Detection of a large TBX5 deletion in a family with Holt-Oram syndrome.

EDITOR—Although point mutations are responsible for many genetic disorders, it is increasingly being appreciated that many disorders can be caused by deletion or duplication of DNA sequences. Alterations in DNA copy number are responsible for several categories of disorders and can range in scale from a chromosome or chromosomal region to just one exon of a single gene. For instance, duplication and deletion of a 1.5 Mb DNA fragment on chromosome 17p11.2 containing the gene PMP22 is the common mechanism for Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), respectively. At the level of individual exons, deletion of exon 17 or duplication of exon 13 of the BRCA1 gene can predispose to breast and ovarian cancer. Cytogenetic studies (including CGH and array based CGH) can screen the whole genome for copy number changes. However, for most cytogenetic methods, the main disadvantage is low resolution, as they can detect deletions and amplifications only when several megabases of DNA are involved. Array based CGH may detect more localised changes, 6 but at present is technically challenging and expensive to implement. Gene dosage assessment at specific genetic loci is possible by using multiplex amplifiable probe hybridisation (MAPH), a simple, inexpensive, and non-microscopic approach to determining locus copy number in a complex genome. In this method, specific short probes flanked by the same primer pair are hybridised with a test genomic DNA sample. After stringent washing, these probes can be recovered and amplified quantitatively by PCR, and products resolved by polyacrylamide gel electrophoresis. The main advantages of this approach are high resolution (down to a few hundred base pairs) and the parallel processing of many DNA samples in a single experiment. We have applied this method to genomic DNA from Holt-Oram syndrome (HOS, MIM 142900) patients in which the mutation at TBX5 was unknown. This syndrome 9 is a rare developmental disorder (prevalence of about 1 in 100 000) with autosomal dominant inheritance, characterised by congenital heart malformations and upper limb defects. Mapped to 12q24.1, TBX5 is a member of the T box gene family, of which the protein products act as transcription factors via a conserved DNA binding domain (the “T domain”). Gene dosage alteration or target DNA binding derangement via TBX5 mutations result in aberrant cardiogenesis. An overall mutation detection rate of 30% was reported by using SSCP (single stranded conformation polymorphism). More than 19 diVerent non-translocation mutations in TBX5 have been shown to cause HOS. These consist of nonsense mutations, small deletions, rearrangements, insertions, and missense mutations. Some of these mutations introduce premature stop codons that are predicted to encode truncated polypeptides; these mutations appear to result in haploinsufficiency. Nevertheless, in many HOS pedigrees in which the disorder is linked to TBX5, a mutation has not been found. We therefore applied MAPH to look for large scale deletions or duplications at TBX5.