Geographical Clustering of Low Density Lipoprotein Receptor Gene Mutations (C292X; Q363X; D365E & C660X) in Cyprus

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1 HUMAN MUTATION Mutation in Brief #302 (2000) Online MUTATION IN BRIEF Geographical Clustering of Low Density Lipoprotein Receptor Gene Mutations (C292X; Q363X; D365E & C660X) in Cyprus Stavroulla L. Xenophontos 1, Alkis Pierides 2, Kyproulla Demetriou 2, Panicos Avraamides 3, Panayiotis Manoli 1, Nafsika Ayrton 1, Nicos Skordis 4, Violetta Anastasiadou 4, George Miltiadous 1, and Marios A. Cariolou 1 1 Molecular Genetics Unit B-DNA Identification Laboratory, The Cyprus Institute of Neurology and Genetics, Nicosia; 2 Nephrology Department, Nicosia General Hospital, Nicosia; 3 Cardiology Department, Nicosia General Hospital, Nicosia; 4 Department of Paediatrics, Archbishop Makarios III Hospital, Nicosia, Cyprus *Correspondence to: M.A. Cariolou, PhD, Cyprus Institute of Neurology and Genetics, Molecular Genetics Unit B-DNA Identification Laboratory, P.O Box 23462, 1683 Nicosia, Cyprus; Fax: ; cariolou@mdrtc.cing.ac.cy Short Title: FAMILIAL HYPERCHOLESTEROLAEMIA IN CYPRIOT FAMILIES Communicated by Mark H. Paalman In Cyprus, no data are yet available on the frequencies of clinically diagnosed FH patients. Further, until now, familial hypercholesterolaemia in Cyprus had not been studied at the molecular level to determine the nature or frequency of LDLR gene mutations. Being a relatively homogeneous population, we anticipated that a few founder mutations would predominate on the island. In the present study, three previously identified LDLR gene mutations were found to cosegregate with high LDL cholesterol levels in 23 unrelated, clinically diagnosed families with FH. Geographical clustering of each of these LDLR gene mutations was indicated, a phenomenon arising from low migration rates and high inbreeding. The latter cultural practices account for the discovery of a homozygous FH sib pair whose parents are carriers of the same mutation. Microsatellite and intragenic haplotype analysis in this FH population, suggested that the families which shared the same LDLR gene mutation have a common origin. This is supported by their relative geographical distribution. Thirty young FH individuals were also offered presymptomatic diagnosis which should facilitate the prevention of premature coronary artery disease. Finally, results from this study support the suggestion that the formation of tendon xanthomata in FH patients may be under environmental influence Wiley-Liss, Inc. KEY WORDS: LDLR, familial hypercholesterolaemia, xanthomata, atherosclerosis, Greek Cypriot INTRODUCTION Familial hypercholesterolaemia (FH; MIM# ), an autosomal dominant disorder, describes a clinical condition characterized by elevation of plasma low density lipoprotein cholesterol, tendon xanthomata and a predisposition to premature atherosclerosis. Typically, FH results from mutations in the low density lipoprotein Received 29 September 1999; Revised manuscipt accepted 27 December WILEY-LISS, INC.

