of smMIPs panels (Retinitis pigmentosa-Leber congenital Abstract Identification of Genes in Pakistani Families with Inherited Eye Diseases xvii amaurosis; RP-LCA) and (Macular degeneration; MD) based panel sequencing was performed in respective families based on their initial diagnosis. Panel based sequencing identified variants in 10 families out of 14 families. In LCA affected family, a known variant p.(Val9Met) was identified in NMNAT1. Two known variants p.(Arg482Trp) and p.(Gln301*) in two different families were identified in TULP1. The TULP1 variant p.(Gln301*) was identified in one loop family C7, but the other related loop of the same family has a novel MERTK variant p.(Gln146*). A novel PRPF8 inframe indel p.(Glu2307del) with low penetrance was identified in family C4. In family C5, a hypomorphic allele p.(Ala615Thr) in HGSNAT was identified with severe RP and a novel nonsense variant p.(Ser550*) in PROM1 genetically explained the phenotype of family C6. One previously known canonical splice site variant c.2493- 2_2495delinsGGC, p.(?) in CNGB1 was identified in a family with CSNB. Three already known variants p.(Arg439Trp), p.(Cys319Arg) and p.(Arg436Trp) in CNGB3 were found in two families achromatopsia (ACHM). The variant p.(Arg439Trp), was identified in homozygous state in affected members of family C10 while heterozygous variants p.(Cys319Arg) and p.(Arg436Trp) were present in trans in the affected members of family C11. Copy number variation (CNV) analysis from smMIPs coverage, identified a novel homozygous deletion in USH2A in the family D1 which presented Usher syndrome. The deletion spans the region of 51.47kb covering coding exons 50-58 in the gene. The exact breakpoints were identified (c.9740- 5487_11389+5457del) through combination of PCR based genome walking and amplicon based PacBio long-read sequencing. The four remaining unsolved families (C1, C8, D2 and D3) were screened through GS for the identification of disease causing variant. A novel variant p.(Ala32Profs*55) in ATOH7 was identified in the family C1 exhibiting congenital blindness. In family D2, a novel COL18A1 indel p.(Pro597Leufs*127) was identified to be associated with Knobloch syndrome, while in the third syndromic IRD family a novel non-coding NDP variant c.-208G>A was identified. Regulatory region variants in NDP are already known to cause Norrie disease. The family C8, diagnosed with night blindness and high myopia remained genetically unexplained in this study even after short read GS. Our approach leads us to the Abstract Identification of Genes in Pakistani Families with Inherited Eye Diseases xviii identification of 11 novel variants with a solve rate of 96% in our cohort of diverse inherited eye disorder families. The work presented here is part of following publications: • Basharat, R., Rodenburg, K., Rodríguez-Hidalgo, M., Jarral, A., Ullah, E., Corominas, J., Gilissen, C., Zehra, S. T., Hameed, U., Ansar, M., & de Bruijn, S. E. (2023). Combined Single Gene Testing and Genome Sequencing as an Effective Diagnostic Approach for Anophthalmia and Microphthalmia Patients. Genes, 14(8), 1573. https://doi.org/10.3390/genes14081573. • Rabia Basharat, Suzanne E. de Bruijn, Muhammad Zahid, Kim Rodenburg, Rebekkah J. Hitti-Malin, Erica G. M. Boonen, Afeefa Jarral, Arif Mahmood, Sharqa Khalil, Jawaid Ahmed Zai, Ghazanfar Ali, Christian Gilissen, Frans P. M. Cremers, Muhammad Ansar, Daan M. Panneman, Susanne Roosing (2023). Application of NGS to genetically diagnose a diverse range of inherited eye disorders in 15 consanguineous families from Pakistan (manuscript under preparation). • Rabia Basharat, Maria Ijaz, Bushra Rao, Muhammad Ansar (2023). CYP1B1; a prevalent cause of PCG in Pakistani families (manuscript under preparation).