Genetic screening protocol for familial hypercholesterolemia which includes splicing defects gives an improved mutation detection rate
Introduction
Familial hypercholesterolemia (FH) was first described in 1920 [1] and is thought to affect about one in 500 individuals, making it one of the commonest single gene disorders. The condition is inherited as an autosomal co-dominant trait, and affected individuals have a defect in the low density lipoprotein receptor (LDLR) [2] which leads to accumulation of LDL cholesterol in plasma. The clinical consequence of FH is a marked predisposition to premature vascular disease, especially coronary artery disease [3]. A phenotypically identical condition (familial defective ApoB, FDB) is caused by defects in the ligand for the LDLR, ApoB. The most common mutation in this gene is R3500Q [4].
The diagnostic physical sign of FH is tendon xanthomata [1], but these are not present in every affected family [5]. Identification and early treatment of affected individuals is clearly desirable, and in the absence of tendon xanthomata, a DNA-based diagnosis provides confirmation of the clinical diagnosis and enables early patient management. In addition, knowledge of the genotype within a family group facilitates the tracking of affected individuals and eliminates the problems associated with equivocal lipid profiles [6].
The LDLR gene is encoded on chromosome 19p13.2, and so far over 800 mutations have been reported (http://www.ucl.ac.uk/fh, http://www.umd.necker.fr). The spectrum of disease causing mutations is quite diverse in most multi-cultural populations. Most previous studies quote mutation detection rates of 80% or less and this may in part be due to a lack of detection of splicing defects. In theory, up to 15% of mutations should be in the intronic splice junction area of the gene [7], and some such mutations have already been described [8], [9], [10]. Recently, Lombardi et al. [11] have shown that at least 16% of Dutch LDLR point mutations affect splicing, and in France intron splicing defects have been reported to account for 24% of defined FH mutations [12]. If genetic testing is to be used as part of a screening program for FH [13], [14], [15] then a detection system capable of identifying the mutations both within the coding region and also within the neighbouring intron regions is highly desirable.
The purpose of the present study was to determine if our previously reported detection rate of LDLR mutations in families with FH of 80% could be increased by the use of primer sets which would enable the detection of splice site mutations in the LDLR gene. We re-examined families previously studied in whom no defect had been identified, and in addition studied 40 new families, including families from a different geographical area (North East England).
Section snippets
Patients
Sixty-eight families with definite FH according to the Simon Broome Register criteria [16] (total cholesterol above 7.5 mmol/l, plus tendon xanthomata in the patient or relative) were studied. The patients attend Lipid Clinics in Northern Ireland (n = 44) and the North East of England (n = 24). They included those families previously screened [5], but in whom no defect in the LDLR gene was identified, and 30 new families. In addition to these patients, 130 possible FH cases (Northern Ireland n = 120
Results
A total of 198 families with FH were studied, of which 68 were classified as definite FH because of the presence of tendon xanthomata in the proband or a relative. Such families were screened for the presence of gene mutations in the LDLR gene and also the ApoB R3500Q mutation. Mutations were identified in 59 of the 68 patients, giving an overall detection rate of 87%. The detection rate was higher in Northern Ireland (40 out of 44, 91%) than in the North East of England (19 out of 24, 79%) (
Discussion
In this study, we report on a method that shows improved detection rate over our previous study and allows splice site mutations of the LDLR gene to be screened. We describe two novel intron splicing defects and two novel missense mutations which occur in exon 13 and exon 15 of the LDLR gene. We studied one of the splice mutations (c.1845 + 11 c > g) in detail. Our results indicate that it is a true mutation rather than a polymorphism as it was not found in the control blood samples (n = 104) and
Acknowledgements
This project was supported by the British Heart Foundation. We would also like to thank Dr. A.I. Polanska, South Tyneside District Hospital, Harton Lane, England for the referral of FH patients to the Regional Genetics Centre, Belfast City Hospital.
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