2 2 Xenophontos et al. receptor gene (LDLR; MIM# , Genbank Accession Number: ). The LDL receptor is a 160 KD, cell surface transmembrane protein which facilitates the uptake of plasma LDL cholesterol into hepatocytes where it is further catabolised. The primary cause for an elevation in low density lipoprotein cholesterol in the blood is a genetic defect in the LDLR gene resulting in familial hypercholesterolaemia (Hobbs et al, 1990; Day et al, 1997). Several international reports indicate that the heterozygote frequency for FH may be one in 500 and for FH homozygosity, one in 1 million. In the present study, we report our findings on the genetic screening of 23 Greek Cypriot families clinically diagnosed as suffering from familial hypercholesterolaemia. Both homozygous and heterozygous probands were studied. Hitherto, over 600 mutations have been reported for the LDL receptor gene, data base web site (Day et al, 1997). METHODS Clinically diagnosed FH probands, referred to us for genetic analysis had elevated total cholesterol (>290mg/dl) and LDL cholesterol ( >200 mg/dl) with normal triglyceride levels (<175mg/dl). Most, had a history of coronary artery disease (CAD) and a family history of hypercholesterolaemia and CAD. Tendon xanthomata were present in 13 families and absent in 10 families. Secondary causes for hypercholesterolaemia such as hypothyroidism, diabetes, renal or hepatic disease were also absent. Familial defective apo B100 hypercholesterolaemia was excluded by PCR analysis as described by Tybjaerg and others (1998). DNA analysis began with amplification of the LDLR exons as described previously (Hobbs et al, 1990). Automated sequencing was carried out with the Perkin Elmer Big Dye Terminator Cycle Sequencing Ready Reaction Kit as described by the manufacturer. Electrophoresis and sequence analysis followed on an ABI PRISM 310 Genetic Analyzer. RESULTS In the present study, one proband from each of the 23 Cypriot FH families was screened with the diagnostic restriction digests for two substitutions (Q363X & D365E) previously found in a Greek Cypriot family (Kotze et al, 1997). We found 5 of the 23 unrelated Cypriot probands (5/23, 22%), to carry this double mutation and on further screening of all their family members, we verified that the two mutations cosegregated with hypercholesterolaemia. Sequence analysis confirmed this, and three generation families with one affected parent confirmed that the changes were allelic. In addition, screening of fifty unrelated, normal individuals confirmed that the missense polymorphism (D365E) was not found in isolation in the normocholesterolaemic, Cypriot population. Our results were in perfect agreement with the original report by Kotze, (1997), where 100 normocholesterolaemic individuals of various ancestries were analysed for both mutations and neither of the mutations were found in these individuals. The second mutation detected in our families was in exon 6, C292X. Direct sequencing of the exon 6 amplicon revealed this truncation mutation in 11/23 unrelated probands (i.e 48%). This mutation was previously detected in two Cypriot FH patients in Greece (Mavroides et al, 1997; Traeger-Synodinos et al, 1998). The third LDLR gene mutation detected was in exon 14, C660X. This is known as the Lebanese mutation. This was found to be quite widespread from the Northern to the Southern world hemispheres (Lehrman et al, 1987; Reshef et al, 1996). In our study, C660X was detected after extensive screening of all exons and the promoter of the LDLR gene in 7 probands from the remaining 7 families with a definite FH diagnosis. One of these families was found to have a homozygous sib pair for this mutation. The mother of these sibs was genetically diagnosed as a carrier of the C660X mutation. However, DNA was not available from the father for genetic analysis, who should be an obligate carrier of the C660X mutation. This truncation mutation, C660X, represents the remaining 30% of LDLR gene lesions in our families. A total of 80 hypercholesterolaemic individuals were therefore genetically diagnosed, thirty of which where 2-25 years of age. Details for these mutations are summarized in table 1. Segregation analysis of the microsatellite marker D19S394 and the three polymorphic sites within the LDLR locus; Stu1, Ava11 and Msp1 in exons 8, 13 and 15 respectively showed that a specific haplotype was associated with each LDLR gene mutation. These were the following: (C292X) exon 6; D19S394 (allele 8); (Stu1 + ); (Ava11 - ); (Msp1 - ). (Q363X) exon 8; D19S394 (allele 4); (Stu1 + ); (Ava11 - ); (Msp1 - ). (C660X) exon 14; D19S394 (allele 6); (Stu1 + ); (Ava11 + ); (Msp1 + ).

