A first pedigree (LIS-900) with m
ephaly with s
yri (MCSG) from Mexico consists of distantly related parents with three affected children and one healthy sibling (). The children showed severe microcephaly, developmental delay, and seizures (see Supplementary Note, Clinical Data
) but no other syndromic features. A second family (LIS-2600) from Turkey showed similar clinical features and has been described before1
(). Subsequent to the identification of the responsible gene, four other families were identified with similar clinical features ( and Supplementary Note, Clinical Data
). Brain MRI of the six families (), revealed small brains, markedly simplified gyral patterns, corpus callosal abnormalities, plus a diversity of additional cortical malformations including polymicrogyria, schizencephaly, and subcortical heterotopia (arrested neurons), sometimes with asymmetry in the same brain (Supplementary Note, Clinical Data and Supplementary Movie 1
Figure 1 Pedigrees and radiographic findings in six consanguineous families with microcephaly and simplified gyri (MCSG). (a–f) Shaded symbols denote affected individuals. (a) LIS-900, a Mexican family with three affected children. (b) LIS-2600, a Turkish (more ...)
Genome-wide linkage screens of LIS-900 showed a single 11.9 Mb region on chromosome 19q13 (D19S431; D19S217) homozygous in all 3 affected patients and not homozygous in the normal sibling (Supplementary Fig. 1
). SNP genotyping of LIS-2600 confirmed the homozygosity and narrowed the interval to ≈ 7.5 Mb () (peak combined multipoint LOD score of 3.84, Supplementary Fig. 2
). SNP analysis of MC-1400 and MC-1600 confirmed the candidate region but did not reduce it ().
Figure 2 Mapping, capture, and sequencing of 148 genes in the MCSG locus. (a) Homozygosity analysis in four pedigrees (LIS-900, LIS-2600, MC-1400 and MC-1600) revealed a 7.5 Mb interval on chr19, bounded by SNP markers rs17581484 and rs4802998. Homozygous SNPs (more ...)
Because of the high gene density of the linked region, we employed an array capture approach2
followed by high-throughput Illumina sequencing in two affected individuals, LIS-903 and LIS-2602. A custom array was designed with oligonucleotide probes targeting the exons of all 148 predicted UCSC genes in the interval () plus non-exonic DNA from a smaller candidate subregion suggested by microsatellite mapping (Supplementary Fig. 2
). DNA bound to the array was amplified and used to generate sequencing libraries. 36–40 million paired-end sequence tags (>1.4 Gb of raw sequence) were generated per patient and mapped (hg18) using BWA ()3
. 50–60% of sequence reads were on-target, and average depth of target coverage was 230X and 224X, with 88% and 90% of bases covered by ≥ 10 reads.
Single nucleotide variants and microinsertions/microdeletions were called and filtered for quality and mapping confidence4
, revealing >2000 potential sequence variants from each proband (). Comparison of SNP calls with Affy 5.0 genotype data showed >99.8% concordance. Variants were filtered out if they were present in dbSNP1304
or the 1000 genomes project (1000G), and categorized by their predicted functional effects using GMCC. Of 2310 variants in LIS-903, 262 (11%) were not in dbSNP130 or 1000G, 93 were potentially pathogenic (4.0%); five met both criteria. Of 2570 variants in LIS-2602, 323 (12%) were absent from dbSNP and 1000G, and 99 (3.8%) were potentially pathogenic; seven variants met both criteria. A single gene, WDR62
, showed novel, pathogenic mutations in both families, making it a strong candidate ().
Variant filtering identifies WDR62 as the causative gene for MCSG
Sanger sequencing confirmed WDR62
mutations in the two families, and identified four additional alleles in four other families (, Supplementary Fig. 3
). A homozygous single basepair insertion in exon 30 (c.3936_3937insC) in LIS-2602 produced a frameshift mutation (). All three affected patients in family LIS-900 showed a homozygous single basepair deletion in exon 4 (c.363delT), creating a frameshift mutation (). Pedigree analysis confirmed appropriate segregation of WDR62
mutations in both families (). In LIS-2500, the affected individual showed a homozygous mutation one basepair downstream of exon 8 (c.1043+1G>A), disrupting the highly conserved GT consensus splicing sequence (, Supplementary Fig. 3
). In MC-1400, the affected child was homozygous for a 4 bp deletion after exon 23 (c.2867+4_c2867+7delGGTG) (, Supplementary Fig. 3
) that removes the conserved +4 and +5 positions of the intron and is predicted to disrupt the splice donor5
tc…; mutation …CAG|gtg----tc…). In MC-1600, the affected child was homozygous for a deletion of 17 bp in the 30th
exon (c.3839_3855delGCCAAGAGCCTGCCCTG), causing a frameshift (, Supplementary Fig. 3
). In PH-16900, all three siblings with microcephaly were homozygous for a missense V65M change (c.193G>A, , Supplementary Fig. 3
) that alters a residue conserved in all vertebrates (Supplementary Fig. 4
). An unaffected sibling (PH-16907, with mild speech delay and articulation difficulties but normal head circumference and normal MRI) was homozygous for the wildtype allele (Supplementary Fig. 3
). Two additional unaffected siblings were homozygous wildtype and heterozygous, respectively (not shown). None of these mutations were found in over 1000 normal chromosomes.
