Expanding the Clinical and Molecular Spectrum of Primary Autosomal Recessive Microcephaly: Novel CDK5RAP2 Gene Variants and Functional Insights on the Intronic Variants

<b>Background/Objectives</b>: Autosomal recessive primary microcephaly is a rare and genetically heterogeneous disorder characterized by congenital non-syndromic microcephaly, with at least 28 causative genes identified to date. Biallelic variants in the <i>CDK5RAP2</i> gene, an ultra-rare cause of autosomal recessive primary microcephaly, lead to Primary Autosomal Recessive Microcephaly 3 (MCPH3). <b>Methods</b>: We present seven patients from six families diagnosed with MCPH3 in light of clinical and molecular findings using whole-exome sequencing (WES). Furthermore, we investigated the effects of the identified intronic variants on splicing through RNA analysis. <b>Results</b>: Almost all patients had severe microcephaly, mild to moderate intellectual disability, speech delay, and cutaneous pigmentary abnormalities. Four patients presented with postnatal short stature, and two showed weight deficiency. Dysmorphic evaluation revealed that the most prominent features included brachycephaly, hypertelorism, epicanthus, high-arched eyebrows, prominent nasal bridge, and micrognathia. We identified five distinct homozygous CDK5RAP2 variants in our patients, including four novel variants. Segregation analysis verified that the parents were carriers. Two of these variants were intronic (c.3148+5G>C and c.383+4dupA), two were frameshift (c.3168del), and one was a nonsense variant (c.1591C>T). Both intronic variants disrupted splicing, generating a premature stop codon and resulting in a truncated protein. <b>Conclusions</b>: This study broadens the mutational landscape of <i>CDK5RAP2</i>. We also sought to demonstrate the functional consequences of the <i>CDK5RAP2</i> intronic variants on gene function using RNA analysis. The identification of four novel variants underscores the importance of molecular diagnostics in patients with primary microcephaly and provides valuable data for genetic counseling and future functional studies.