Expanding the Mutational Spectrum of Congenital Microcephaly in Pakistani Families

Sundas Farooq¹Maria Asif^2,3Bonsu Ku⁴Madiha Shadab¹Muzammil Ahmed Khan⁵Muhammad Muzammal⁵Raja Waqar⁶Rameez Nisar¹Zahid Latif¹Falak Sher Khan⁷Sanwal Aslam⁸Michal Ruth Schweiger³Ansar A. Abbasi^1,9Muhammad Sajid Hussain^3*

• ¹Department of Zoology, Mirpur University of Science and Technology (MUST), Mirpur, Azad Jammu and Kashmir, Pakistan
• ²Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
• ³Cologne Center for Genomics (CCG), University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany
• ⁴Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology125 Gwahak-ro, Yuseong-gu,, Daejeon, Republic of Korea
• ⁵Gomal Center of Biochemistry and Biotechnology, Gomal University, Dera Ismail Khan, Khyber-Pakhtunkhwa, Pakistan
• ⁶Department of Zoology, University of Kotli, Kotli, Azad Jammu and Kashmir, Pakistan
• ⁷Department of Biological Sciences, University of Sialkot, Sialkot, Pakistan
• ⁸School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, China
• ⁹Department of Zoology, University of Azad Jammu and Kashmir, Muzaffarabad, Azad Jammu and Kashmir, Pakistan

Autosomal recessive primary microcephaly (MCPH) is a genetically heterogeneous neurodevelopmental disorder characterized by a markedly reduced head circumference (−3 to −5 standard deviations) at birth, with relatively preserved brain architecture. Affected individuals often present with mild to moderate intellectual disability, and the condition is more prevalent in populations with high rates of consanguinity, such as Pakistan. To date, pathogenic variants in at least 32 genes have been associated with MCPH, with ASPM and WDR62 accounting for the majority of cases (68% and 14%, respectively). In this study, we investigated four consanguineous families with congenital microcephaly and identified three novel variants in CPAP, WDR62, and ASPM. In Family 1, we identified a novel missense variant (c.3947C>A; p.(Thr1316Lys) in CPAP (NM_018451.4) located within the highly conserved TCP domain, which mediates interactions with other MCPH proteins, including STIL and CEP135. Family 2 harbored a previously unreported splice-site variant, c.2867+5G>T, in WDR62 (NM_001083961.2). In Families 3 and 4, we identified one novel (c.3188T>G; p.(Leu1063*)) and one previously reported (c.9730C>T; p.(Arg3244*)) pathogenic variant in ASPM (NM_018136.4). Computational analyses and structural modeling indicated that all these variants are likely deleterious, disrupting normal protein function. Our findings expand the mutational spectrum of CPAP and WDR62 and reinforce ASPM as the most frequently mutated gene underlying MCPH in the Pakistani population.