3 Familial Hypercholesterolaemia in Cypriot Families 3 Unrelated non-fh individuals (54 alleles), were also typed for the microsatellite marker D19S394 to compare the frequencies within the two populations. The D19S394 alleles found to be in linkage disequilibrium with the LDLR gene mutations specified above, were relatively rare in this normal population. From this screening a total of eleven alleles were identified in the Greek Cypriot population. Table 2 indicates the relative frequencies of alleles 4, 6 and 8 found in unrelated FH and non-fh chromosomes. Table 1.Summary of LDLR Gene Mutations, Frequency and Distribution in Cypriot Families with Diagnosed FH Mutation Exon No. Nucleotide Position Base Change Amino Acid Change Detection Assay No. Families/ Percentage Family Origin C>A C292X Sequencing 11/23 (48%) Central Highlands CAD/ Xanthoma Present & 1158 C>T C>G Q363X D365E PvuII Digest AvaII Digest 5/23 (22%) North NE & SE Absent C>A C660X Hinf I Digest 7/23 (30%) North West Present Table 2. Frequency of D19S394 Alleles in Cypriot FH Families and Unrelated Non-FH Chromosomes D19S394 Allele a LDLR Mutation 8 Exon 6 C292X 4 Exon 8 Q363X D365E 6 Exon 14 C660X Unrelated-FH Chromosomes Non-FH Chromosomes b 11/11 (100%) 4/54 (7%) 5/5 (100%) 7/54 (13%) 7/7 (100%) 5/54 (9%) D19S394 Allele segregating with the LDLR mutation a, Number of D19S394 alleles (8, 4 or 6) in unrelated individuals b DISCUSSION Haplotype analysis suggests that in each group of FH families, the LDLR gene mutations are identical by descent, (IBD) as opposed to being so by state, (IBS). No recombination is observed in any of the phase known meioses between the marker D19S394 and any of the FH mutations or normal homologues, i.e complete linkage disequilibrium is seen between the respective LDLR mutation and the linked D19S394 allele. As expected, the

4 4 Xenophontos et al. homozygous FH sib pair with the exon 14 truncation mutation were homozygous for the linked, D19S394 allele 8. Even though DNA was not available from the father for analysis, the homozygous haplotype for the sib pair indicates that their parental FH chromosomes have a common origin. The geographical distribution of the families bearing the same LDLR mutations supports a common origin and geographical clustering of these mutations. In addition, the fact that these three mutations do not occur in CpG rich regions, which are mutation hotspots, excludes the recurrence hypothesis (Krawczak et al, 1998). Further, as the first two mutations in exons 6 and 8 have only been found in Greek Cypriots, (Mavroides et al, 1997; Traeger-Synodinos et al, 1998), it is possible that they originated in Cyprus, whilst the exon 14 C660X mutation, being most common in Lebanon, suggests that genetic drift from Lebanon is the most probable route by which this mutation was introduced in Cyprus. A comparison of the phenotype of the exon 14 mutation (C660X), in homozygotes and heterozygotes has been made possible in this study. The homozygous sib pair exhibit all the serious clinical characteristics of the affected genotype, whilst the parents, in their fourth decade of life have no indications of coronary artery disease while of course, under hypolipidaemic drug therapy. At this point, the only successful therapy for the Cypriot FH homozygous sib pair is regular, long term plasmapheresis every fortnight. Whilst drug therapy can reduce cholesterol levels in the heterozygotes to near normal concentrations, it has negligible effects in the homozygotes, as no receptor molecules are functional. Plasmapheresis has been effective in reducing serum cholesterol levels in the affected sib pair to average values under 300mg/dl and at these levels, the cutaneous xanthomata have cleared completely. An interesting observation in the present study is that xanthomata were not detected in the 5 families we examined with the exon 8 mutations (Q363X & D365E), yet, in the original FH family xanthomata were observed ( Kotze et al, 1997). The presence or absence of xanthomata, in our families, was determined by ultrasonography carried out on both Achilles tendons of affected members. Furthermore, one of our 5 families bearing the exon 8 double mutation, is related to the Cypriot family reported in the above publication. ( Kotze et