Application of Genomic Techniques in Identifying Genes in Families Segregating Hereditary Retinal Dystrophies

Abstract

Vision is an important sensory process that enables us to see things. This process is based on the price structure and function of retinal tissues and mutations in any gene playing role in these tissues can cause retinal dystrophies (RDs). RDs are further divided into different types based on the clinical presentation of the patients but major types include Leber Congenital Amaurosis (LCA), Retinitis Pigmentosa (RP), Cone- Rod Dystrophy (CRD and Macular Degeneration (MD). The onset, progression and severity of every RD phenotype depends on the nature of gene involved and type of mutation. Despite the great progress on the identification of RD genes, the underlying genetic defect are still unknown in large number of cases/families which require additional genetic studies. This study was initiated to recruit fifteen families with different types of RD and perform genetic analysis to identify the underlying mutations. Analysis of the clinical information of fifteen families, recruited in this study, showed distribution in three groups. Majority of the families (8 families) belonged to LCA, whereas four families were placed in RP group while the remaining three families were placed in other RD group. Genetic analysis of these families with genome wide genotyping, homozygosity mapping, exome sequencing and Sanger sequencing resulted in the identification of underlying genetic defects in 13 out of 15 families, indicating a diagnostic yield of 86.6%. Novel pathogenic variants were identified in four families including a large deletion present on chromosome 14. The remaining nine mutations were either reported in literature or public databases. Novel mutations were identified in families A, B,C and M and in genes SPATA7, CRB1, LCA5 and ABCA4, respectively. In family A, an 81.85Kb deletion was detected on chromosome 14 (hg38; chr14:88470782-88388933) which spans all the coding exons of SPATA7 gene. The deletion breakpoints were identified by primer walking based on the information from genotype data and BAM file of individuals used for exome sequencing. The loss of SPATA7 gene was confirmed through the amplification of exon 5 of from cDNA samples of two affected individuals. In family B a mutation (c.2424T>A; p.Tyr808Ter) was identified in CRB1 gene, that could possibly lead to loss of function due to nonsense-mediated decay. Third novel homozygous mutation (c.1550_1551delGA) was found in the family C in LCA5 gene. The dinucleotide deletion results in a frameshift and therefore causes an early termination of the specific protein Abstract vii (p.Arg517Ilefs*3). Fourth novel mutation was identified in family M which was clinically diagnosed as Stargardt disease. In this case, a novel splice-site variant (c.3328+1G>C) was identified in ABCA4 gene through whole exome sequencing. In silico data analysis showed that variant to be likely pathogenic and may result in intron retention. In the remaining nine families known mutations were identified in AIPL1 (Family D & E), NMNAT1 (Family F), CRB1 (Family G), CCDC66 (Family I), TTC8 (Family J), TENM1 (Family K), ABCA4 (Family N) and BEST1 (Family O). But in one LCA family (Family H) and one RP family (Family L) pathogenic mutations could not be identified and may require further studies. This study provides insight to the genetic diversity of inherited retinal disorders in the Pakistani population and reports the identification of four novel mutations in families segregating heterogeneous RDs. Genetic screening of such families that belong to remote areas with less resources and health facilities will help in accurate diagnosis and family counselling for further disease management. The work presented in this study has been partly published in the following publications; 1. 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Sajid S, Rabia Basharat, Ehsan Ullah, Muhammad Arif Nadeem Saqib, Memoona Rasheed, Muhammad Ansar. Identification of Disease-Causing Mutations Using Homozygosity Mapping and Whole Exome Sequencing of Abstract viii Index Cases of Inherited Retinal Dystrophy Families. Molecular Vision (Manuscript under revision)