© 1997 American Heart Association, Inc.
Articles |
No Evidence of Linkage Between Familial Combined Hyperlipidemia and Genes Encoding Lipolytic Enzymes in Finnish Families
From the National Public Health Institute, Department of Human Molecular Genetics (P.P., M.P., A.-C.S., L.P.); the Department of Medicine, University of Helsinki (K.V.K.P., M.-R.T., L.S., A.-T.L.); the National Public Health Institute, Department of Biochemistry (M.A., S.M.-R., S.E., C.E.); and the Department of Medicine, University of Turku (I.N., J.S.A.V.), Finland, part of the EUFAM group.
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Abstract |
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Abstract Familial combined hyperlipidemia (FCHL) is characterized by different lipid phenotypes (IIa, IIb, IV) and elevated apolipoprotein B (apo B) levels in affected family members. Despite intensive research, the genes involved in the expression of this complex disorder have not been identified, probably because of problems associated with phenotype definition, unknown mode of inheritance, and most probably genetic heterogeneity. To explore the genetics of FCHL in the genetically homogeneous Finnish population, we collected 14 well-documented Finnish pedigrees with premature coronary heart disease and FCHL-like dyslipidemia. The lipolytic enzymes lipoprotein lipase (LPL), hepatic lipase (HL), and hormone-sensitive lipase (HSL) were selected as initial candidate genes because of their central roles in apo B and triglyceride metabolism. On the basis of the pedigree structures, a dominant mode of inheritance was adopted for linkage analyses, and serum total cholesterol and/or triglyceride levels exceeding the 90th percentile level were set as diagnostic criteria (criterion 1). In pairwise linkage analyses with intragenic markers, no evidence for linkage was found. Instead, the significantly negative LOD scores suggested exclusion of all three loci for single major gene effect. LOD scores were -14.63, -5.03, and -5.70 for the three LPL polymorphisms (
=0.00); -9.40, -6.30, and -4.74 for the three HL polymorphisms (=0.00); and -15.29 for the HSL polymorphism (=0.00). The results were very similar when apo B levels over the 90th percentile were used as criteria for affected status (criterion 2). Also, when linkage calculations were carried out using an intermediate or recessive mode of inheritance, the results of pairwise linkage analysis remained negative. Furthermore, when haplotypes were constructed from multiple polymorphisms of the LPL and HL genes, no segregation with the FCHL phenotype could be observed in the 14 Finnish families. Data obtained by the affected sib-pair method supported these findings, suggesting that the LPL, HL, or HSL genes do not represent major loci influencing the expression of the FCHL phenotype.
Key Words: familial combined hyperlipidemia • lipolytic enzymes • linkage analysis • genetics • complex disease
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Introduction |
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Familial combined hyperlipidemia, first described more than 20 years ago,1 2 is considered to be one of the most frequent genetic dyslipidemias associated with premature CHD.3 Because of the lack of specific biochemical or genetic markers, the diagnosis of FCHL has traditionally been based on detailed analysis of the lipid status in individual family members. According to FCHL family criteria, different lipid phenotypes (IIa, IIb, IV) and elevated apo B levels should exist in affected family members.1 4 In addition, at least one first-degree relative should have a different lipoprotein phenotype than the proband.
The number of genes influencing FCHL has been estimated by complex segregation analysis. Evidence for one or two major loci affecting serum triglyceride levels5 and for heterogeneity has been presented.6 Difficulties in defining the complicated individual FCHL phenotype, especially without detailed analyses of all family members, also suggest that FCHL is a complex disorder with a heterogenic background. In most earlier studies, FCHL has been defined as a dominant disease with an age-dependent penetrance1 or with full penetrance already in childhood.7 8 However, as long as the genes behind FCHL remain unknown, neither the mode of inheritance nor the penetrance of FCHL is unambiguously clear.
