Molecular Genetic Analysis of Families with Neurodevelopmental Disorders

Abstract

The human brain is a highly complex organ comprised of billions of interconnected neurons. The development of the brain involves multiple processes including neurogenesis, neuronal migration, neuronal differentiation, neuronal connections, synaptic pruning and gyrification. Any kind of discrepancy in these developmental processes can lead to neurodevelopmental disorders which may affect the brain and impair cognition. The most common neurodevelopmental disorders (NDDs) are autism spectrum disorder, attention deficit hyperactivity disorder, epilepsy, developmental dyslexia, intellectual disability (ID) and microcephaly (MCPH). In this study, eight families suffering from two major NDDs i.e., ID and microcephaly, were recruited from different provinces of Pakistan. These families comprise 23 individuals suffering from either ID or MCPH. Out of 23 affected individuals, 15 were males (65%) and 8 were females (35%). In family A-F patients were suffering from syndromic ID while patients from families G and H had microcephaly as a major feature. Whole genome genotyping and exome sequencing (WES) techniques were used to identify the genetic cause of the NDDs in these families (A-G) except for family H where only homozygosity mapping was performed. The affected members of family A presented with intellectual disability and retinitis pigmentosa (IDRP) syndrome. Exome sequencing identified a nonsense variant (c.747C>A) in exon 7 of the AGPAT3 gene, and western blot analysis of overexpressed wild-type (Wt) and mutant AGPAT3 (Tyr249Ter) in HEK293T cells showed a loss of mutant protein. Knockdown of Agpat3 in the mouse brain clearly showed deficits in neuronal migration, which suggested that the absence of AGPAT3 affects neuronal function. These data indicate that AGPAT3 is essential for neuronal migration and function. Furthermore, the localization study of Wt and AGPAT3 (Tyr249Ter) in U2OS cells revealed the absence of mutated protein from golgi and endoplasmic reticulum (ER) membranes, while Wt protein was distributed in golgi and ER membranes. These results confirmed the absence of mutant protein in cell lines, and that the loss of function of AGPAT3 can affect neuronal function which may cause IDRP syndrome in family A. Patients from family B were suffering from ID, facial dysmorphism and psychological disorders. Genetic analysis found a causal variant (c.451A>C) in the exon 2 RAB11FIP1 gene. Knockdown of Rab11fip1 in the mouse brain significantly reduced the neuronal migration in the upper cortical layers of the brain. Affected Abstract Molecular Genetic Analysis of Families with Neurodevelopmental Disorders xii individuals from family C presented ID with night blindness and seizures. The genetic analysis revealed the involvement of a missense variant (c.3365C>T) in the PLCB3 gene. The variant is predicted to be disease-causing by various in silico tools. In families D and E patients presented with ID caused by a splice site mutation (c.1958-2A>G) in AGAP2 and a missense mutation (c.245G>T) in exon 6 of SYNE1, respectively. The finding concerning AGAP2 was reported previously only once in an ID family. However, SYNE1 was already shown to be involved in cerebral ataxia and ID. Affected individuals from family F were also suffering from syndromic ID with postaxial unilateral and bilateral polydactyly. Genetic analysis of family F did not reveal any disease-causing variants in reported ID genes and Bardet Biedl Syndrome (BBS) genes. Affected members of family G and H were suffering from severe microcephaly. WES in family G could not detect any disease-causing variant in the investigated genes. Similarly, in family H targeted sequencing and homozygosity mapping did not reveal any causative gene defect in this family. Genetic analysis can be expanded by whole genome sequencing in such families to find pathogenic variants. In summary, five mutations in four novel ID candidate genes and one reported gene have been identified in five out of eight families with NDDs. These genes are thought to perform various functions in brain development. The work presented in this thesis has been published in the following articles; Malik M.A, Saqib M.A.N, Mientjes E, Acharya A, Alam M.R, Wallaard I, Schrauwen I, University of Washington Center for Mendelian Genomics, Bamshad M.J, Nickerson D.A, Santos-Cortez R.L.P, Elgersma Y, Leal S.M, Ansar M (2023). A loss of function variant in AGPAT3 underlies intellectual disability and retinitis pigmentosa (IDRP) syndrome. European Journal of Human Genetics, 2023 Dec;31(12):1447-1454. https://doi.org/10.1038/s41431-023-01475-w. Rasheed M, Khan V, Harripaul R, Siddiqui M, Malik M.A, Ullah Z, Zahid M, Vincent J. B, Ansar M (2021). Exome sequencing identifies novel and known mutations in families with intellectual disability. BMC Medical Genomics, 14(1), 211. https://doi.org/10.1186/s12920-021-01066-y. Malik M.A, Ansar M. Genetic players of intellectual disability and microcephaly in Pakistani population; A review. (Manuscript under preparation).