Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome.

We demonstrate the successful application of exome sequencing to discover a gene for an autosomal dominant disorder, Kabuki syndrome (OMIM%147920). We subjected the exomes of ten unrelated probands to massively parallel sequencing. After filtering against existing SNP databases, there was no compelling candidate gene containing previously unknown variants in all affected individuals. Less stringent filtering criteria allowed for the presence of modest genetic heterogeneity or missing data but also identified multiple candidate genes. However, genotypic and phenotypic stratification highlighted MLL2, which encodes a Trithorax-group histone methyltransferase: seven probands had newly identified nonsense or frameshift mutations in this gene. Follow-up Sanger sequencing detected MLL2 mutations in two of the three remaining individuals with Kabuki syndrome (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.

Kabuki syndrome is a rare, multiple malformation disorder characterized by a distinctive facial appearance (Supplementary Fig. 1), cardiac anomalies, skeletal abnormalities, immunological defects, and mild to moderate mental retardation. Originally described by Niikawa et al.5 and Kuroki et al.6 in 1981, Kabuki syndrome has an estimated incidence of 1 in 32,0007 and about 400 cases have been reported worldwide. The vast majority of reported cases have been sporadic, but parent-to-child transmission in more than a half a dozen instances8 suggests that Kabuki syndrome is an autosomal dominant disorder. The relatively low number of cases, the lack of multiplex families, and the phenotypic variability of Kabuki syndrome have made the identification of the gene(s) underlying Kabuki syndrome intractable to conventional approaches of gene discovery, despite aggressive efforts.

We sequenced the exomes of ten unrelated individuals with Kabuki syndrome, seven of European ancestry, two of Hispanic ancestry, and one of mixed European/Haitian ancestry (Supplementary Fig. 1, Supplementary Table 1). Enrichment was performed by hybridization of shotgun fragment libraries to custom microarrays, followed by massively parallel sequencing1–3. On average, 6.3 gigabases of sequence were generated per sample to achieve 40× coverage of the mappable, targeted exome (31 megabases). As previously, our analyses focused primarily on nonsynonymous (NS) variants, splice acceptor and donor site mutations (SS) and coding indels (I), anticipating that synonymous variants were far less likely to be pathogenic. We also predicted that variants underlying Kabuki syndrome are rare, and therefore likely to be novel. Novelty was defined here by absence from all datasets used for comparison, including dbSNP129, the 1000 Genomes Project, exome data from sixteen individuals previously reported by us2,3, and ten exomes sequenced as part of the Environmental Genome Project (EGP).

Under a dominant model in which each case was required to have at least one novel NS/SS/I variant in the same gene, only a single candidate gene (MUC16) was shared by all ten exomes (Table 1, row 4; Supplementary Table 2). However, MUC16 was considered likely to be a false positive due to its extremely large size (14,507 aa). Potential explanations for our failure to find a compelling candidate gene in which novel variants are observed in all affected individuals included: (a) that Kabuki syndrome is genetically heterogeneous, and therefore not all affected individuals will have mutations in the same gene; (b) that we failed to identify all mutations in the targeted exome; (c) that some or all causative mutations were outside of the targeted exome, e.g., in non-coding regions or unannotated genes. To allow for a modest degree of genetic heterogeneity and/or missing data, we conducted a less stringent analysis by looking for candidate genes shared among subsets of affected individuals. Specifically, we searched for subsets of x out of 10 exomes having ≥ 1 novel variant in the same gene, for x = 1 to 10. For x = 9, 8, and 7, novel variants were shared in three genes, six genes, and sixteen genes, respectively (Table 1, row 4). However, there was no obvious way to rank these candidates.

Table 1

Number of genes common to any subset of x affected individuals

The number of genes with at least one non-synonymous variant (NS), splice-site acceptor/donor variants (SS) or coding indel (I) are listed under various filters. Variants were filtered by presence in dbSNP or 1000 genomes ("Not in dbSNP129 or 1000 genomes") and control exomes ("Not in control exomes") or both ("Not in either"); control exomes refer to those from 8 Hapmap3 4 FSS3, 4 Miller2 and 10 EGP samples. The number of genes found using the union of the intersection of x individuals is given.

a. Subset analysis (any x of 10)	1	2	3	4	5	6	7	8	9	10
NS/SS/I	12,042	8,722	7,084	6,049	5,289	4,581	3,940	3,244	2,486	1,459
Not in dbSNP129 or 1000 genomes	7,419	2697	1057	488	288	192	128	88	60	34
Not in control exomes	7,827	2865	1025	399	184	90	50	22	7	2
Not in either	6,935	2227	701	242	104	44	16	6	3	1
Is loss-of-function (nonsense/frameshift indel)	753	49	7	3	2	2	1	0	0	0

