Familial hypercholesterolemia (FH) is a metabolic disorder

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1 Pvu II Polymorphism of Low Density Lipoprotein Receptor Gene and Familial Hypercholesterolemia Study of Italians Antonio Daga, Marina Fabbi, Tiziana Mattioni, Stefano Bertolini, and Giorgio Corte Familial hypercholesterolemia is a metabolic disorder inherited as an autosomal dominant trait characterized by an increased plasma low density lipoprotein (LDL) level. It has been demonstrated that the disease is caused by several different mutations in the LDL receptor gene. Although early identification of individuals carrying the defective gene could be useful in reducing the risk of atherosclerosis and myocardial infarction, the available techniques for determining the number of the functional LDL receptor molecules are not sufficiently accurate. The recent isolation of the LDL receptor gene now makes it possible to use restriction fragment length polymorphisms to study the inheritance of the defective allele in families with familial hypercholesterolemia. In the present study, we report the use of a Pvu II restriction fragment length polymorphism to follow the inheritance of familial hypercholesterolemia in a total of 79 patients from 37 different families. This restriction fragment length polymorphism allowed unequivocal diagnosis in 32.5% of the cases. Furthermore, in the Italians studied, the absence of a polymorphic Pvu II cutting site (P1 allele) was found to be strongly associated with familial hypercholesterolemia. (Arteriosclerosis 8: November/December 1988) Familial hypercholesterolemia (FH) is a metabolic disorder characterized by a raised plasma low density lipoprotein (LDL) concentration, xanthomas of skin and tendons, and premature atherosclerosis. The disease is inherited as an autosomal dominant trait with a heterozygote frequency of about 1 in 500 in Europeans and Americans. Heterozygosity is associated with a high risk of myocardial infarction in the fourth decade of life. Homozygotes are much more severely affected and often experience myocardial infarction in the first or second decade. Over the past few years, it has been clearly demonstrated that the primary defect in FH is a mutation in the gene coding for the LDL receptor that prevents a normal clearance of plasma LDL. 1 Several reports have recently detailed some of these mutations that affect different parts of the gene. 2-5 Early identification of FH heterozygotes, especially in those cases where the cholesterol level overlaps that of normal subjects, is highly desirable so that the risk of future myocardial infarction can be reduced by diet and life style change. However, the techniques currently available for determining the number of functional LDL recep- From the Atherosclerosis Prevention Center, the Departments of Internal Medicine and Biochemistry, University of Genoa, and the National Institute for Cancer Research, Genoa, Italy. This work was partially supported by CNR Progetto Finalizzato Oncologia Grant 85/ and by a Ministero Pubblica Istruzione grant. Address for correspondence: Stefano Bertolini, M.D., University of Genoa, Atherosclerosis Prevention Center, Department of Internal Medicine, Viale Benedetto XV,6, Genova, Italy. Received July 14, 1987; revision accepted July 8, tor molecules on cultured cells are not accurate enough to give unambiguous results. 678 The recent isolation of a cdna clone for the human LDL receptor gene has now made it possible to investigate the presence of restriction fragment length polymorphisms (RFLPs), which can be used for early diagnosis of FH. 9 Some RFLPs of the LDL receptor gene that might be used to follow the inheritance of the affected gene in informative families have been found. Thus, Humphries et al and Leppert et al. 12 have recently shown that the two alleles defined by a polymorphic Pvu II site located in the intron between exons 15 and 16 cosegregate with the disease in informative families. In the present study, we report the distribution of the Pvu II alleles in 112 normolipidemic Italian subjects ages 2 to 99 years old and in 79 FH patients from 37 different families. Methods Subjects The subjects were selected according to the following criteria: Normolipidemic controls had cholesterol levels <5.2 mmol/l with LDL cholesterol <3.5 mmol/l (younger than 30 years old); cholesterol levels s5.7 mmol/l with LDL cholesterol <4.0 mmol/l (30 years old or older); triglyceride levels si.8 mmol/l; negative family history of hyperlipidemias, coronary heart disease, or other clinical signs of atherosclerosis. FH patients had cholesterol levels s6.7 mmol/l with LDL >4.6 mmol/l (younger than 16 years old); cholesterol levels s7.5 mmol/l with LDL >5.2 mmol/l (16 years old or older); a total triglyceride level not exceeding 2 mmol/l; plus two of the following: tendon xanthomas in the proband or in one or more first 845