No definite metabolic defect has been identified in FCHL, although hepatic overproduction of apo B and prolonged postprandial lipemia have been suggested as possible explanations.9 10 11 12 Consequently, any gene encoding a protein participating in cholesterol and triglyceride metabolism can be considered a candidate gene. One suggestive linkage result has been obtained to the apo A-I/C-III/A-IV gene complex,13 although this result was not confirmed in another study.14
We used the special advantages of the genetically homogeneous Finnish population to pursue the genetic background of FCHL. The family material from the isolated population and strict criteria for FCHL should result in selection of a FCHL phenotype with high genetic influence. Fourteen Finnish FCHL pedigrees with both angiographically confirmed premature CHD and FCHL-like dyslipidemia were chosen for the study. The genes encoding the lipolytic enzymes LPL, HL, and HSL were selected as candidate genes because of the indicative findings of earlier studies15 16 17 18 19 20 21 and their central roles in apo B and triglyceride metabolism. No evidence for linkage to any of these genes could be established, and our data actually excluded these lipolytic enzymes as the primary cause of FCHL in Finnish families, although the possible genetic heterogeneity has to be taken into account when evaluating these results in other, more mixed populations.
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Methods |
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Subjects
The aim of the data collection was to obtain large, carefully documented pedigrees with both premature CHD and FCHL-like dyslipidemia. The patient recruitment was done in the Helsinki and Turku Central University Hospitals in Finland. The study design was approved by the ethical committees of the participating centers, and each subject gave oral informed consent before participating in the study. By August 1995, 14 FCHL families had been identified.
The data collection consisted of three phases. In the first phase, probands were selected prospectively from patients undergoing elective coronary angiography for clinically suspected coronary artery disease. In addition, three probands were selected from an angiography register. Inclusion criteria for the proband were the following: age 30 to 55 years for men and 30 to 65 years for women, 
50% stenosis in one or more coronary arteries, serum total cholesterol and/or serum triglycerides higher than or equal to that of the age- and sex-specific 90th percentile, and at least five accessible first-degree relatives. Exclusion criteria for the proband were type I diabetes, hypothyreosis, and hepatic or renal disease. In the second phase, the proband and all first-degree relatives were examined. Families with at least two different lipid phenotypes (IIA, IIB, or IV) in affected members were included in the final phase, in which all accessible relatives and their spouses were examined. The pedigrees of the families are shown in the Figure
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Biochemical Analyses
For each family member >5 years old, blood was drawn for the measurement of serum lipids and for DNA isolation. For members >15 years old, blood pressure was measured, abdominal distribution of body fat was estimated by measurement of waist and hip circumferences, weight and height were recorded, BMI was calculated as weight/height2, and standard questionnaires were completed for the estimation of smoking and drinking habits, previous medical history, and medication. Subjects using lipid-lowering agents were asked to interrupt the medication for 4 weeks before blood sampling. The lipid measurements were performed in the scientific laboratory of the Helsinki University Central Hospital. Blood samples were taken after 12 hours of overnight fast. Serum samples were stored frozen at -70°C until analyzed. Serum total cholesterol and triglycerides were determined with automated enzymatic methods.22 Serum apo B was measured by an immunochemical assay (Orion Diagnostica). The apo B measurements in the FINMONICA study, which were used as reference values (see below), were performed with apo B antiserum and a Cobas instrument (Roche). The two methods used were compared, and the difference in levels was estimated by linear regression analysis on a small subsample (n=211) of FINMONICA. After evaluation and omission of possible outliers, the difference was corrected accordingly. The lipoprotein fractions VLDL, d<1.006; IDL, 1.006<d<1.019; LDL, 1.019<d<1.063; HDL2, 1.063<d <1.125; and HDL3, 1.125<d<1.210 were separated by sequential flotation in an ultracentrifuge as described.23 Apo E phenotyping was done in serum by isoelectric focusing.24 Blood glucose concentrations were measured by the glucose dehydrogenase method (Gluc-DH, Merck). Serum free insulin concentrations were determined by a radioimmunoassay with the Phadeseph Insulin RIA kit (Pharmacia). Familial hypercholesterolemia was excluded from each pedigree by determining the LDL-receptor status of the proband by the lymphocyte culture method.25
