We report a de novo microdeletion of ~250 kb at 20p12.1 in a 15‐year‐old girl with the KS, using array CGH with a 1 Mb resolution. This was a fortunate finding, as the resolution of the array is lower than that of the deletion size. Several observations suggest that C20orf133
is a causative gene for the observed phenotype in the index patient. First, the deletion in this patient occurred de novo in the macro‐Appr‐Pase‐like domain of the putative protein, thereby probably disrupting the enzymatic activity. Second, the expression pattern of the C20orf133
gene during mouse embryonic development supports its importance in the development of various tissues and organs, such as those affected in this patient. The gene contains a macro functional domain, which links it with the chromatin structure. Recently, several congenital malformation syndromes have been described as caused by haploinsufficiency of a gene involved in chromatin remodelling.42,43,44,45,46,47
In Mus musculus
, both in situ hybridisation and RT‐PCR showed that C20orf133
is expressed during embryonic development of the brain, in particular the ventricular zone. Just before birth, at E18.5, C20orf133
remains expressed in the brain, with relatively high levels of expression in discrete regions: the subventricular zone of the striatum and olfactory lobe, the cortical plate, the cerebellar primordium and the inferior colliculus of the tectum. The high levels of expression in the brain across a wide range of developmental stages and its persistence during adulthood is consistent with a role for the gene in mental development, and may reflect a requirement for C20orf133
in axonal outgrowth and functioning of the adult cortex. The gene is also expressed in other embryonic tissues that are typically affected in patients with KS. High levels of expression in embryonic kidney/urinary tract have been found. One study found renal malformations in 28% of tindividuals with KS,48
but such malformations might be underdiagnosed because they sometimes remain asymptomatic. In the current study, expression was seen in several craniofacial regions, which, given the specific facial characteristics of patients with KS, can be expected. C20orf133
was expressed in the mesenchymal components of the tooth‐bud condensations at E14.5. Interestingly, dental anomalies such as hypodontia, malocclusion, microdontia and small dental arches, were seen in 68% of patients with KS in one study.48,49
Expression of the gene in the cells lining the vestibulocochlear and cochlear duct at stage E14.5, possibly explaining hearing loss was reported in up to 82% of patients with KS.48
In the developing mouse eye, strong hybridisation was seen in the cuboid epithelium of the lens and the inner nuclear layer of the retina, which could explain the frequency of colobomata and cataracts in KS.3
Expression in the heart is low, despite congenital heart disease being reported in 42% of patients with KS.48
Interestingly, in our index patient, no heart defect was seen after physical and ultrasound investigation. An overview of the mouse expression data, the phenotype of the index patient and the features of KS are provided in table 1.
gene contains a macro domain (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/
). Previously identified as displaying Appr‐1”‐P processing activity, the macro domain may play roles in distinct ADP‐ribose pathways. In vivo evidence suggests that ADP ribosylation is an important post‐translational modification that has a role in DNA repair, transcriptional activation and repression, telomere and chromatin biology, long‐term memory formation, DNA binding, and DNA and/or RNA unwinding, among other processes.50
To date, no genes containing macro domains have been implicated in mental retardation and/or developmental delay. However, it is striking that several syndromes with mental retardation and multiple congenital anomalies result from haploinsufficiency of genes involved in DNA repair, transcription, chromatin biology and long‐term memory formation, exactly the same processes in which macro domain‐containing proteins are known to play a role. Possibly, the macro domain family of proteins may represent a novel class of dose‐sensitive genes that may cause developmental disorders when mutated.
Genevieve et al
reported that one of the patients with KS in their study had a clinical overlap with CHARGE syndrome.51
This syndrome was recently shown to be caused by mutations in or deletions of CHD7
, a member of the chromodomain helicase DNA‐binding genes, which have a unique combination of functional domains, including two N‐terminal chromodomains, an SNF2‐like ATPase/helicase domain and a DNA‐binding domain.52
ADP–ribose binding affects the function of the Alc1 protein, a protein with homology to the Snf2 ATPase/helicase.53
Therefore, the alteration of the phosphorylation status of ADP–ribose by C20orf133
may have an influence on the activity of this Swi/Snf chromatin remodelling factor and thus explain the clinical overlap between some patients with CHARGE syndrome and KS.
Screening for mutations, deletions or duplications in 20 other patients with KS did not reveal any mutations within the C20orf133 candidate gene. Possibly, mutations outside the coding region as well as epigenetic changes might cause the KS phenotype. We also cannot exclude the presence of as yet unknown genes within the region, which might be affected by this deletion. However, it seems likely that KS is genetically heterogeneous, and that mutations in other genes may result in phenocopies. The accumulating findings of several different chromosomal rearrangements in patients with KS summarised above may pinpoint several loci that may cause KS.
Recently, other syndromes associated with multiple congenital anomalies and mental retardation have been shown to be genetically heterogeneous. For example, Noonan syndrome can be caused by mutations in the protein–tyrosine phosphatase nonreceptor‐type 11 (PTPN11
or by mutations in the V‐Ki‐Ras2 Kirsten rat sarcoma 2 (KRAS2
) viral oncogene homologue,46
both components of the Ras pathway, or by mutations in the Son of Sevenless Drosophila
homologue 1 gene (SOS1
The CHARGE syndrome phenotype can, in addition to mutations in the CHD7
gene, also be caused by a mutation in the semaphoring‐3E gene (SEMA3E
Rubinstein–Taybi syndrome can be caused by mutations in the gene encoding the transcriptional coactivator CREB‐binding protein (CREBBP
or by mutations in the E1A‐binding protein 300‐kDa (EP300
In conclusion, C20orf133 is a viable candidate gene for the phenotype in the index patient. Further evaluation is needed to determine to what extent this gene could be involved in the aetiology of KS. In addition, this study further illustrates the value of array CGH to localise genes causing developmental disorders. The fortuitous finding of a microdeletion below the 1 Mb resolution of our array demonstrates that high‐resolution array CGH will enable the functional identification of more genes involved in the aetiology of KS and other clinical genetic syndromes.
Electronic database information
- NCBI accession numbers of the sequences used for alignment are NM_080676.5 (Homo sapiens), XM_001136712.1 (Pan troglodytes), AB173156 (Macaca fascicularis), CAM14292 (Mus musculus), BC060026.1 (Xenopus laevis) and NP_956843 (Danio rerio).