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Exome Sequencing Study Yields Kabuki Syndrome Gene

By Andrea Anderson

NEW YORK (GenomeWeb News) – Exome sequencing has been used to successfully uncover a gene involved in another rare genetic disease.

Last year, a University of Washington-led research group reported that they had successfully applied exome sequencing to find mutations behind a rare, single-gene disorder called Miller Syndrome. This time, the team identified a causative gene called MLL2 behind another rare condition known as Kabuki syndrome using exome sequencing on 10 unrelated individuals/families with the condition. Their follow-up works suggests the gene is mutated in about two-thirds of Kabuki cases.

Now, team members are focusing their exome analyses on individuals with Kabuki syndrome who don't carry obvious MLL2 mutations in hopes of finding additional genetic changes involved in the condition, Jay Shendure, a genome sciences researcher at the University of Washington, told GenomeWeb Daily News.

"We're operating under the working hypothesis that it is another gene and collecting a set of cases that don't have mutations in this [MLL2] gene," he explained, "with the idea that we would focus our efforts on potentially finding a second gene by that approach."

Shendure and his co-workers published their findings in the early, online edition of Nature Genetics yesterday.

The study was funded through an American Recovery and Reinvestment grant from the National Human Genome Research Institute to Shendure and fellow University of Washington researchers Michael Bamshad, Deborah Nickerson, and Wendy Raskind.

Kabuki syndrome, first identified in the early 1980s, affects roughly one in every 32,000 individuals. The autosomal dominant condition is associated with unusual facial traits such as elongated eyes and arched eyebrows. Affected individuals may also have heart, kidney, or skeletal malformations as well as slight mental retardation, though symptoms and their severity vary from one individual to the next.

Although Kabuki syndrome can be passed on from parents to their children, the researchers noted, such cases are rare and most cases occur spontaneously, making it difficult to map genetic transmission within families.

For the current study, researchers captured the coding regions of 10 genomes — representing 10 individuals with Kabuki syndrome — with custom target enrichment arrays. They then used the Illumina Genome Analyzer II to do massively parallel paired-end sequencing of these exomes to about 40 times coverage.

The capture arrays used in the current study were expanded compared with those used to study Miller syndrome, Shendure noted, and included coding sequences from all of RefSeq.

Study participants were not related to one another and came from several ancestral backgrounds, including European, Hispanic, and Haitian ancestry.

When the team screened variants in the exomes against the dbSNP129 database, data from the 1000 Genome Project, and 16 previously sequenced exomes, they found numerous changes in a gene called MUC16.

But, they quickly realized that these MUC16 changes were false positive mutations not involved in Kabuki syndrome.

"The gene that popped out from the standard analysis that we'd been doing was MUC16, which we pretty clearly classified as an artifact," Shendure noted. "It didn't work the way we had defined it before."

The researchers speculated that this could, perhaps, reflect non-coding or structural culprits behind Kabuki syndrome, genetic heterogeneity within the condition, and/or missing data, he explained.

To address these possibilities, the team relaxed their analyses and looked at how specific types of mutations related to Kabuki cases that had been classified by facial traits, developmental patterns, symptom severity, and so on.

"One thing we did was to really look at the phenotypes and rank the individuals based on how closely they resembled the classic phenotype of Kabuki syndrome," Shendure said. "The other [approach] was to look at nonsense or loss-of-function mutations separately."

Indeed, they found that individuals with the most highly ranked or "classic" Kabuki syndrome cases carried nonsense mutations in MLL2, a 54 exon gene coding for a histone methyltransferase in the so-called Trithorax family, which can participate in epigenetic programming.

Though the gene has been linked to some cancers, Shendure noted, studies in model organisms suggest MLL2 also contributes to developmental processes.

In their subsequent analyses, which included targeted Sanger sequencing, the researchers found that nine of the 10 individuals with Kabuki syndrome carried changes to MLL2 that are predicted to hamper the gene's function.

These loss-of-function mutations included nonsense or frameshift mutations in seven affected individuals that were detected by high-throughput sequencing and MLL2 mutations in two more individuals found through Sanger sequencing.

"Most of these mutations are loss-of-function mutations, which suggests that this may have something to do with dosage of the histone methyltransferase," Shendure said.

Meanwhile, targeted Sanger sequencing in dozens of additional individuals with Kabuki syndrome suggests about two-thirds of affected individuals carry MLL2 mutations. That has researchers speculating that additional non-coding or structural changes in and around that gene and/or mutations in another gene could also cause Kabuki syndrome.

They now plan to focus in on individuals without obvious MLL2 mutations to get to the bottom of this potential genetic heterogeneity — likely using a combination of exome sequencing and high resolution microarrays.

"It is clear that there may be additional genes in which variants cause Kabuki syndrome, as approximately one-third of cases did not have MLL2 mutations," co-corresponding author Michael Bamshad, a pediatrics researcher at the University of Washington, said in a statement. "To find these, it will be important to sequence the exomes of additional, well-characterized cases of Kabuki syndrome in which we do not find we don't see MLL2 mutations."