The lipid criteria used for classification of study subjects 25 to 60 years old were derived from FINMONICA, a large population-based survey done in 1992.26 Polynomial regression analyses were used to assess the influence of age on total cholesterol, triglyceride (n=6022), and apo B (n=1197) values in both sexes separately. The best model for each analysis was chosen on the basis of Mallow's Cp statistics. With these models, the whole population was adjusted to the age of 25, and the 90th percentile points were determined. These percentile points were then extrapolated to the other age groups. In these analyses, the triglyceride values were loge transformed. The FINMONICA data were confined to subjects <65 years old, and therefore, percentile values for 60- to 65-year-olds were applied to the whole age group >60 years old. There was no possibility to use Finnish data for calculating the percentile levels for persons >65 years old because of the lack of a large population-based survey in this age group. Therefore, in the linkage calculations, we allowed 0.9% phenocopies and used the penetrance of 90% to avoid the possible slight bias due to this estimation of percentile levels used for persons >65 years old (details of these parameters used in the linkage calculations are given in the first paragraph of the "Results" section). The fractile cutpoints for subjects <25 years old were derived from the Cardiovascular Risk Factors in Young Finns study follow-up samples of 1986 and 1992 (n=2236).27 For classification, the highest untreated lipid measurement was used.
Statistical analyses of clinical parameters were performed with version 6.10 of the SAS software28 and version 6.1 of the SPSS for Windows software.29 The probability values were calculated under the assumption of potential two-sided differences. Differences between groups were assessed with a 
2 test for categorical variables and either with the Kruskal-Wallis one-way nonparametric ANOVA or parametric ANOVA for continuous variables. The data collection is ongoing, and the present study is a pilot study for the large European Multicenter Study on Familial Dyslipidemias (EUFAM Study) launched in 1996.
DNA Markers
Of the three analyzed intragenic polymorphisms in the LPL gene, the first was a highly polymorphic (heterozygosity, 0.84) dinucleotide repeat marker with 8 alleles,30 31 the second a biallelic HindIII polymorphism in intron 8,32 and the third a biallelic PvuII polymorphism in intron 6.33 The HL gene was studied with three intragenic polymorphisms: one dinucleotide repeat polymorphism with 5 alleles (heterozygosity, 0.52)34 and two single nucleotide variations in codons 13335 and 45736 of exons 4 and 8, respectively. For HSL, a highly polymorphic intragenic dinucleotide repeat marker with 12 alleles (heterozygosity, 0.82)37 was analyzed. The allele frequencies for the HindIII and PvuII polymorphisms and for the two biallelic HL polymorphisms were determined by genotyping 85 healthy Finnish volunteers, none of whom were related to any of the 14 FCHL families. The allele frequencies for all the dinucleotide polymorphisms were estimated by genotyping 40 control samples unrelated to study subjects.
Genotyping
DNA was isolated according to the method of Vandenplas et al,38 with minor modifications. The primers used for amplification of the polymorphic DNA fragments and for the solid-phase minisequencing assay are shown in Table 1. The oligonucleotides were synthesized on an Applied Biosystems 381A DNA synthesizer. One of the primers for amplification of the microsatellite markers was labeled with fluorescein during synthesis using the Fluor prime reagent (Formica). The 5' end of one of the primers for amplification of the single-nucleotide polymorphisms was biotinylated during its synthesis with a biotinyl phosphoramidite reagent (Amersham). The polymorphic DNA fragments were amplified by PCR39 at optimized conditions in microtiter plate wells. The single-nucleotide polymorphisms were analyzed by the solid-phase minisequencing method.40 In this method, variable nucleotides are identified by a single-nucleotide primer extension reaction catalyzed by a DNA polymerase on a solid support. The PvuII polymorphism was detected by digestion of a PCR product covering this region with PvuII enzyme (Biolabs). The digested reaction products were separated on a 1.5% agarose gel. The electrophoresis separating the dinucleotide alleles of different sizes was carried out with an automated laser fluorescence DNA sequencer (Pharmacia) with 6% Hydrolink gels.41 Two internal size standards produced by PCR were included in each lane. The alleles were identified by the Fragment Manager (Pharmacia) conversion program and the ALP software (A. Brown, MRC Human Genetics Unit, Edinburgh, Scotland).