We speculated that genotypic and/or phenotypic stratification would facilitate the prioritization of candidate genes identified by subset analysis. Specifically, we assigned a categorical rank to each Kabuki case based on a subjective assessment of the presence of, or similarity to, the canonical facial characteristics of Kabuki syndrome (Supplementary Fig. 1) and the presence of developmental delay and/or major birth defects (Supplementary Table 1). The highest ranked case was one of a pair of monozygotic twins with Kabuki syndrome. We then categorized the functional impact (i.e. nonsense versus nonsynonymous substitution, splice-site disruption, frameshift versus in-frame indel) of each novel variant in candidate genes shared by each subset of two or more ranked cases. Manual review of these data highlighted distinct, novel nonsense variants in MLL2 in each of the four highest ranked cases. On sequential analysis of phenotype-ranked cases with a loss-of-function filter, MLL2 is the only candidate gene remaining after addition of the second individual (Table 2, row 5, column "+2"). No novel variant in MLL2 was found in the Kabuki case ranked 5th, such that the number of candidate genes drops to zero after the fourth individual (Table 2, row 5). However, a 4-bp deletion was found in the case ranked 6th and nonsense variants in the cases ranked 7th and 9th. Thus, exome sequencing identified a nonsense substitution or frameshift indel in MLL2 in seven of the ten Kabuki cases.

Table 2

Number of genes common in sequential analysis of phenotypically ranked individuals

Variants were filtered as in Table 1. Exomes were added sequentially to the analysis by ranked phenotype, e.g. column "+ 3" shows the number of genes at the intersection of the three top ranked cases. (Supplementary Fig. 1). The gene with at least one NS/SS/I in all individuals is MUC16 which is very likely to be a false positive due to its extreme length (14,507 aa).

b. Sequential analysis	1	+2	+3	+4	+5	+6	+7	+8	+9	+10
NS/SS/I	5,282	3,850	3,250	2,354	2,028	1,899	1,772	1,686	1,600	1,459
Not in dbSNP129 or 1000 genomes	687	214	145	84	63	54	42	40	39	34
Not in control exomes	675	134	50	26	13	13	8	5	4	2
Not in either	467	89	34	18	9	8	4	4	3	1
Is loss-of-function (nonsense/frameshift indel)	25	1	1	1	0	0	0	0	0	0

Retrospectively, if we apply a loss-of-function filter to the subset analysis of exome data (Table 1, row 5), at x = 7, MLL2 is the only candidate gene. We also developed a post hoc ranking of candidate genes based on functional impact of variants present (“variant score”) and the rank of the cases in which each variant was observed (“case score”). When applied to the exome data as a combined metric, MLL2 emerges as the top candidate (Supplementary Fig. 2).

In parallel with these analyses, we applied genomic evolutionary rate profiling (GERP)9 to exome data. GERP uses mammalian genome alignments to define a rejected substitution (RS) score for each variant, regardless of functional class. We have previously shown that the quantitative ranking of candidate genes by the RS scores of their novel variants can facilitate the exome-based analysis of Mendelian disorders10. In subset analysis with GERP-based ranking, MLL2 remains on the candidate list up to x = 8, ranking 3rd in a list of 11 candidate genes at this threshold (Table 3, Supplementary Fig. 3). Interestingly, the additional MLL2 variant contributing to this analysis (such that MLL2 is still considered at x = 8) is a synonymous substitution with an RS score of 0.368 in the 5th ranked case.

Table 3

Analysis of exome variants using genomic evolutionary rate profiling

The number of genes with at least a single novel variant with an rejected substitution (RS) score10 > 0 in at least x individuals is given. A gene rank is assigned based on the average GERP score9 over all observed novel variants with RS score > 0 in all affected individuals.

c. GERP Score analysis (at least x of 10)	1	2	3	4	5	6	7	8	9	10
Variant RS score > 0	7,176	2,360	754	269	106	39	20	11	3	1
MLL2 Rank	3,732	1,232	399	136	47	14	6	3	NA	NA

We sought to confirm all novel variants identified in MLL2, particularly because loss-of-function variants identified through massively parallel sequencing have a higher prior probability of being false positives. All seven loss-of-function variants in MLL2 were validated by Sanger sequencing. We further analyzed the three cases in which we did not initially find a loss-of-function variant in MLL2, first by array comparative genomic hybridization (aCGH) to determine any gross structural changes, and then by Sanger sequencing of all exons of MLL2 in case of false negatives by exome sequencing. Since an average of 96% of coding bases in MLL2 were called at sufficient quality and coverage for single-nucleotide variant detection, we anticipated that any missed variants were more likely to be indels instead, because of the higher coverage required for confident indel detection in short-read sequence data. Indeed, although aCGH did not find any structural variants in the region, Sanger sequencing did identify frameshift indels in two of these three cases (ranked 8th and 10th).

