Expanding our knowledge of conditions associated with the ASXL gene family

Genome-wide sequencing has identified de novo truncating mutations in ASXL3 in four patients with intellectual disability, feeding problems and distinctive facial features. Their presentation resembles that of Bohring-Opitz syndrome, which is associated with de novo nonsense mutations in ASXL1. This newly defined phenotype provides an important clinical resource for comparison with future cases in which mutations are found in ASXL3. The phenotypes for patients with mutations in each gene will undoubtedly be further delineated as more patients are reported.

Dysmorphologists and clinical geneticists have worked over the past 40 years to defi ne clinical syndromes by their similar facial features, physical attributes and clinical course. Cytogenomic and molecular testing have helped to defi ne the underlying genetic etiology for many of these syndromes and clarifi ed why related genes in the same pathway sometimes lead to similar clinical pheno types. Now, with the advent of genomewide sequencing, dysmorphologists have a powerful new tool at their disposal. Th rough sequencing, clinicians and researchers are able to study a cohort of patients with mutations in the same gene and to characterize further an identifi able syndrome. Once published, other similar cases can be tested, and gene variants discovered through whole genome or exome sequencing can be compared with the previously published phenotype to defi ne the relevant clinical features further.
Th is exciting story is repeated with the new report by Bainbridge et al. in Genome Medicine [1], which des cribes the discovery of de novo truncating mutations in ASXL3 in four patients with similar clinical presen ta tions. Th e fi rst two patients were identifi ed through exome sequen cing of the proband and both parents. Th e subsequent two cases were found through sequencing of the aff ected child only. A custom exoncapture strategy was used, followed by highthroughput sequencing with the Illumina HiSeq platform. All coding and nearintronic diff erences between the patients and the human reference genome were evaluated. Th e unaff ected family members in the fi rst two cases were used as controls to localize only de novo changes in the probands. De novo mutations were defi ned as those present in a patient but absent from all other members of their family, fl agged as 'high quality' , and supported by at least fi ve reads with an allele percentage of 0.4 to 0.6%. Th e four de novo mutations that were found coded for premature stop codons and probably create a truncated ASXL3 protein. Th e muta tions in each case were previously unreported and included insertions and deletions, which likely represent lossoffunction alleles, given that the mRNAs containing these stop codons are degraded by nonsensemediated decay [1].
After identifying mutations in ASXL3 in these four patients, Bainbridge et al. used several large datasets to look for previously reported ASXL3 mutations. Th ey found four other truncating mutations in ASXL3 in phenotypically normal patients. Th ree of these mutations were located in the 3' end of ASXL3 [1], bringing into question whether these ASXL3 mutations are benign variants. Th e authors developed a model to determine the probability of observing multiple de novo mutations in ASXL3 in their four phenotypically similar patients. Th e results of their analysis show that this probability is approximately 4.0 × 10 17 . Th is makes it highly unlikely that these changes occurred by chance, but functional studies are still required to confi rm the pathological nature of these ASXL3 mutations [1].

Defi ning ASXL
Th e additional sex combs (Asx) gene has long been known to maintain homeotic gene activation and silencing in Abstract Genome-wide sequencing has identifi ed de novo truncating mutations in ASXL3 in four patients with intellectual disability, feeding problems and distinctive facial features. Their presentation resembles that of Bohring-Opitz syndrome, which is associated with de novo nonsense mutations in ASXL1. This newly defi ned phenotype provides an important clinical resource for comparison with future cases in which mutations are found in ASXL3. The phenotypes for patients with mutations in each gene will undoubtedly be further delineated as more patients are reported.  Drosophila, and three orthologs (Asxl1, Asxl2, Asxl3) are recognized in mice. ASXL1 is known to act on homeobox (Hox) genes as a repressor as well as an enhancer, and is important for development. Not much is known about the developmental function of the ASXL gene family in humans, but in Drosophila, Asx regulation is highly variable and tightly controlled directly after fertilization [2]. In humans, ASXL3, like ASXL1 and ASXL2, codes for a putative polycomb protein that probably forms a complex with other proteins and acts as a histone methyl transferase [3].

R E S E A R C H H I G H L I G H T
De novo ASXL1 mutations have been identified in 9 out of 15 published cases of BohringOpitz syndrome (BOS) in which they were tested [4,5]. Somatic ASXL1 muta tions have also been linked to myeloid malig nancies and myelodisplastic syndromes [6]. Following the example of other gene networks, such as the RAS signaling pathway genes and the RTKP13KAKT signaling pathway genes, it is plausible that somatic ASXL1 mutations could cause cancer, while germline mutations lead to developmental disorders [4,7,8]. To date, there have been two published cases of a neoplastic condition in BOS: a child with a medullo blastoma at 5 years old [9] and bilateral nephro blasto matosis found at autopsy in a 5monthold infant [10]. Both patients were negative for mutations in ASXL1 and ASXL3 [1,4]. It remains to be seen if patients with ASXL1 or ASXL3 mutations require tumor surveillance.
Although all four subjects discussed by Bainbridge et al. shared clinical characteristics with each other and with BOS, these features are relatively nonspecific. The patients with an ASXL3 mutation had intrauterine  [4] and the two reported in [5]. The second column includes the four cases reported in [1]. This table is adapted from [5]. When a specific feature is not reported, the patient is not considered in the calculation. mutations were also found to be negative for ASXL3 muta tions [1,4]. Undoubtedly, the publication of this article will lead to further reporting of patients with muta tions in the ASXL gene family, and the clinical features of this ASXL3 mutation syndrome will become further defined. In the meantime, wholegenome sequencing will continue to provide insight into novel mutations causing previously undefined clinical conditions.