2 846 ARTERIOSCLEROSIS V O L 8, No 6, NOVEMBER/DECEMBER 1988 degree relatives, hypercholesterolemic children in the family, cholesterol levels s7.5 mmol/l in two or more family members, and family history of coronary heart disease in one or more first or second degree relatives younger than 50 years old. All the subjects gave their informed consent for this study kb 14 Laboratory Analyses DNA was prepared from 10 ml of fresh whole blood by a sodium dodecyl sulfate (SDS) lysis method.13 DNA was digested with the Pvu II restriction enzyme, according to the manufacturer's directions (Bethesda Research Laboratories, Gibco BRL, Scotland). DNA fragments were separated by size on a 0.8% agarose gel and were transferred to Gene Screen Plus filters (New England Nuclear, Du Pont, West Germany) by the Southern blotting technique.14 The LDL receptor probe, pldlr-2hh1, kindly provided by David W. Russell, is a 1.9 kb fragment (bp 1573 to 3486) of the 3' end of the LDL receptor cdna clone. The fragment was excised from a 1 % low melting agarose gel and was labeled by the nick translation technique with 32p(a)dCTP at 3000 Ci/mmol (Amersham International, England).13 5 x 106 cpm/ml of the probe was incubated with filters for 36 hours at 65 C in a 6x SSC, 1 % SDS, 5 x Denhart, 100 /ug/ml herring DNA solution. Filters were washed with 0.2 x SSC, 1% SDS at 65 C and exposed to X-ray film (Kodak Xomat AR) for 2 to 4 days at -80 C. Statistical Analyses Statistical comparison of allele frequencies was done by the chi-squared test. The polymorphism information content was calculated according to the method of Botstein et al Results As previously described, 1016 digestion with Pvu II generates three different fragment patterns in normal subjects (Figure 1). One fragment (3.6 kb) is common to all individuals, while the 16.5 and 14.0 kb derive from two different alleles (P1 and P2, respectively). The polymorphic Pvu II site has been located in the intron separating exon 15 and 16 and is due to an adenine-to-guanine transition in the right arm of the 5' Alu repeat of a cluster of two tandem Alu sequences.16 The frequencies of the two alleles were determined in 112 unrelated normolipidemic Italian individuals ages 2 to 99 years old (mean age 41.2 years) who had plasma cholesterol, 4.61 ±0.53 mmol/l; LDL cholesterol, 2.75±0.67 mmol/l; and triglyceride 0.92±0.39 mmol/l. Table 1 reports the observed frequencies and the distribution of genotypes, which are close to the expected values if the population is in Hardy-Weinberg equilibrium. Table 1 also reports a comparison with the frequencies observed in previous studies in different populations. It is evident that, in the Italian population, the two alleles are more evenly represented, since the frequency of allele P2 (0.402) was nearly twice that in the English population (0.226). Afrikaners17 are, in this respect, more similar to 2.5 Figure 1. Pvu II restriction fragment length polymorphism detected with the low density lipoprotein receptor probe, LDLR 2HH1. From left to right:,,. Italians than to English (0.346). Analysis of the allele frequencies with the chi-squared test showed that the difference between the Italian and English populations is statistically significant (#2=11.03, p<0.005). The Pvu II RFLP was then determined in 79 individuals with heterozygous FH from 37 different families, ages 3 to 66 years old (mean age 40.9 years) who had plasma cholesterol, 9.31 ±1.76 mmol/l; LDL cholesterol, 7.38± 1.79 mmol/l; triglyceride, 1.18±0.46 mmol/l. An abnormal restriction fragment, which was associated with the disease and which could not be assigned to either the P1 or

3 LDL RECEPTOR GENE POLYMORPHISM Daga et al. 847 FH2 FH3 FH5 1/1 1?1 1/1 1 1 I I I V - 1/1 1% 1/1 1#/1 2/2 1/1 171 FH6 B 1/1 172 FH9 jfc-6 l/ 2/2 A 6 4 FH17 T^ 2/2 1% 172 2/ /1 I FH32 \ jtf-po ^K3 FH28 i72 I 2/2 1T2 2/2 a 172 1/1 1?2 1/1 Normal male and female FH proband (heterozygous) * ^ Individual Ml Myocardial Infarction SD- Sudden Death FH 33 Q- 21 Q - s o OrBO 1/1 I 172 Figure 2. Cosegregation of the P1 allele with familial hypercholesterolemia in 17 families. 172 > 1 # /2 O B 0 0