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Linkage Analysis
The linkage analyses were computed with the Linkage package programs (version 5.1, updated by J. Ott)42 43 and the POLYPREP program44 with FASTLINK, version 2.3P.42 45 46 47 These programs calculate the overall likelihood of the data on two alternative assumptions: first, that the two loci are linked with the given recombination fraction (
), and second, that they are not linked. The logarithm to the base 10 of the ratio of these two likelihoods is the LOD score. Then, the most likely distance between two loci is the recombination fraction () at which the LOD score peaks. An odds ratio of >1000:1 in favor of linkage (expressed on a logarithmic scale as an LOD score of >3.0) is considered a statistically significant demonstration of linkage in monogenic autosomally inherited disorders in humans. For a complex disorder with a heterogenic and/or polygenic background, the estimation of a critical value for a positive LOD score is rather difficult. Generally, an LOD score of >3.6 would be needed to support linkage.48 However, in the present study we performed the calculations using six models (two affection status models each with dominant, intermediate, and recessive modes of inheritance), and thus, a correction of the LOD score has to be performed according to the following formula49 : c=3+log10(n), where c is the correction coefficient and n is equal to the number of models used in the calculations. Thus, if n=6, the LOD score has to be 3.78 to be considered in favor of linkage. On the other hand, an odds ratio of <1:100 (LOD score more negative than -2.0) is agreed to indicate the exclusion of linkage in traditional human genetics. The program MLINK of the Linkage package was used to calculate the two-point linkage analysis between FCHL and each intragenic marker of LPL (three markers), HL (three markers), and HSL (one marker) and between FCHL and the combined intragenic LPL and HL marker haplotypes. The recombination fraction between the three LPL markers was assumed to be zero, because no recombination events were observed in the LPL haplotypes of 14 FCHL families and because the markers are all located within the LPL gene. The same assumption was used for all three HL markers. The program POLYPREP,44 which contains the program HOMOG, was run to test the possible heterogeneity of FCHL. The program HOMOG analyzes the odds for genetic heterogeneity that underlie a disease with respect to a single marker locus. The program POLYPREP44 also contains the SIBPAIR program, which was used to perform the sib-pair analysis on nuclear families. In affected sib-pair analysis, two sibs show identical-by-descent sharing for zero, one, or two copies of any locus, with a 25%-50%-25% distribution expected under random segregation. Excess allele-sharing can then be measured with a simple 2 test.50 51 52 53
The linkage analyses were calculated under the assumption of three modes of inheritance (dominant, intermediate, and recessive). For the affection status, we used two different diagnostic criteria. In the first (criterion 1), we used total cholesterol and/or triglyceride values over the age- and sex-specific 90th percentile in the determination of affection status. To avoid false exclusions, we considered it genetically safer to code subjects with only apo B levels over the 90th percentile but with normal cholesterol and triglyceride levels as unknown, rather than healthy, in criterion 1. Worth noting is that the number of these subjects was only 10 in the calculations. We also made the calculations with criterion 1 by coding these 10 only apo B–positive subjects as healthy, which represents the traditional FCHL criteria. The results were equally negative. These data are not included in the present article but are available upon request (P.P.). In the second model (criterion 2), we considered only subjects with apo B levels over the 90th percentile to be affected so as to clarify the role of factors influencing apo B alone in the genetics of FCHL, and subjects with only elevated total cholesterol and/or triglycerides but with normal apo B concentrations were coded as unknown. If a spouse of a family member in the second generation in any of the 14 families was also affected according to the lipid criteria, the offspring of these two were not included in the calculations to avoid bilineal introduction of the trait.
