Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome

Abstract

We demonstrate the successful application of exome sequencing1–3 to discover a gene for an autosomal dominant disorder, Kabuki syndrome (OMIM %147920). The exomes of ten unrelated probands were subjected to massively parallel sequencing. After filtering against SNP databases, there was no compelling candidate gene containing novel variants in all affected individuals. Less stringent filtering criteria permitted modest genetic heterogeneity or missing data, but identified multiple candidate genes. However, genotypic and phenotypic stratification highlighted MLL2, a Trithorax-group histone methyltransferase4, in which seven probands had novel nonsense or frameshift mutations. Follow-up Sanger sequencing detected MLL2 mutations in two of the three remaining cases, and in 26 of 43 additional cases. In families where parental DNA was available, the mutation was confirmed to be de novo (n = 12) or transmitted (n = 2) in concordance with phenotype. Our results strongly suggest that mutations in MLL2 are a major cause of Kabuki syndrome.

Disease gene identification strategies for exome sequencing

“…After this, typically between 150 and 500 private non-synonymous or splicesite variants are prioritized as potential pathogenic variants (see Figures 1 and 2). 4,[28][29][30][31][32][33][34] It is important to emphasize that prioritization may discard the pathogenic variant. A variant that is present at low frequency in a heterozygous state in the normal population may be removed even though it causes disease if present in a homozygous state.…”

“…Ng et al 71 Hoischen et al 33 Ng et al 30 Simpson et al 72 Isidor et al 73 Vissers et al 74 Albers et al 75 Dickinson et al 76 Sirmaci et al 77 Agrawal et al 78 De novo (Figure 3e Byun et al 79 Haack et al 80 Worthey et al 44 Götz et al 69c Erlich et al 45 Ozgul et al 81 a,b,c Indicate studies that use a combination of two strategies.…”

How genetically heterogeneous is Kabuki syndrome?: MLL2 testing in 116 patients, review and analyses of mutation and phenotypic spectrum

“…PCR amplification for all the 54 exons spanning the MLL2 gene was performed using primer sequences and the PCR conditions previously described. 4 PCR amplification was carried out on Veriti thermal cycler or 2720 thermal cycler (Applied Biosystems, Paisley, UK) using the Reddy Mix Custom PCR Master Mix (ABgene, Epsom, UK, catalogue no: AB-0575/DC/LD/b) according to the manufacturer's instructions. Amplified products were cleaned using Agencourt AMPure XP (Beckman Coulter Genomics, Takeley, UK) system on an automated Beckman Coulter Liquid Handler, Biomek 3000, as per the manufacturer's instructions.…”

“…4 Still, the underlying cause cannot be identified in 20 to 45% of patients with a presumed diagnosis of KS, suggesting possible genetic heterogeneity. [5][6][7][8] To investigate the spectrum of mutations associated with KS, we sequenced MLL2 in 116 patients with clinically suspected KS.…”

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

“…Classical tools for the detection and characterization of germline and clonally amplified somatic variants are being replaced by a new array of massively parallel sequencing based methods. The characterization of genome-wide genetic variants in individual humans has become an important tool in the diagnosis of congenital malformations[125, 126], especially with recent advances in non-invasive prenatal genetic testing[127], and has led to the discovery of novel genes and pathways associated with human disease[128, 129]. Applications in cancer research and clinical oncology are even more pertinent, where MPS has already impacted oncogene discovery, clinical diagnosis, pharmacogenomics, and the monitoring of disease progression.…”