Mapping of Genes Responsible for Autosomal Recessive Primary Microcephaly

Abstract

Autosomal recessive primary microcephaly (MCPH; microcephaly primary hereditary) is a congenital condition caused by impairment of growth and development of foetal brain. The only associated characteristic phenotype is non-progressive intellectual disability of varying degree. Therefore, MCPH is a principal disorder to hunt for genes having critical role in prenatal brain growth. MCPH is genetically heterogeneous with 11 loci and 10 genes been mapped to date. In the present study 11 families segregating MCPH were ascertained for genetic and molecular characterization. Prior to which clinical parameters including measurement of occipital head circumference, pedigree analysis, estimation of intelligence quotient (IQ with amended Wechsler scale), computed tomography (CT) scan, and biometric data collection, were investigated. These assessments clearly specify that under study families segregate nonsyndromic primary microcephaly with autosomal recessive mode of inheritance. After then linkage analysis based on homozygosity mapping was performed. Whole genome SNP genotyping with 250K Nsp 1 array was carried out after exclusion mapping in selected individuals of family A. Data analysis using homozygosity mapper identified three homozygous linkage regions on chromosome 1, 10 and 16 while and analysis with dChip rule out the loci on chromosome 1 and 10. Furthermore microsatellite based genotyping of all available family members was also carried out for three putative loci. Parametric linkage analysis yielded a maximum multipoint LOD score of 3.2 at markers D16S3042 and D16S3128. This has led to the mapping of a novel locus at chromosme16p13.3-13.2 spanning 4.85 Mb region. The identified HBD interval was flanked by rs7192880 and rs11648289 and harbors 46 protein coding genes. However sequencing of Rbfox1 and WDR58 lying within the linkage interval did not identify any pathogenic sequence variant. Microsatellite based genotyping revealed linkage of four families (B-E) to MCPH2 on chromosome 19q13.1–13.2. Multipoint linkage analysis carried out by pooling the genotype data of these families yielded a maximum LOD score of 9.5 at markers D19S554 and D19S223 tightly linked to WDR62 gene. Subsequently Sequence analysis Mapping of Genes Responsible for Autosomal Recessive Primary Microcephaly Abstract of 32 coding exons and splice junction sites of WDR62 gene led to the identification of two novel (c.3232G>A/ p.Ala1078Thr; c.1942 C>T/ p.Q648X) and two known (c.1313G>A/ p.Arg438His; c.3936_3937insC/ p.Val1314ArgfsX18) sequence variants segregating with disease phenotype. Molecular genetic analysis of six MCPH families (F-K) mapped linkage at MCPH5 locus/ASPM on chromosome 1q31. ASPM is the most prevalent gene, responsible for >50 MCPH cases worldwide. Sequence analysis of 28 coding exons and splice junction sites of ASPM gene found two novel (c. 6686-6689delGAAA/ p.R2229TfsX9; c. 77delG/ p. G26AfsX41) and three recurrent (c.9159delA/ p. K3054fsX5; c.1260- 1266delTCAAGTC/ p.Ser420fsX31, c. 3978G>A/ W1326X) mutations. AFLP analysis in two families bearing (c. 3978G>A/ W1326X) mutation revealed common disease associated haplotype suggested founder mutation in Pakistani population. The present work also supports the high prevalance of MCPH in Pakistani families. It also supports the genetic heterogeneity of MCPH in Pakistani population. The identified mutations extend the body of evidence implicating the role of two genetic players (ASPM and WDR62) in disease associated patho-mechanisms.