A novel single base pair duplication in WDR62 causes primary microcephaly

Background Primary microcephaly is a disorder of the brain resulting in a reduced head circumference that can come along with intellectual disability but with hardly any other neurological abnormalities. Case presentation In this study we report on three Pakistani males from a consanguineous family with 2, 4 and 25 years, diagnosed with autosomal recessive primary microcephaly. By genotyping, Sanger sequencing and using bioinformatical approaches the disease causing mutation was identified and evaluated. Conclusion By using a 250K SNP array, we were able to detect an 11Mb large autozygous region in the MCPH2 locus on chromosome 19q13.12. Sequencing of the associated gene, WDR62, revealed the frameshift causing single base pair duplication, c.2527dupG. This mutation is predicted to affect the structural features of WDR62 which in turn changes the conformation and function of the protein. Aspartic acid (D) at position 843 was found to be conserved among various ortholog species. The present findings will be helpful in genetic diagnosis of patients and future studies of WDR62.

Although sloping foreheads and reduced intelligence are very common, they are not listed as a basic criteria for the diagnosis of microcephaly [19,22]. The loci for MCPH2 on chromosome 19q13.12 has already been discovered in 1999 by Roberts et al. [23] but although excessive sequencing of this locus has been performed since then the corresponding gene, WDR62, remained undiscovered until only recently [5,13,16]. In human, two alternative transcripts are expressed. The full-length WDR62 gene consists of 32 exons resulting in a genomic size of 50230bp. It encodes for a 1523 amino acid long protein that comprises 15 WD-40 repeats, one CpG signal and a polyadenylation signal [13,19,[24][25][26]. The homodimerization region can be found on the C-terminal domain which shows no sequence homologies to any known oligomerization signals [27]. First studies on WDR62 revealed it as a JNK scaffold protein that associates with the two JNK-signalling pathway proteins JNK (c-Jun N-terminal kinase) and MKK7 (MAP kinase kinase 7) and is recruited to stress granulaes upon cellular stress induction [28]. Although Bilgüvar et al [5] showed in expression experiments that WDR62 was rather a nuclear than a centrosomal protein, Bhat et al. [24] proved its centrosomal localization during mitosis as well as its nuclear localization but also suggested that the localization of WDR62 strongly depends on cell cycle phase and cell type. Only recently, WDR62 was identified as a phosphoprotein associated with mitotic spindle poles during prophase to metaphase transition but lacked its centrosomal localization during ana-and telophase. Depletion experiments led to a mitotic delay and to abnormal spindle formation as well as an increased formation of multipolar spindles [29].
A wide range of cortical malformations have been described for mutations in this gene, including microcephaly, pachygyria with cortical thickening, hypoplasia of the corpus callosum, polymicrogyria, simplified gyral patterns, cerebral hypoplasia, band heterotopias, lissencephlay and schizencephaly [5,13,16,17,19,24,30]. Due to the increased incidence of autozygous regions in children from consanguineous families, the probability of carrying a disease causing identical-by-decent mutation is increased in patients with autosomal recessive disorders [30]. Following this assumption, we were able to identify a novel homozygous mutation in WDR62 in a consanguineous Pakistani MCPH2 family.

Sample collection
After obtaining informed consent, blood was drawn and DNA was isolated from 9 family members, including two affected brothers and their affected relative, according to standard protocols. the manufacturer's protocol. The physical distance of the LOH region was determined via University of California Genome Browser UCSC [25]. Microsatellite markers in this region (D19S414, D19S220 and D19S420) were selected from ABI PRISM® Linkage Mapping Set v2.5 for fine-mapping the disease locus and to analyze the segregation among the available family members. The resulting data were analyzed with Peak Scanner Software v1.0 (Applied Biosystems).

Sanger sequencing
WDR62 exons and their flanking intron junctions were determined with UCSC Browser. Primers were designed using ExonPrimer (http://ihg.helmholtzmuenchen.de/ihg/ ExonPrimer.html) and synthesized by Microsynth. Primer sequences are available on request.
WDR62 exons and their flanking intron sequences were amplified with HotStarTaq Master Mix Kit (Qiagen) using a standard amplification protocol, the sequencing reaction was set up with Big Dye Terminator 3.1 (Applied Biosystems) and remaining dye nucleotides were removed with Sephadex™ G-50 superfine (GE Healthcare). Analysis of the amplicons was performed on ABI 3130xl Genetic Analyzer.

Computational analysis
To predict the secondary structure of WDR62 protein, the online server Psipred [31] was used. MSA (Multiple Sequence Alignment) was performed using T-Coffee [32]  The third 25 years old patient, MCP1-6 (Figure 1a C), has a head circumference of 39.37 cm and a height of 170 cm. As for patient MCP1-2, aggressiveness and a watery mouth have been observed. The computed tomography (CT) scan of this individual revealed a reduced volume of the right cerebral hemisphere and prominent extra axial cerebrospinal (CSF) spaces with ill-defined gryal and nuclei pattern (Data not shown here). However, no local area of brain attenuation and intracerebral blood was observed. Due to the non-cooperative behavior of two other affected individuals (MCP1-2 and MCP1-5) detailed magnetic resonance imaging (MRI) scan could not be performed.
to consist of 61 coils, 60 strands and 1 helix compared to 64 coils, 58 strands and 7 helixes in the wild type. This not only affects the conformation but also the function of the protein. T-Coffee result (Figure 3b) shows that Aspartic acid (D, replaced by Glycine as a result of the mutation) at position 843 (indicated by an arrow head) is highly conserved among various orthologous species which indicates the importance of this amino acid.

Conclusion
According to Mahmood et al. [19] ASPM (MCPH5 locus) and WDR62 (MCPH2) are the two most common genes for primary microcephaly found mutated in more than 55% of the affected families. 4% of all MCPH cases are only due to mutations in WDR62 [35].
Due to the high prevalence of MCPH2 in primary microcephaly cases among consanguineous families more mutations in this gene will probably be revealed in the upcoming years. However, the high frequency of WDR62 mutations in consanguineous primary microcephaly patients will especially simplify the clinical counseling and diagnostic screening of non-consanguineous primary microcephaly patients.

Consent
We have obtained the written informed consent from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review from the Editor of this journal.