Atherosclerosis 180 (2005) 155–160

The relationship of molecular genetic to clinical diagnosis of familial hypercholesterolemia in a Danish population Dorte Damgaarda,∗ , Mogens L. Larsena , Peter H. Nissenb , Jesper M. Jensena , Henrik K. Jensenc , Vibeke R. Soerensena , Lillian G. Jensend , Ole Faergemana a

Department of Medicine and Cardiology, Aarhus Sygehus, Aarhus University Hospital, Tage Hansens Gade 2, DK-8000 Aarhus C, Denmark b Department of Clinical Biochemistry, Aarhus Sygehus, Aarhus University Hospital, Denmark c Department of Cardiology, Skejby Sygehus, Aarhus University Hospital, Aarhus, Denmark d Department of Clinical Genetics, Aarhus Sygehus, Aarhus University Hospital, Denmark Received 10 August 2004; received in revised form 22 November 2004; accepted 3 December 2004 Available online 12 January 2005

Abstract The genes encoding the LDL receptor and apoB were screened for mutations associated with familial hypercholesterolemia (FH) in 408 patients referred to the Lipid Clinic in 1995–2003. The study aimed at testing the ability of three different sets of clinical criteria to predict the results of molecular genetic analysis, and secondly test whether population-based age- and sex-specific percentiles of LDL-cholesterol offer useful supplemental information in the selection of patients for molecular genetic analysis. The patients were retrospectively categorised according to Simon Broome Register Group criteria, Make Early Diagnosis to Prevent Early Death criteria (MEDPED) and the Dutch Lipid Clinic Network criteria, and the distribution of patients was compared to the results of the molecular genetic analysis. The study illustrates a classical dilemma. Mutation detection rates (and specificities) are high only if sensitivity is very low and vice versa: to find most mutation carriers, even patients with only possible FH must be examined by molecular genetic testing leading to mutation detection rates as low as 30–40%. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Familial hypercholesterolemia; Genetics; Low-density lipoprotein receptor gene; LDL receptor gene; Apolipoprotein B gene; ApoB gene; Screening

1. Introduction The molecular basis for familial hypercholesterolemia (FH) is apparently very well understood [1–3] and, increasingly, the disorder is defined in molecular rather than clinical terms. Accordingly, diagnosis by molecular genetic analysis has gradually become widely applied, but reported mutation detection rates vary considerably [4,5]. In our clinic, for example, mutation detection rates have dropped from about 80% [6] to about 30% over the past 10 years, reflecting investigation of patients with ∗

Corresponding author. Tel.: +45 8949 7601; fax: +45 8949 7619. E-mail address: [email protected] (D. Damgaard).

0021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2004.12.001

increasingly milder clinical phenotypes. The strength of the relationship between genotype and phenotype in FH has therefore emerged as a matter of practical and theoretical concern. Several sets of clinical criteria have been employed to identify patients with FH [7–9]. In the United Kingdom a mutation in the LDL receptor gene was found in 32% of the patients with definite FH according to the Simon Broome Register criteria [5], and the Dutch Lipid Clinic Network group found a mutation in the LDL receptor gene in 80% of the patients with definite FH [4]. The US MEDPED Program used total cholesterol and LDL-cholesterol cut-points and calculated that a cut-point providing a specificity of 98% was expected to give a sensitivity of 54% for detection of an LDL

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receptor gene mutation in the general population of the USA [8]. To our knowledge, direct estimation of sensitivities and specificities of different clinical diagnostic criteria for FH to predict results of molecular genetic analysis of index patients has not been done. In this study, we therefore retrospectively tested the ability of the three sets of clinical criteria, developed in the United Kingdom, the USA and the Netherlands, to predict the results of routine molecular genetic analysis of the LDL receptor and apoB genes in Danish index patients. We also tested whether population-, age- and sex-specific percentiles of LDL-cholesterol constitute useful supplemental information for selection of patients for molecular genetic analysis. Our results suggest that the relationship of genotype to phenotype in the case of heterozygous FH is more tenuous than generally appreciated.