Figure 3 Six WDR62 mutations reported in association with microcephaly with simplified gyri. (a) Alterations are shown in genomic, coding DNA, and protein contexts. The human WDR62 gene consists of 32 exons shown as boxes, and encodes a protein of 1518 amino acids (more ...) WDR62
encodes a 1518 amino acid protein with multiple WD40 repeats ()6
, but little is known about its function. A proteomic study identified WDR62 as a binding partner of the centrosomal protein CEP1707
, which is of interest given the involvement of other centrosomal proteins in microcephaly8–14
. Another report identified WDR62 as a binding partner for c-Jun N-terminal kinase (JNK), suggesting that it potentiates JNK activity15
. In situ
hybridization with a probe to mouse WDR62
showed widespread expression in the developing brain at embryonic day 14.5 (E14.5) (), with highest expression in forebrain. Expression was seen in the ventricular zone and the cortical plate (), consistent with roles in progenitor cells and postmitotic neurons.
Figure 4 WDR62 expression in developing mouse brain and subcellular localization. (a) in situ hybridization of E14.5 mouse brain with antisense probe to mouse WDR62. Sense strand (not shown) showed no specific hybridization. (b) Higher power view. Strong WDR62 (more ...)
WDR62 demonstrates strikingly cell cycle-dependent localization. Immunofluorescence staining of endogenous WDR62 in HeLa cells with an anti-WDR62 antibody revealed punctate, perinuclear expression during interphase, suggesting localization to the Golgi apparatus and confirmed by GM130 immunoreactivity (). In contrast, in HeLa or HEK cells in M phase, WDR62 is found at the spindle poles as demonstrated by double-labeling with gamma-tubulin and dynein (). Close inspection of recombinant HA-tagged WDR62 protein demonstrated subcellular localization closely matching that of CEP170 () and surrounding LIS1 (), a pattern similar to that previously demonstrated for ASPM16,17
and other microcephaly proteins12,13,18,19
. Bilguvar and colleagues20
recently described mutations in WDR62 with brain malformations and reported nuclear localization of WDR62 in cortical progenitor cells. While we cannot rule out nuclear localization in some cells, our data, as well as previously published data7
suggest a major centrosomal role.
Postmortem analysis of LIS-2601, an affected sibling in the LIS-2600 family1
, suggests roles for WDR62 in neuronal proliferation and migration. The 27-week fetus showed a small cranial cavity enclosing a profoundly small brain (50g, compared to a normal of 127 +/− 20 g for this age and size fetus). The surface of the hemispheres was largely smooth (not shown), with poorly defined Sylvian fissures and few sulci1
. The cerebellum appeared remarkably preserved (not shown), whereas the cerebral cortex was severely abnormal (). The outermost cortical layer (layer I) appeared generally normal, but remaining cortical neuronal layers were thinner than normal (). There was an almost complete lack of small- to medium-sized pyramidal neurons in their normal location (layers II and III), suggesting profoundly defective neurogenesis. Neurons located beneath layer I were primarily medium- to large pyramidal neurons, followed by a cell-sparse zone and a third layer of multipolar neurons at the deepest layer of the cortex, an appearance consistent with layers V and VI, respectively (). Neurons in the medium-to-large pyramidal layer retained an immature radial columnar organization and also exhibited abnormal, non-radial clumping (). In some sections, the pial surface was interrupted by microscopic extrusions of cells into the subarachnoid space, forming heterotopia (). Heterotopia were also sometimes found in the intermediate zone, arranged in streaks particularly in posterior frontal cortex (), corresponding to the localization of subcortical heterotopia seen on MRI. There were also clusters () of small round cells in the outer subventricular zone (SVZ) suggestive of progenitor cells. These findings support a proliferative defect in brains of patients with WDR62
mutations, potentially affecting the outer SVZ (precursors to upper layer cortical neurons)21
as well as defective neuronal migration, in some ways evocative of the dual roles in proliferation and migration seen for LIS122,23
Figure 5 Histopathologic analysis of a 27 week human fetus with MCSG. (a) H&E stained coronal section from the forebrain of a 27 week gestational age fetus with microcephaly with simplified gyri (LIS-2601). Section is at the rostral end of the caudate (more ...)
Our results suggest that mutations in WDR62
cause microcephaly, simplified gyral pattern, callosal abnormalities, and a wide range of additional cortical abnormalities, including polymicrogyria, schizencephaly, and subcortical heterotopia. WDR62
lies within the MCPH2
a gene-rich locus that has resisted gene identification for > 10 years, and appears to be allelic to MCPH2
(see accompanying paper). The remarkable diversity of cortical malformations associated with WDR62
mutation suggests that it acts at a critical hub of human cerebral development.