Partial ATRX gene duplication causes ATR‐X syndrome

Mutations in the ATRX gene (OMIM #300032), located at Xq13, are responsible for causing ATR-X syndrome (OMIM #301040) [Gibbons et al., 1995]. This is a relatively common clinical consideration in males that present with mental retardation (MR) and hypotonia. Affected males have severe developmental delay with little or no language and seizures are present in approximately 30%. Additional dysmorphic facial features, genital anomalies and alpha thalassemia often help guide clinicians in making this diagnosis [Stevenson, 2007]. Numerous ATRX point mutations have been associated with ATR-X and nearly all are found in either the zinc finger or helicase functional domains [Gibbons et al., 2008]. Additionally, Thienpont et al. [2007] have reported two ATR-X families with partial duplications of ATRX that result in the lack of a functional transcript which ultimately results in no detectable protein. In this letter, we detail the identification of another partial duplication in a third ATR-X family with three affected males. The duplication in our family (K8922) appears very similar to those reported by Thienpont et al. [2007] and also disrupts the normal expression of the ATRX gene. The duplication was discovered using a customized synthetic Multiplex Ligation-dependent Probe Amplification (MLPA) kit designed to target X-linked mental retardation (XLMR) genes of clinical interest. The synthetic MLPA kit included five X-linked genes (ATRX, L1CAM, FGD1, NEMO, and CDKL5) as well as three other genes which functioned as internal controls. Two cohorts of males with MR and either hypotonia (n1⁄4 95) or suspected X-linked inheritance (n1⁄4 205) were screened. The probands were also tested with a commercially available MLPA kit (MRX-P106 from MRC-Holland) that included probes for 14 nonsyndromal XLMR genes. Data from the synthetic MLPA kit indicated that dosage alterations of the five XLMR genes tested appear to be uncommon as only two individuals were identified with results consistent with an X-linked duplication. The first individual carried a clinical diagnosis of ATR-X syndrome and was found to have a partial duplication of ATRX. There were two additional affected males in the pedigree and obligate carrier females had highly skewed X-inactivation ratios that supported the diagnosis even though previous ATRX sequencing had been reported as normal. The second proband was identified with a duplication of NEMO which was subsequently shown to be part of a larger duplication involving the MECP2 gene, which was consistent with his clinical course [Van Esch et al., 2005; Friez et al., 2006]. There were no additional dosage alterations detected in the 14 XLMR genes analyzed by the MRX-P106 kit. Array CGH on two X-chromosome specific platforms (Nimblegen, Madison, WI and Oxford Gene Technology, Oxford, UK) and qPCR were done to more accurately determine the breakpoints of the ATRX duplication. Taken together, these results indicated that the duplication is 248 kb in length and spans ATRX exons 2–31 (Fig. 1). RNA analysis by Northern blotting failed to identify a full-length transcript although RT-PCR was able to detect smaller nested fragments that could be used for sequencing. The apparent absence of transcript (based on Northern versus qPCR) is of interest based on the finding that truncating ATRX mutations are likely ‘‘rescued’’ to some degree and appear as hypomorphs rather than complete loss-of-function mutations [Gibbons et al., 2008]. In similar fashion to other key X-linked genes, a complete loss of ATRX function due to gross rearrangement of the gene is most likely lethal. Thienpont et al. [2007] indicate a complete loss of protein, but the sensitivity of their assay is unclear and the same can be said for our attempt at detecting full-length RNA transcript. Based on our initial ATRX sequencing data generated from cDNA we recognize