4 848 ARTERIOSCLEROSIS VOL 8, No 6, NOVEMBER/DECEMBER 1988 Table 1. Comparison of Genotype Distribution and Allele Frequency of Pvu II Restriction Fragment Length Polymorphisms in Normoiipidemic Individuals from Different Populations Genotype distribution (n) Number of <illeles (frequency) P1 P2 134 (0.598) 90 (0.402) 96 (0.774) 28 (0.226) (0.764) (0.236) 85 (0.654) 45 (0.346) Italian (n=112) English (n=62) 1 American (n=19) 16 Afrikaner (n=65) Superscripts refer to references. Table 2. Comparison of Genotype Distribution and Allele Frequency of Pvu II Restriction Fragment Length Polymorphisms in Normoiipidemic and Familial Hyperchoiesterolemic Individuals Genotype distribution (n) Number of alleles (frequency) P1 P2 134(0.598) 90 (0.402) 55 (0.786) 15(0.214) 18 0 Normal controls (n=112) FH probands (n=35) FH=familial hyperchoiesterolemic. the P2 allele, was observed in two families. The frequencies of the two alleles were estimated in the remaining 35 unrelated patients. As reported in Table 2, FH was strongly associated with the P1 allele at a frequency of The genotype (57.1 % of probands, 58.9% of all patients) was more frequent than in the normal population (35.7%), while the was not found. Statistical analysis showed that the frequencies of the alleles in the FH proband group was significantly different from that of normal controls (* 2 =8.17, p<0.005). Only 25 of the 35 probands had a sufficient number of family members available for study. When the RFLP was used to follow the inheritance of the LDL receptor gene in these 25 FH families, the disease was found to unambiguously cosegregate with the P1 allele in 23 families (Table 3). Figure 2 shows the complete pedigree of seventeen families. Table 4 summarizes the independent haplotypes found in the 25 families. This finding had already been reported, 10 but was not considered significant because of the low frequency (20%) of the P2 allele in the English population and the small number (two) of families examined. In our study, with the observed frequency of P2 in the Italian population (0.402, see Table 2), one would expect that in 40% (10 of 23) of informative families the FH phenotype would segregate with P2. Discussion A number of recent reports indicate that the mutation in the LDL receptor gene in FH patients is not unique. 2 " 5 Thus, several different mutations, often involving the multitude of Alu sequences present in the gene, have been found in different families with FH. However, in most cases, even several restriction enzymes have failed to demonstrate any abnormality in the restriction fragments, either because the deletion involved was too small or because a point mutation was responsible for the defect. Thus, in our study, we found an abnormal restriction fragment inherited with the disease in only 2 of the 37 families examined (data not shown). Both mutations appeared in a preliminary characterization to be different from the ones already described. (These will be reported in detail elsewhere.) It seems unlikely then, that a restriction pattern characteristic of FH that could be used to unequivocally diagnose the disease can be found. Nevertheless, once the allele of the affected gene is identified, the RFLP generated by the polymorphic Pvu II site between exons 15 and 16 of the LDL receptor gene can be used to follow the gene's inheritance. This is especially useful in populations like the Italian and the Afrikaner, in which the two alleles are evenly represented. It is, in fact, possible to follow the inheritance of the defective allele from heterozygous patients (42.9% in our study) and thus unequivocally diagnose FH in early infancy in nearly one-third of the patients (Table 5). One interesting point is the association between the P1 allele and FH. The single point mutation generating the extra Pvu II site does not seem, by itself, to adversely affect the LDL receptor gene function, since the plasma concentrations of total cholesterol and LDL cholesterol were not significantly different among the,, and groups. Thus, the most likely explanation is that a mutation causing FH occurred on a chromosome that lacked the cutting site for Pvu II, and that this mutation makes a significant contribution to the pool of mutation causing FH in the Italian population. This would suggest the existence of a founder effect as is seen in the Afrikaner population. 18 Although Pvu II identifies an individual at risk in only 32.5% of FH cases, it is certainly a step toward an early and unequivocal diagnosis of this disease. If different RFLPs of the LDL receptor that can be used to split the Pvu II alleles are identified, it will be possible to more accurately follow the inheritance of the disease and to eventually offer an early diagnosis in most, if not all, families with FH. Some promising RFLPs have, indeed, been recently reported with Ava II, Apa LI, and NCO

