Mutations in WDR62 gene in Pakistani families with autosomal recessive primary microcephaly

Primary autosomal recessive microcephaly (MCPH) is a congenital disorder, initially characterized by architecturally normal brain but of reduced size associated with mild to moderate mental retardation. However recent studies have broadened the phenotypic spectrum of MCPH gene mutations by associating additional cortical anomalies [1‐4]. Autosomal recessive primary microcephaly is a heterogeneous disorder with seven causative genes reported to date. These include MICROCEPHALIN at MCPH1 [5], CDK5RAP2 at MCPH3 [6]CEP152 at MCPH4 [7], ASPM at MCPH5 [8], CENPJ at MCPH6 [6], STIL at MCPH7 [9] and WDR62 at MCPH2 [1‐3].

In Pakistani population, mutations in WDR62 are second most common cause of MCPH. WDR62 (NM_001083961) gene maps to chromosome 19q13.12 and encodes two alternative WDR62 transcripts in humans, however full length transcripts comprise 32 coding exons encoding a 1,523 amino acid protein. Mutations in WDR62 in five Pakistani families have been reported by Nicholas et al. [2]. In present work, we report identification of a novel and three known mutation in WDR62 gene in four additional Pakistani families with autosomal recessive primary microcephaly.

By homozygosity mapping, four families resulted in linkage with the MCPH2 locus harboring the WDR62 gene. Three families (MCP26, MCP35, MCP67) were located in villages near Lahore in the Punjab province of Pakistan, while fourth family (MCP3) originates from Abbottabad in the Khyber Pakhtunkhwa province. The evaluation of the affected individuals of all families (Figure 1) revealed presence of microcephaly and mental retardation. In these patients, microcephaly was noticed at birth and their head circumferences were between -3 and -7 standard deviations. The affected individuals of these families exhibited similar developmental trajectory without any signs of facial dysmorphism, seizures and autism. Interestingly affected male individuals (IV-5, IV-6) of family MCP67 have abnormal sleeping habits. The computerized tomography (CT) scan of the affected individual IV-5 of family MCP67 revealed reduced volume of right cerebral hemisphere and prominent extra axial cerebrospinal (CSF) spaces with ill defined gryal and nuclei pattern (Figure 2). However no local area of brain attenuation and intracerebral blood was observed. Due to the non cooperative behavior and effectiveness of available sedative methods on this individual (IV-5) detailed magnetic resonance imaging (MRI) scan could not be performed.

DNA sequencing analysis of the entire coding region of WDR62 gene identified two truncating mutations (of which one novel) and two previously reported missense mutations (Figure 3). The novel mutation in family MCP67 is a single base pair transversion in exon 15 of the gene (c.1942 C>T) resulting in a premature stop codon (p.Q648X). In family MCP26 an already reported homozygous single base pair insertion (c.3936_3937insC) in exon 30 was observed that changed the reading frame, creating a premature stop codon 50 base pair downstream (p.Val1314ArgfsX18) (Figure 3). Additionally two missense mutations were identified in exon 10 (c.1313G>A; p.Arg438His) and 11 (c.1531G>A; p.Asp511Asn) in families MCP35 and MCP3, respectively. These mutations were homozygous in affected individuals and found heterozygous in obligate carriers and were not found in 100 control individuals of Pakistani origin.

To date 18 mutations have been reported in WDR62 gene (Table 1) which are associated predominately with primary microcephaly but additional features like pachygyria with cortical thickening and hypoplasia of the corpus callosum, schizencephaly and polymicrogyria were also found in few patients [1‐4]. The current study describes identification of two protein truncating and two missense mutations in WDR62 gene in four consanguineous Pakistani families originating from Punjab and Khyber Pakhtunkhwa province.

The p.Val1314ArgfsX18 truncating mutation identified in family MCP26 had already been reported in a Turkish family [3], as well as in affected members of Caucasian family [2] with thickened cerebral cortex and simplified gryal pattern leading to severe microcephaly. The affected individuals of family MCP26 also have severe clinical features, but unfortunately CT and MRI scan were not available to make better comparison. In family MCP67, harboring the truncating mutation p.Q648X, the affected subjects also have severe clinical course and ill-defined gyral pattern on brain imaging, in line with the hypothesis that truncating mutations in WDR62 gene are associated with severe microcephaly and associated brain malformations [2]. Indeed, truncating mutations may lead to nonsense mediated decay which results in complete loss of Wdr62 protein, causing the absence of Wdr62 at the spindle poles of the dividing cells. However the affected individuals of families MCP3 and MCP35, carrying known missense mutations, showed milder symptoms and simplified gyral patterns (Data not shown).

Cerebral cortex, the most complex region of mammalian nervous system is formed by the orderly generation of postmitotic neurons during neurogenesis through proliferation and differentiation of neural progenitors at the ventricular and subventricular zones that line the cerebral cavity [12, 13]. Initially neural progenitor cells divide symmetrically to generate a pool of neural precursors for lateral expansion of neocortex. Subsequently asymmetric neurogenic division perpendicular to their apical-basal axis (horizontal cleavage) renews the precursor cell along with generation of post mitotic neuron [14]. The switch of mammalian neuroepithelium (NE) cells from symmetric, proliferative to asymmetric, neurogenic divisions has been proposed to involve a change in cleavage plane orientation hence depends upon spindle pole positioning [14].

Wdr62 is a spindle pole protein expressed in neuronal precursor cells undergoing mitosis in the mammalian embryonic neuroepithelium [2‐4] and hence vital for positioning the cleavage furrow for symmetric proliferative division to increase the number of neuronal precursor cells [2]. Therefore, mutated wdr62 may results in depletion of progenitor pool affecting the proliferation rate and thus produce reduced neuron numbers causing primary microcephaly. Nicholas et al. [2] furthermore hypothesized that WDR62 is also involved in neuronal migration and mutated wdr62 may cause cortical lamination defect along with impairing centrosomal function.

This study supports the notion that WDR62 mutations results in brain malformations in addition to primary microcephaly irrespective of family origin and ethnicity. Furthermore it also indicates that beside ASPM, WDR62 gene is a relevant contributor for autosomal recessive primary microcephaly in Pakistan, being responsible for about 4% cases.