All 54 protein-coding exons and intron-exon boundaries of MLL2
were screened by Sanger sequencing in a cohort of 110 kindreds with Kabuki syndrome. This cohort included 107 simplex cases (including a pair of monozygotic twins) and three familial (i.e., parent-offspring) cases putatively diagnosed with Kabuki syndrome. Seventy novel MLL2
variants that were inferred to be disease-causing were identified in 81/110 (74%) kindreds ( and Supplementary Table II
online). These eighty-one mutations included 37 nonsense mutations (32 different sites and five sites with recurrent mutations), three in-frame deletions or duplications (2 different sites and 1 site with a recurrent mutation), 22 frameshifts (22 different sites), 16 missense mutations (11 different sites and four sites with recurrent mutations) and 3 splice consensus site (or intron-exon boundary) mutations. None of these variants were found in dbSNP (build 132), the 1000 Genomes Project pilot data, 190 chromosomes from individuals matched for geographical ancestry. In total, pathogenic variants were found at seventy sites. Additionally, there were ten sites at which recurrent mutations were observed.
Genomic structure and allelic spectrum of MLL2 mutations that cause Kabuki syndrome
For 25 simplex cases in which we identified MLL2
mutations, DNA was available from both unaffected parents, and in each case the mutation was confirmed to have arisen de novo
(Supplementary Table II
online). These included 14 nonsense, five frameshift, three missense, two splice site mutations and one deletion. De novo
events were confirmed at six of the 10 sites where recurrent mutations were noted. In addition to the 81 kindreds in which we identified causal MLL2
mutations, we found two MLL2
variants in each of three simplex cases. In each case, neither MLL2
mutation could unambiguously be defined as disease-causing (Supplementary Table II
online). In one case, we found both a 21 bp in-frame insertion in exon 39 and a 1 bp insertion in exon 46 predicted to cause a frameshift. However, the unaffected mother also carried the 21 bp insertion suggesting that this is a rare polymorphism, and that the 1 bp deletion is the pathogenic mutation responsible for Kabuki syndrome.
Apparent disease-causing variants were discovered in nearly half (i.e., 22/54) of all protein-coding exons of MLL2 and in virtually every region known to encode a functional domain (). However, the distribution of variants appeared non-random as 13 and 12 novel variants were identified in exons 48 and 39, respectively. These sites accounted for 25, or more than one-third, of all the novel MLL2 variants and 31/81 mutations that cause Kabuki syndrome in our cohort. Eleven of the 12 pathogenic variants in exon 39 were nonsense mutations and occurred in regions that encode long polyglutamine tracts.
Four of the families studied herein had two individuals affected with Kabuki syndrome. A pair of monozygous twins with a c.15195G>A mutation were concordant for mild developmental delay, congenital heart disease, preauricular pits and palatal abnormalities, but discordant for hearing loss, and a central nervous system malformation. Concordance for mild developmental delay between an affected parent and child was observed in two families with MLL2 mutations, one with a nonsense mutation, c.13579A>T, p.K4527X, and the other with a missense mutation, c.16391C>T, p.T5464M that was also found in a simplex case. No MLL2 mutation was found in the remaining affected parent and child pair.
To examine the relationship between genotype and phenotype, we first compared the frequency of developmental delay, congenital heart disease, cleft lip and/or palate, and structural renal defects between MLL2 mutation-positive vs. MLL2 mutation-negative cases. No significant difference was observed between groups for three of these four phenotypes (). However, renal anomalies were observed in 47% (31/66 cases) of MLL2 mutation-positive cases compared to 14% (2/14 cases) of MLL2 mutation-negative cases and this difference was statistically significant (χ=5.1, df=1, p=0.024). In 35 cases in two clinical cohorts for whom more complete phenotypic data were available, short stature was observed in 54% (14/26) of MLL2 mutation-positive cases compared to 33% (3/19 cases) of MLL2 mutation-negative cases. We also divided the MLL2 mutation-positive cases into those with nonsense and frameshift mutations and those with missense mutations and compared the frequency of developmental delay, congenital heart disease, cleft lip and/or palate, and structural renal defects between groups. No significant differences were observed between groups ().
Phenotypic traits grouped by MLL2 mutation status (a) and type (b)
In 26 independent cases of Kabuki syndrome, including one parent-offspring pair, no MLL2 mutation was identified. Both persons in the mother-child pair had facial characteristics consistent with Kabuki syndrome (), mild developmental delay, and no major malformations. The mother is of Cambodian ancestry and her daughter is of Cambodian and European American ancestry. In general, most of the MLL2 mutation-negative Kabuki cases had facial characteristics () similar to those of the MLL2 mutation-positive Kabuki cases, and a similar pattern of major malformations () with the exception of fewer renal abnormalities.
Facial photographs of mother and daughter with Kabuki syndrome in whom no causative mutation in MLL2 was identified. Both have mild developmental delay and no known major malformations.
Facial photographs of four children diagnosed with Kabuki syndrome in whom no causative mutation in MLL2 was found.
We screened the MLL2
mutation-negative cases by aCGH for large deletions or duplications that encompassed MLL2
. Abnormalities were found in four cases. In one case, a 1.87 kb deletion of chromosome 5 (hg18, chr5:175,493,803-177,361,744) that included NSD1
and had breakpoints in flanking segmental duplications identical to the microdeletion commonly found in Sotos syndrome, was found. This suggests that this individual has Sotos syndrome, not Kabuki syndrome [Kurotaki et al., 2002
]. A second case had a novel 977-kb deletion of chromosome 19q13 (hg18, chr19:61,365,420–62,342,064) encompassing 20 genes. The majority of genes within the deleted region are zinc finger genes, some of which are known to be imprinted in both human and mouse. A third case had a complex translocation t(8;18)(q22;q21). Finally, a fourth case was found to have extra material for the entire chromosome 12. Average log2 ratio across chromosome 12 was 0.49, most likely representing mosaic aneuploidy of chromosome 12. No aCGH abnormalities were observed in 21 cases and aCGH failed for one case.