5 LDL RECEPTOR GENE POLYMORPHISM Daga et al. 849 Table 3. Cosegregation of Familial Hyperchoiesterolemia with P1 Allele in 25 Families Parent Offspring Family FH2 FH3 FH5 FH6 FH7 FH9 FH10t FH11f FH13 FH14 FH15 FH17 FH20 FH21 FH22 FH24 FH25 FH27 FH28 FH29 FH31 FH32 FH33 FH34 FH35 Affected (sister) (sister) (brother) (sister) (cousin) (cousin) (sister) (sister (sister) (sister) (brother) (sister) Unaffected Affected (1), (1) (1) (1)* (1) (1),(1) (1)* (1)* (2) (1) (2) (2) (3)* (1) (1)* (2) (1) (1) (1) (1) (1) (1) (1) (1), (1) (1), (1) (1)* Unaffected (1) (1) (3) (2)* (1) (1)* (1) (1) (2)* (1) (1), (1) (1)* (1)* (1) The number in parenthesis is the number of subjects. 'Cases in which Pvu II RFLP allows unequivocal diagnosis of health or disease without knowledge of plasma cholesterol level. tambiguous families. Acknowledgments We thank David W. Russell, Michael Brown, and Joseph Goldstein for supplying the LDL receptor probe. Table 4. Families D-P1 23 Independent D-P2 D-? 0 2 Haplotypes d-p1 58 D=the disease allele; d=the normal allele. Observed d-p2 39 in 25 d-? 26 Table 5. Probability that Offspring Are Informative for Familial Hyperchoiesterolemia in Italian Population Genotype frequency Genotype frequency Frequency Informative offspring of affected parent of unaffected mate of mating Frequency (0.571) All genotypes (1) (0.428) (0.357) (0.428) (0.482) (0.428) (0.161) Total Polymorphism information content value=

6 850 ARTERIOSCLEROSIS VOL 8, No 6, NOVEMBER/DECEMBER 1988 References 1. Goldstein JL, Brown MS. The LDL receptor defect in familial hypercholesterolemia. Med Clin Am 1982;66: Lehrman MA, Goldstein JL, Brown MS, Russell DW, Schneider W. Internalization-defective LDL receptors produced by genes with nonsense and frameshift mutations that truncate the cytoplasmic domain. Cell 1985;41: Lehrman MA, Russell DW, Goldstein JL, Brown MS. Exon-Alu recombination deletes 5 kilobases from the low density lipoprotein receptor gene, producing a null phenotype in familial hypercholesterolemia. Proc Natl Acad Sci USA 1986;83: Lehrman MA, Schneider WJ, Sudhof TC, Brown MS, Goldstein JL, Russell DW. Mutations in LOL receptor: Alu-Alu recombination deletes exons encoding transmembrane and cytoplasmic domains. Science 1985;227: Horsthemke B, Kessllng AM, Seed M, Wynn V, Williamson R, Humphries SG. Identification of a deletion in the low density lipoprotein receptor gene in a patient with familial hypercholesterolemia. Hum Genet 1985;71: Lees AM, Lees RB. Low density lipoprotein degradation by mononuclear cells from normal and dyslipoproteinemic subjects. Proc Natl Acad Sci USA 1983;80: Johansen KB, Faergeman O, Andersen GE. Serum lipoprotein and lymphocyte LDL receptor studies in parents and children with heterozygous familial hypercholesterolemia. Scand J Clin Lab Invest 1982;42: Cuthbert JA, East CA, Bilheimer DW, Lipsky PE. Detection of familial hypercholesterolemia by assaying functional low density lipoprotein receptors on lymphocytes. N Engl J Med 1986:314: Yamamoto T, Davis CG, Brown MS, et al. The human LDL receptor: a cystein-rich protein with multiple Alu sequences in its mrna. Cell 1984;39: Humphries SE, Kessling AM, Horsthemke B, et al. A common DNA polymorphism of the low density lipoprotein (LDL) receptor gene and its use in diagnosis. Lancet 1985; 1: Humphries SE. Familial hypercholesterolemia as an example of early diagnosis of coronary artery disease risk by DNA techniques. Br Heart J 1986;56: Leppert MF, Hasstedt SJ, Holm T, et al. A DNA probe for the LDL receptor gene is tightly linked to hypercholesterolemia in a pedigree with early coronary disease. Am J Hum Genet 1986:39: Maniatis T, Frltsch EF, Sambrook J. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, 1982: Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975; 98: Botstein D, White RL, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphism. Am J Hum Genet 1980;32: Hobbs HH, Lehrman MA, Yamamoto T, Russell DW. Polymorphism and evolution of Alu sequences in the human low density lipoprotein receptor gene. Proc Natl Acad Sci USA 1985;82: Brink PA, Steyn LT, Bester AJ, Steyn K. Linkage disequilibrium between a marker on the LDL receptor and high cholesterol levels. South Afr Med J 1986:70: Brink PA, Steyn LT, Coetzee GA, Van der Westhuyzen DR. Familial hypercholesterolemia in South African Afrikaners. Pvu II and Stu I DNA polymorphisms in the LDLreceptor gene consistent with a predominating founder gene effect. Hum Genet 1987;77: Leitersdorf E, Hobbs HH. Human LDL receptor gene: two Apa LI RFLPS. Nucleic Acid Res 1987;15: Hobbs HH, Esser V, Russell DW. Ava II polymorphism in the human LDL receptor gene. Nucleic Acid Res 1987; Kotze MJ, Langenhoven E, Dietzsch E, Retief AE. A RFLP associated with the LDL receptor gene. Nucleic Acid Res 1987;15:376 Index Terms: LDL receptor gene familial hypercholesterolemia DNA polymorphism preventive medicine