The mutation spectrum in Holt-Oram syndrome.

EDITOR—Holt-Oram syndrome (HOS) is a developmental disorder characterised by malformations of the radial ray of the forelimb and by congenital heart disease. The syndrome shows a marked variability in phenotype, with radial ray defects ranging from minor thumb abnormality through to severe reduction defect or phocomelia. The cardiac manifestations of HOS are similarly varied, and patients can present with a variety of structural heart abnormalities, atrial septal defects (ASDs) and ventricular septal defects (VSDs) being the most common, or conduction defects evident on ECG profiles. Previous studies have shown no correlation between the severity of a patient’s cardiac and skeletal abnormalities. Intrafamilial variation can be wide. HOS shows autosomal dominant inheritance and mutations in the T box transcription factor gene (TBX5) have been shown previously to be responsible for this disorder. 4 There is also evidence for genetic heterogeneity. The mechanism by which mutations in TBX5 cause a dominant phenotype is not understood at present, and it is anticipated that knowledge of the type of mutations causing HOS may shed light on this. Knowledge of a large number of mutations and the relation of a person’s genotype to phenotype is also useful for genetic counselling. In the face of a growing demand for a molecular diagnostic test for HOS, it is also helpful to have a quantitative estimate of the ability of current methods to detect mutations in TBX5. Twenty five cases with a clinical diagnosis of Holt-Oram syndrome have been tested for this study, bringing to 47 the total number of cases studied by us. Minimal diagnostic criteria were as described previously: bilateral radial ray defect, plus either cardiac abnormality or family history of cardiac abnormality. Cases were referred by a variety of clinicians and underwent full clinical assessment including x ray, electrocardiography (ECG), and echocardiography. Information regarding the clinical features of these patients came from the referring doctor. The patients represented both sporadic (8) and familial (17) cases of Holt-Oram syndrome. Mutational analysis was carried out using SSCP methods initially, followed by fluorescence sequencing of exons showing a non-standard SSCP banding pattern. The methods used were described in Li et al. Since that study, further analysis of the genomic structure of TBX5 has recognised that the previously reported exon B is in fact two exons. Extensive resequencing of both genomic and cDNA forms of TBX5 has also been undertaken, leading to revisions in the previously reported TBX5 sequence (the new sequence has accession number AF221714). This sequence is the same across the coding region as that produced by others. Of 17 new familial cases tested, eight showed linkage to chromosome 12 and the other families were too small to assess linkage. Table 1 shows that five mutations were identified in familial cases. Only one of these families was suYciently large to show meaningful linkage to chromosome 12q markers. Of eight new sporadic cases studied, three have yielded mutations. Thus, in the 34 familial cases studied by us, eight mutations have been identified, and six mutations have been identified in 13 sporadic cases. The precise natures of all the new mutations identified are detailed in table 2. The clinical features of HOS in all 47 cases are consistent with the previously described phenotype and show the wide spectrum of cardiac and skeletal abnormalities in this syndrome (see Bruneau et al for details of the complexity of cardiac abnormalities in HOS patients). Most patients show at least one defect of cardiac septation (an atrial or ventricular septal defect, or atrioventricular block) and abnormalities of the thumb. Radial hypoplasias and aplasias are present in sporadic cases PpHs, H20s, H22s, and H16s and in the familial cases H6f and Ghf, although not in every aVected member. Twenty five non-translocation mutations have been reported, including those presented here. These mutations are of 19 distinct types, with six mutations being identical to previously described forms identified in unrelated subjects (this paper and Basson et al). Of the 19 distinct non-translocation, disease causing mutations in TBX5 currently known, five are truncations, five amino acid substitutions, three splice site changes, and six reading frame shifts. We have observed a significant diVerence in the proportion of cases in which a mutation was detected in our group of sporadic cases as opposed to our group of familial cases. Forty six percent (6/13) of sporadic cases studied in our laboratory have yielded mutations by SSCP screening, whereas only 24% (8/34) of familial cases have done so. The overall mutation detection rate of 30% may be low for a variety of reasons. An inadequate mutation detection method would explain these results, yet the system in use by us is a standard one used on a variety of projects, all of which produce detection rates nearer the theoretical level for these techniques (approximately 95%). The largest PCR product used in the present analysis is only 326 bp long, well within the size acceptable for this kind of analysis. Mutation detection is also fully repeatable in our hands. There are four other possible explanations for this low mutation detection rate: (1) clinical misdiagnosis in our patients, (2) genetic heterogeneity of Holt-Oram syndrome, (3) the presence of mutations in the untranslated and promoter regions of TBX5, which have not been tested in this analysis, and (4) deletion of whole exons of TBX5, which would not be recognised by SSCP.