2. Patients The spectrum of LDL receptor gene mutations in Danish patients with xanthomatous FH has been published [6]. This early work, characterised by a high mutation detection rate, fostered routine genetic counselling and molecular genetic analysis of patients referred for FH to the Lipid Clinic of Aarhus Sygehus University Hospital, and all such patients, examined between January 1995 and December 2003, were included in the present study. Our laboratory performs DNA analysis of the LDL receptor and apoB genes for patients seen at other hospitals, but only patients seen in our own lipid clinic were included. A specially trained nurse (“genetic field worker”) obtained a full family history and drew a family tree. In general, DNA analysis was performed if the patient fulfilled two of three of the following criteria: (1) total cholesterol >8 mmol/L, LDL-cholesterol >6 mmol/L and triglycerides 95th percentile (%) Patients with LDL-cholesterol > 90th percentile (%)

Relatives

Mutation carriers (n = 135)

Non-carriers (n = 273)

Mutation carriers (n = 205)

Non-carriers (n = 180)

97.0 94.7 99.2

95.6 70.5 91.2

87.3 67.0 76.5

94.4 6.5 14.7

0.35). These findings are similar to those made in the Dutch FH population [20,21].

5. Discussion Our study illustrates that the relationship of the FH genotype to the FH phenotype is not straightforward. Why was the mutation detection rate, even in patients with a clinical diagnosis of definite FH, as low as 61.3 or 62.9% in our study, and indeed, why is it as low as 32% in other studies [5]? One explanation is inadequacy of the molecular genetic screening strategy employed: i.e. failure of the molecular screening technique (SSCP) to detect all mutations; intronic mutations of the LDL receptor gene; apoB mutations other than R3500Q; PCSK9 gene mutations [22,23] or mutations in other monogenic FH loci. Another possibility is that even the definite FH phenotype may not always be due to a mutation in only one gene (monogenic disease). As described by others [24], four patients in the present study, with a phenotype not easily distinguishable from that of the majority of patients, had mutations in the LDL receptor gene as well as in the apoB gene (double heterozygous FH), and it is quite possible that mutations in three or more genes may be responsible for severe hypercholesterolemia and tendon xanthomas with a phenotype indistinguishable from that of FH due to mutations in one allele of the LDL receptor gene. In principle, either inadequacy of the molecular genetic analyses or a more complex, polygenic background for the FH phenotype, must be invoked to explain that almost 40% of our patients with definite FH (Table 4) did not have an identifiable mutation in either the LDL receptor gene or the apoB gene. Unfortunately, we are unable to assess the relative importance of these two explanations. For good reasons, most of current literature is devoted to molecular biology, but it is worthwhile recalling that, just before the era of molecular medicine [25], Jensen and Blankenhorn, in 1972, concluded from a review of family studies of FH that polygenic inheritance is more probable than transmission by a single dominant gene [26]. That conclusion might still pertain to an important proportion of FH families, and we believe that a clinical definition of FH, i.e. severe primary hypercholesterolemia segregating (monogenic), or even aggregating (polygenic) in families, might very well be preferable to a molecular genetic definition. The clinical definitions of FH employed by

the Simon Broome Register and the Dutch Lipid Clinic Network are both very useful, and it is apparent from Table 4 that the criterion of possible FH must be included if we want to identify most single gene mutation carriers. A corollary of that approach is acceptance of single gene mutation detection rates as low as 30–40%. The statement, “treat the phenotype but counsel the genotype” [27], is as relevant as ever, and it is not safe to assume that patients with an FH phenotype, but with an unknown genotype, are at lower risk of coronary artery disease than patients with a known genotype, because most studies of morbidity and mortality have been performed in patients with a clinical rather than a molecular genetic diagnosis of FH [14,28,29]. Acknowledgements The work was supported by the Danish Heart Foundation (grants: 00-1-3-42-22804, 00-1-3-42-22804B, 01-1-3-13A22888 and 03-1-3-39-22062) and by grants from The Institute of Experimental Clinical Research at the University of Aarhus, Aarhus University Hospital Research Fund, Kirsten Anthonius’ grant and Aarhus University Research Fund. We are grateful for the expert technical assistance of Mrs. Gitte Glistrup Nielsen and Mrs. Anette Stenderup from our research laboratory and Mrs. Kirsten Kruse Olsen and Mrs. Kirsten Hald from the Department of Clinical biochemistry. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atherosclerosis. 2004.12.001. References [1] Goldstein JL, Hobbs HH, Brown MS. Familial hypercholesterolaemia. In: Scriver CR, Beaudet AL, editors. The metabolic and molecular basis of inherited disease, vol. III. New York: McGraw Hill; 2001. p. 2863–914. [2] Innerarity TL, Mahley RW, Weisgraber KH, et al. Familial defective apolipoprotein B-100: a mutation of apolipoprotein B that causes hypercholesterolemia. J Lipid Res 1990;31:1337–49. [3] Rader DJ, Cohen J, Hobbs HH. Monogenic hypercholesterolemia: new insights in pathogenesis and treatment. J Clin Invest 2003;111: 1795–803.

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