Ultimately, loss-of-function mutations in MLL2 were identified in nine out of ten cases in the discovery cohort (Fig. 1), making it a compelling candidate for Kabuki syndrome. For validation, we screened all 54 exons of MLL2 in 43 additional cases by Sanger sequencing. Novel nonsynonymous, nonsense or frameshift mutations in MLL2 were found in 26 of these 43 cases (Fig. 1 and Supplementary Table 3). In total, through either exome sequencing or targeted sequencing of MLL2, 33 distinct MLL2 mutations were identified in 35 of 53 families (66%) with Kabuki syndrome (Fig. 1 and Supplementary Table 3). In each of twelve cases for which DNA from both parents was available, the MLL2 variant was found to have occurred de novo. Three mutations were found in two cases each: one mutation was confirmed to have arisen de novo in one of the cases, indicating that some mutations are recurrent. Novel MLL2 mutations (K4527X and T5464M) were also identified in each of two families in which Kabuki syndrome was transmitted from parent-to-child. None of the additional MLL2 mutations were found in 190 control chromosomes from individuals of matched geographical ancestry.

Figure 1

Genomic structure and allelic spectrum of MLL2 mutations that cause Kabuki syndrome

MLL2 is composed of 54 exons that encode untranslated regions (orange) and protein coding sequence (blue) including 7 PHD fingers (yellow), FYRN (green), FYRC (green), and a SET domain (red). Arrows indicate the locations of 32 different mutations found in 53 families with Kabuki syndrome including: 20 nonsense, 7 indels, and 5 amino acid substitutions. Asterisks indicate mutations that were confirmed to be de novo and crosses indicate cases for which parental DNA was unavailable.

Our results strongly suggest that mutations in MLL2 are a major cause of Kabuki syndrome. MLL2 encodes a large 5,262 residue protein that is part of the SET family of proteins, of which Trithorax, the Drosophila homologue of MLL, is the best characterized11. The SET domain of MLL2 confers strong histone 3 lysine 4 methyltransferase activity and is important in the epigenetic control of active chromatin states12. Murine loss of Mll2 on a mixed 129Sv/C57BL/6 background slows growth, increases apoptosis and retards development leading to early embryonic lethality, due in part to mis-regulation of homeobox gene expression13. However, no morphological defects have been reported in Mll2+/− mice13.

Most of the MLL2 variants identified in Kabuki cases are predicted to truncate the polypeptide chain before translation of the SET domain. Accordingly, though it is not certain whether Kabuki syndrome results from haploinsufficiency or a gain-of function at MLL2, haploinsufficiency seems to be the more likely mechanism. Deletion of chromosome 12q12-q13.2, which encompasses MLL2, has been reported in a child with characteristics of Noonan syndrome14. However, we re-analyzed this case using oligo aCGH (including 21 probes that cover MLL2) and found the distal breakpoint to be located ~700 kb proximal of MLL2 (data not shown). Interestingly, all of the pathogenic missense variants identified herein are located in regions of MLL2 that encode C-terminal domains. This suggests that missense variants elsewhere in MLL2 could be better tolerated or, alternatively, are embryonic lethal.

For the 18 of 53 cases for which no novel protein-altering variant was found, it is possible that non-coding or other missed mutations in MLL2 are responsible instead. Alternatively, Kabuki syndrome could be genetically heterogeneous, and further analysis of these cases by exome sequencing may elucidate additional genes for Kabuki syndrome and potentially explain some of the phenotypic heterogeneity seen in this disease. Notably, 9 of 10 individuals in the discovery cohort (90%), but only 26 of 43 individuals in the replication cohort (60%), were ultimately found to have mutations in MLL2. It is therefore possible that the careful selection of canonical Kabuki cases for the discovery cohort enriched for a shared genetic basis. This underscores the importance of access to deeply phenotyped and well-characterized cases.

In summary, we applied exome sequencing of a small number of unrelated cases to discover that mutations in MLL2 underlie Kabuki syndrome. As predicted in previous analyses2,3, allowing for even a small degree of genetic heterogeneity or missing data significantly confounds exome analysis by increasing the number of candidate genes consistent with the model of inheritance. To facilitate the prioritization of genes under such criteria, we stratified data by ranked phenotypes and found that MLL2 was prominent in the higher ranked cases. However, nine of the ten Kabuki cases in the discovery cohort were ultimately found to have MLL2 mutations, such that stratification by phenotype was of less importance than originally appeared to be the case. Nonetheless, the sequential analysis of ranked cases may have reduced the probability of confounding due to genetic heterogeneity. All of the MLL2 mutations found in the discovery set via exome sequencing were loss-of-function variants. As a result, MLL2 ranked highly among candidates assessed by predicted functional impact. Such a pattern will likely occur for some, but not all, Mendelian phenotypes subjected to this approach. We anticipate that the further development of strategies to stratify data at both the genotypic and phenotypic level will be critical for exome and whole genome sequencing to reach their full potential as tools for discovery of genes underlying Mendelian and complex diseases.