al, 1997). Ultrasonography of the Achilles tendon is considered an important clinical diagnostic procedure for FH (Kiovunen et al, 1994). Therefore, one might conclude that, since these two families share an ancestral parent and an identical genetic lesion but yet they do not share the same clinical phenotype, (i.e development of xanthomata), the different habits and locations where the families reside, i.e South Africa vs Cyprus may be responsible for this phenotypic variation. Similar findings were reported for Chinese FH patients (Pimstone et al, 1998). The fact that none of the carriers of the LDLR exon 8 double mutation (Q363X & D365E), were found to carry the two different mutations in trans, suggests that the mutation events must have occurred simultaneously. We observed, through a three generation pedigree, that both mutations are transmitted via one parental chromosome. As the (D365E) change has not been detected in isolation from the Q363X mutation, we cannot draw any conclusions as to its role in hypercholesterolaemia. Further, the amino acids interchanged by this mutation (i.e aspartic acid by glutamic acid) have the same side group and are both acidic. Little change in LDLR structure may result from this substitution, suggesting that it may have negligible effects on LDLR function. In conclusion, the information generated in this study should serve as a foundation for the implementation of a nationwide screening programme in Cyprus facilitating presymptomatic diagnosis of many FH individuals. ACKNOWLEDGMENTS We thank the patients and their families who participated in this study and Professor Moses Elisaf for helpful discussion. Funding for this project was provided by the Ministry of Health of the Republic of Cyprus. REFERENCES Day INM, Whitall RA, O'Dell SD, Haddad L, MK Bolla, V. Gudnason, and S.E. Humphries (1997) Spectrum of LDL Receptor Gene Mutations in Heterozygous Familial Hypercholesterolaemia. Human Mutation 10: Hobbs HH, Russell DW, Brown MS, Goldstein JL. (1990) The LDL receptor locus and familial hypercholesterolaemia: Mutational analysis of a membrane protein. Ann Rev Genet 24:

5 Familial Hypercholesterolaemia in Cypriot Families 5 Koivunen-Niemela T, Viikari J, Niinikoski H, Simell O, Alanen A (1994) Sonography in the detection of achilles tendon xanthomata in children with familial hypercholesterolaemia. Acta Paediatr 83;11: Kotze MJ, de Villiers JNP, Loubser O, Thiart R, Scholtz CL, Raal F.J (1997) A double mutant LDL receptor allele in a Cypriot family with heterozygous familial hypercholesterolemia. Hum Genet 100: Krawczak M, Ball EV, Cooper DN. (1998) Neighbouring-nucleotide effects on the rates of germ-line single-base-pair substitution in human genes. Am J Hum Genet 63 (20): Lehrman MA, Schneider WJ, Brown MS, Davis CG, Elhammer A, Russell DW, Goldstein JL (1987) The Lebanese allele at the low density lipoprotein receptor locus. Nonsense mutation produces truncated receptor that is retained in endoplasmic reticulum. J Biol Chem 5; 262(1): Mavroidis N, Traeger-Synodinos J, Kanavakis E, Matsaniotis N, Humphries SE, Day IN, Kattamis C (1997) A high incidence of mutations in exon 6 of the low-density lipoprotein receptor gene in Greek familial hypercholesterolemia patients, including a novel mutation. Hum Mutat 9;3: Pimstone SN, Sun Xi-M, du Souich C, Frohlich JJ, Hayden MR, Soutar AK. (1998) Phenotypic Variation in Heterozygous Familial Hypercholesterolemia. A Comparison of Chinese Patients with the same or similar mutations in the LDL receptor gene in China or Canada. Arterioscler Thromb Vasc Biol 18: Reshef A, Henrik N, Triger L, Hensen TS, Eliav O, Schurr D, Safadi R, Gare M, Leitersdorf E (1996) Molecular Genetics of familial hypercholesterolemia in Israel. Hum Genet 98: Traeger-Synodinos J, Mavroidis N, Kanavakis E, Drogari E, Humphries SE, Day INM, Kattamis C, Matsaniotis N (1998) Analysis of low density lipoprotein receptor gene mutations and microsatellite haplotypes in Greek FH heterozygous children: six independent ancestors account for 60% of probands. Hum Genet 102: Tybjaerg-Hansen A, Steffensen R, Meinertz H, Schnohr P, Nordestgaard BG (1998) Association of mutations in the apolipoprotein B gene with hypercholesterolemia and the risk of ischaemic heart disease. N Engl J Med 338: