PMC3916514_01
WES identifies a novel frameshift mutation in WDR62. (A) Family pedigree showing the four affected siblings (solid symbols) and unaffected family members (open symbols). Squares represent males, circles females, asterisk denote available DNA. Patient II-1 was selected for WES (black arrow). (B) WES in Patient II-1 revealed a homozygous single-base pair insertion (c.1821dupT) in WDR62 producing a frameshift mutation (p.Arg608Serfs*26). (C) The mutation was validated by Sanger sequencing in all affected siblings (only II-1 and II-2 are shown); parental DNA was unavailable for this study. The mutation is absent in an unaffected control (WT). (D) Reported WDR62 mutations in MCPH2 families. WDR62 is located on chromosome 19q13.12 and contains 32 exons (NM_001083961.1) encoding 1523 amino acids. For clarity, reported WDR62 mutations are annotated as peptide changes (grey). Number sign indicates the compound heterozygous mutation present in one reported family. The homozygous single-base pair insertion identified in the French-Canadian family is also shown (red).
1471-2377-14-22-1.jpg
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F1
WES identified ten exome-wide homozygous variants including a rare variant in WDR62, encoding a protein essential to cerebral cortex development and known to cause microcephaly (Figure 1B) and thus felt to be a strong candidate gene for the disorder in this family. The WDR62 mutation identified is a novel homozygous single-base pair insertion NM_001083961.1:c.1821dupT) producing a frameshift mutation (p.Arg608Serfs*26) in exon 14 (Figure 1C).
PMC3916514_02
WES identifies a novel frameshift mutation in WDR62. (A) Family pedigree showing the four affected siblings (solid symbols) and unaffected family members (open symbols). Squares represent males, circles females, asterisk denote available DNA. Patient II-1 was selected for WES (black arrow). (B) WES in Patient II-1 revealed a homozygous single-base pair insertion (c.1821dupT) in WDR62 producing a frameshift mutation (p.Arg608Serfs*26). (C) The mutation was validated by Sanger sequencing in all affected siblings (only II-1 and II-2 are shown); parental DNA was unavailable for this study. The mutation is absent in an unaffected control (WT). (D) Reported WDR62 mutations in MCPH2 families. WDR62 is located on chromosome 19q13.12 and contains 32 exons (NM_001083961.1) encoding 1523 amino acids. For clarity, reported WDR62 mutations are annotated as peptide changes (grey). Number sign indicates the compound heterozygous mutation present in one reported family. The homozygous single-base pair insertion identified in the French-Canadian family is also shown (red).
1471-2377-14-22-1.jpg
PMC3916514_02_1471-2377-14-22-1.jpg
F1
WES identified ten exome-wide homozygous variants including a rare variant in WDR62, encoding a protein essential to cerebral cortex development and known to cause microcephaly (Figure 1B) and thus felt to be a strong candidate gene for the disorder in this family. The WDR62 mutation identified is a novel homozygous single-base pair insertion NM_001083961.1:c.1821dupT) producing a frameshift mutation (p.Arg608Serfs*26) in exon 14 (Figure 1C).
Exome sequencing workflow. Exome sequencing was performed on isolated DNA from Patient II-1. The data was filtered to identify non-synonymous sequence variants including frameshift and non-frameshift indels, single nucleotide variants (SNV) including stop-gain and missense SNVs, and splicing and splicing-extending variants. Variants with a frequency of less than 1% in the 1000 genomes pilot release (Nov 2010) and in 100 in-house controls were then identified. The first method of analysis focused on rare homozygous variants (Method 1) and a second, simpler approach, focused only on rare variants in disease genes known to cause microcephaly/epilepsy (Method 2). Both methods identified the homozygous mutation in WDR62 as the cause of the neurodevelopmental disorder in this family.
1471-2377-14-22-2.jpg
PMC3916514_02_1471-2377-14-22-2.jpg
F2
WES data was analyzed as previously described and non-synonymous variants (frameshift and non-frameshift indels, single nucleotide variants (SNV) including stop-gain and missense SNVs, and splicing and splicing-extending variants) were identified as rare when they had a frequency of less than 1% in the 1000 genomes pilot release (Nov 2010) and in 100 in-house controls. We then compared two approaches to examine this subset of WES variants (Figure 2). Following our preliminary filtering steps that identified 183 rare non-synonymous variants, we examined the sequencing data for homozygous variants as the pedigree was consistent with autosomal recessive inheritance and the parents were distantly related (see Method 1, Figure 2). We next examined the WES data using a second approach to identify all rare variants in genes known to be associated with epilepsy and microcephaly (see Method 2, Figure 2); the same mutation in WDR62 was identified as the only candidate.
PMC3916514_03