Genetic Studies in Pakistani Families with Hereditary Neurological Disorders

Abstract

Hereditary neurological disorders are a broad class of diseases caused by certain genetic factors. These factors may vary from monogenic to a group of genetic risk alleles and environmental factors. The current study was designed to find the genetic basis of particular neurological disorders in consanguineous families from Pakistan including intellectual disability with epilepsy, muscular dystrophy, motor neuropathy and congenital insensitivity to pain. Intellectual disability (ID) is a large and diverse group of genetic disorders in syndromic and non-syndromic forms. It is a common neurological disease having an onset usually before the end of second decade of life. There are around 1396 genes involved in ID [1] and some of them involved in Pakistani population are AP1S2, ARHGEF6, MECP2, AGTR2, PTCHD1, MECP2, TSPAN7 and ATRX. Distal hereditary motor neuropathy (dHMN) is broadly used terms for progressive neurological disorders which cause degeneration of lower motor neurons. It follows autosomal dominant, recessive and X-linked patterns of inheritance. Sigma non-opioid intracellular receptor-1 (SIGMAR1) gene (MIM# 601978) is reported to have a probable role in amyotrophic lateral sclerosis (ALS), fronto temporal dementia, Silver-like syndrome and distal hereditary motor neuropathy (dHMN). Limb girdle muscular dystrophy (LGMD) is a collective term for description of muscular dystrophies having similar physical presentations of atrophy and weakness of proximal muscle, raised creatinine kinase (CK) levels and dysmorphic muscle biopsies. Limb girdle muscular dystrophy 2A (LGMD2A) is the most prevalent sub type of LGMD in the world xii and is the first ever molecularly diagnosed muscular dystrophy. It occurs due to the mutation in calpain-3 gene (CAPN3) (MIM# 114240, 15q15.2-q21.1. Patients show scapular winging, asymmetric weakness, and muscular atrophy with no cardiac system compromised. Congenital insensitivity to pain (CIP; MIM 243000) is a form of hereditary sensory and autonomic neuropathies (HSAN). Mutations in SCN9A, SCN11A and PRDM12 are responsible for causing CIP; in which patients are insensitive to pain, touch and may also lose sense of smell. Other symptoms include tongue biting, bruises on skin, early loss of teeth and fractured bones. Five autosomal recessive Pakistani families with multiple affected individuals were recruited for the present study (Families A-E). A summary of their results is given below. Family A: Affected individuals showed epilepsy and intellectual disability. SNP genotyping revealed 2.5 Mb homozygous region at 6q21 between SNP marker rs2023705 and rs925755. Whole exome sequencing was done to find out candidate variant. Initially there were seven genes (ZPLD1, FAT2, GTF3C6, SYNE1, TRPA1, HMCN2 and SDHD). Candidate genes were analyzed on the basis of bioinformatics tools and GTF3C6 was found to be the most promising. All the affected individuals were found to have (rs755615657, c.86G>T; p.Gly29Val). In Family B: Affected individuals showed mild form of intellectual disability. Upon the exome analysis and data filtration affected individuals were found to have (DZIP3 c.2162G>T; p.Ser721Ile) and parents were found to be heterozygous. xiii Family C: Affected individuals of this family showed distal hereditary motor neuropathy. SNP genotyping was done on all the five affected individuals of Family C, and homozygous regions were found where there is possibility of presence of candidate genes. Affected individuals were found to share 12.08 Mb of homozygous region (chr9:23,788,680-35,867,901) bordered by rs1410846 and rs10814288 and was present on 9p21.3-9p13, harboring 112 genes including previously reported SIGMAR1. We identified deletion of T nucleotide in splice donor site in intron 1 of the SIGMAR1 gene (rs796065352, chr 9:34,637,543 G/T). This result predicted to cause a breakage in splice donor site resulting in alternative splicing event causing an in-frame deletion of 60 base pairs in exon 1 (NM_005866.3; c.92_151del), resulting in shortened SIGMAR1 31_50del including an integral part of transmembrane domain. This prediction is previously validated in vitro and we further did co-segregation analysis which validated the results. All the affected individuals were found to have these results and this was absent in 150 control chromosomes from Pakistani population. Further in Family D: All affected individuals showed distal muscle wasting and weakness of the upper and lower limbs, brisk deep tendon reflexes, and proximal muscle wasting and weakness. SNP genotyping of whole genome was done on all the three affected individuals to find the homozygosity (homozygous) region for the identification of candidate genes. All the affected individuals were found to share a homozygosity region of 25.1 Mb spanning between rs16971558 and rs999047 (chr15:41,026,397-66,213,698 [hg19]) on chromosome 15. The locus is found to have 489 genes including the strong candidate gene CAPN3. For the identification of variant within the locus we performed whole exome sequencing on one affected individual (II:5). After data filtering using standard parameters xiv including potential pathogenicity, call quality, localization within interval and population frequency, we identified substitution A>G nucleotide in splice donor site of exon 2 of the CAPN3 gene (chr15: 42,651,698- chr15:g.42384556A>G [hg 38]), which is predicted to cause an alternative splicing event. In Family E: Medical history was collected and diagnosis of congenital insensitivity to pain was established by a neurologist. Whole exome sequencing and data analysis from DNA of this affected boy revealed an already reported homozygous mutation (Ala353_Ala359dup) in PRDM12 gene (data not shown). In this family we found a trio and the mutation was present in PRDM12 gene which was previously reported to cause the same condition. For further analysis, PRDM5, PRDM8, PRDM12 and PRDM13 were selected because of their involvement in neurological disorders, out the four only PRDM12 was found to be signaling protein. Functional annotation of PRDM12 was also done. For finding the interaction of PRDM12 with other proteins elucidating role in pain perception we used STRING analysis which showing interaction of PRDM12 with PRDM8 (MIM# 616639), PRDM11 (MIM# 616347), PRDM13 (MIM# 616741) and ZNF408 (MIM# 616454). Early clinical evaluation is crucial in making a correct choice of genetic testing in families affected with hereditary neurological disorders. From these results it is certain that homozygosity mapping coupled with whole exome sequencing / targeted panel sequencing in families having multiple affected individuals is the best strategy for mutation detection. In future, functional studies of mutations in GTF3C6, DZIP3, SIGMAR1, and PRDM12 along with further studies on CAPN3 would provide molecular insights for neurological pathways paving way for novel therapeutic strategies.