We describe a strategy that combines QM‐PCR, bidirectional sequencing, RT‐PCR, and missense analysis to identify mutations for 155 of 194 families with a confirmed clinical diagnosis of HHT. We previously showed that genetic testing, if applied in a systematic and optimised programme, renders care more effective and less expensive than clinical management alone.28
Test sensitivity for the cost–benefit study was estimated at 75% before our strategy was clinically implemented. An actual test sensitivity of 80% suggests that cost savings from systematic genetic testing for HHT families may be even greater than previously indicated.
QM‐PCR is invaluable in detecting whole exon or multiexon deletions and duplications that cannot be identified by sequencing and represent 7% of ENG mutations and 7% of ACVRL1 mutations found in our series. To our knowledge, this is the first report of whole exon or multiexon deletions in any HHT2 families. QM‐PCR analysis is also a more efficient means of identifying intraexonic deletions and insertions than sequencing, because several exons are screened simultaneously. Of the mutations identified, 43% of ENG mutations and 19% of ACVRL1 mutations could be detected by QM‐PCR. It is often difficult to distinguish missense mutations from relatively rare polymorphic variants that have not been identified in the general population. We evaluated the use of SIFT analysis to predict variations that affect protein function based on evolutionary conservation. We confirmed that 82 of 102 mutations are predicted to affect protein function. In the case of ACVRL1, there is a very good correlation between putative disease causing mutations and prediction based on evolutionary conservation; SIFT predicted that 62 of 68 missense variants affect the protein function, while the other six are tolerated. In the case of ENG, 20 of 34 missense variants were predicted to affect protein function, while six were tolerated. There were far fewer proteins in the ENG alignment than in ACVRL1. Nine non‐informative predictions were observed for ENG; five of these were for variants in the leader peptide, for which very few sequences were available for comparison. For variant G331S, RNA analysis revealed a splice site mutation, which overrides the SIFT prediction. This leaves only three non‐informative SIFT predictions for ENG. SIFT predictions are subject to non‐trivial false positive and false negative rates and must be used with caution.
Our strategy failed to find mutations for 39 clinically confirmed probands. Some of these patients are likely to have mutations in the putative HHT3 gene, not yet identified but located on chromosome 5.15
A few families might have undetected MADH4
mutations, as this gene was only analysed for a subset of clinically confirmed HHT families for whom no ENG
mutation was found. New evidence suggests that HHT patients should all be tested for MADH4
mutations if no ENG
mutation is found, because individuals who carry MADH4
mutations may not present with the classic signs of JPHT (gastrointestinal tract involvement or juvenile polyposis) but as HHT patients.29
We also cannot rule out the possibility that distant mutations, not readily detected by our strategy and perhaps in unidentified regulatory regions, may affect any of the above genes. To confirm the locus involved in disease, linkage analysis requires several consenting individuals from informative families. Such data are not often available. Sequence analysis of the ACVRL1 promoter and exon 1 may increase the detection sensitivity. To date, we have sequenced the ENG promoter for 32 samples without finding any mutations.
It is possible that our cohort includes patients who do not have HHT. Clinical diagnosis of HHT is complicated by large variation in visceral manifestations, even among individuals with the same mutation, and by age dependent manifestations of visible signs. This ambiguity encourages specialists to refer patients for genetic testing who have a suspected diagnosis of HHT, some of whom may not have a genetic predisposition. The number of individuals in the cohort who truly carry a mutation that leads to HHT cannot be determined with certainty; this complicates the calculation of test sensitivity. In our series, no mutation was found for 63 families after complete analysis. For 24 families, clinical diagnosis was deemed to be uncertain, either by the referring physician or because the patient had fewer than three Curaçao criteria.30
A study of HHT patients in the Netherlands reported sensitivity of 90% after sequence analysis alone.31
The difference in sensitivity may result from several factors other than the molecular diagnostic strategy. In the Dutch study, a relatively homogeneous cohort of patients was studied for as long as 30 years by the same team of physicians, using standardised diagnostic criteria. Our cohort of patients was very heterogeneous in terms of location and physicians involved, and we could only eliminate those who did not meet the Curaçao criteria. In addition, founder mutations appeared at a greater rate in the Dutch study than in ours.
Despite these complications, molecular testing for HHT families has several real benefits. Once a familial mutation is identified, relatives at risk can be tested conclusively by one efficient and relatively inexpensive test. Individuals with a mutation are identified for intensive clinical surveillance, while those without a mutation may safely be removed from clinical screening.