The prevalence of MCPH5, the most frequently reported type, is 1/10,000 in consanguineous populations, such as Pakistan's sporadically, its probability is 1/1,000,000 in non-consanguineous European population ( 1, 2). Microcephaly (MCPH, OMIM#251200) is a neurodevelopmental disorder characterized by a small head circumference, non-progressive intellectual disability, and small brain size compared with the age- and sex-matched population. Collectively, this study added 12 more families to the mutational paradigm of ASPM and expanded the Pakistani MCPH cohort. Furthermore, the previously known mutations p.Arg3244 * and p.Arg1019 * were inherited in two families with Punjab ethnic profile. The founder mutation p.Trp1326 * was segregating in 10 families, which further confirmed the evidence that it is the most prominent mutation in Pashtun ethnicity living in Pakistan and Afghanistan. Variant filtering and in silico analysis identified three known mutations in ASPM, a MCPH5-associated gene. Candidate variants were validated through Sanger sequencing in all available family members. In the current study, whole exome sequencing (WES) was performed on probands from 10 families sampled from South Waziristan and two families from rural areas of the Pakistani Punjab. MCPH5 is the most frequently reported type, accounting for up to 68.75% alone in a genetically constrained population like Pakistan. Many of these genes are reported in Pakistani population, but due to a high rate of consanguinity, a significant proportion of MCPH cohort is yet to be explored. MCPH manifests as an isolated condition or part of another clinical syndrome so far, 25 genes have been linked with MCPH. Microcephaly (MCPH) is a genetically heterogeneous disorder characterized by non-progressive intellectual disability, small head circumference, and small brain size compared with the age- and sex-matched population. 5National Institute for Biotechnology and Genetic Engineering (NIBGE-C), Faisalabad, Pakistan Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan.4Shenzhen Real Omics Biotech Co., Ltd., Shenzhen, China.3University of Chinese Academy of Sciences, Beijing, China.2Shenzhen Key Laboratory of Biomimetic Materials and Cellular Immunomodulation, Institute of Biomedicine and Biotechnology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.1Department of Bioinformatics and Biotechnology, Government College University Faisalabad, Faisalabad, Pakistan.Niaz Muhammad Khan 1 †, Basharat Hussain 2,3 †, Chenqing Zheng 4 †, Ayaz Khan 5, Muhammad Shareef Masoud 1, Qingquan Gu 4, Linhui Qiu 2, Naveed Altaf Malik 5, Muhammad Qasim 1 *, Muhammad Tariq 5 and Junlei Chang 2 *