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Results |
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Table 2
shows the clinical parameters on the family members by criterion 1. In addition to lipid levels already higher by definition, the affected group also exhibited higher glucose (P=.019), insulin (P=.008), and BMI (P=.001) values than the unaffected group. The number of diabetic individuals was 12 of the 61 affected family members and 3 of the 53 unaffected ones (P=.027). With regard to obesity (BMI 28), the numbers were 22 of 61 and 5 of 53 (P=.001), respectively. The affected individuals did not smoke (P=.42) or drink (P=.62) more than the unaffected ones. To exclude other confounding effects, the percentages of apo E phenotypes in the affected and unaffected groups were determined: E2/2, 0.00/0.00; E3/2, 4.74/0.00; E4/2, 2.38/5.71; E3/3, 45.24/62.86; E4/3, 45.24/25.71; and E4/4, 2.38/5.71 (P=.21).
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Although the familial hypercholesterolemia was initially excluded by testing the probands by the lymphocyte culture method, all patients were examined, and none of them had tendon xanthomata. Furthermore, the presenting mean cholesterol level of the probands was 6.58 mmol/L (SD, 1.09; range, 5.00 to 8.40), which is lower than cholesterol levels typically seen in familial hypercholesterolemia patients.
The prevalence of FCHL in Finland is not precisely known. We used the gene frequency of 0.3% for the dominant model and 0.6% and 7.5% for intermediate and recessive models, respectively, on the basis of the proposed FCHL prevalence of 1.0% to 2.0%.1 54 Furthermore, the calculations used the phenocopy probability of 0.9% with all modes of inheritance. We estimated the penetrance of FCHL by calculating the number of affected siblings divided by the total number of siblings multiplied by 2 in each generation. If a spouse of a family member in the second or third generation in any of the 14 families was also affected according to the lipid criteria, the offspring of these two were not included in the calculations of the penetrance to avoid bilineal introduction of the trait. When we used criterion 1 (cholesterol and/or triglycerides over the age- and sex-specific 90th percentile), the penetrances in generations I, II, III, and IV were 125% (62.50), 109% (54.41), 100% (50.00), and 50% (25.00) and with criterion 2 (apo B over the 90th percentile), 133% (66.67), 109% (54.55), 58% (29.03), and 0% (0.00), respectively. The percentage of affected subjects in every generation is given in parentheses. Worth noticing, however, is that the number of subjects in generation IV was limited: only four with criterion 1 and one with criterion 2. To be cautious, we adopted values slightly lower than the observed penetrance for individuals <20 years old even with criterion 1. We used a penetrance of 70% (criterion 1) and 60% (criterion 2) among family members <20 years old and 90% (with both criteria) among all subjects >20 years old in the calculations.
In our highly selected material of 14 Finnish FCHL families, the segregation of the trait closely resembled the dominant type of inheritance (Figure
). However, we performed the linkage calculations under three modes of inheritance (dominant, intermediate, and recessive) to allow for the different inheritance pattern for individual genes in this potentially polygenic trait. The pairwise linkage analysis under the dominant mode of inheritance resulted in the exclusion of all three loci for single major gene effect in this material (Tables 3 and 4). The results of all families of pairwise linkage analysis between FCHL and the LPL polymorphisms were -14.63 (dinucleotide repeat polymorphism), -5.03 (HindIII), and -5.70 (PvuII) at the recombination fraction ()=0.00 by criterion 1. With the HL gene, the dominant model using criterion 1 gave total LOD scores of -9.40 for the dinucleotide repeat polymorphism, -4.74 for the variable nucleotide in codon 133, and -6.30 for the variable nucleotide in codon 457 (=0.00). The result of all families with the dinucleotide repeat polymorphism of the HSL gene was -15.29 (=0.00) by criterion 1. Also, the linkage data on individual families revealed that none of the individual families produced significantly positive LOD scores, although the uninformativeness of the marker, especially of the four biallelic LPL and HL markers, resulted in so many homozygotes in some families that the results of these individual families remained uninformative with these particular markers (Table 3). When we used criterion 2 in the calculations, the LOD scores obtained were similar to the results with criterion 1 at the recombination fraction 0.00 (Table 4). Furthermore, at the recombination fraction 0.05 (a distance of 
5x106 bp), the pairwise linkage analyses of the 14 families with all polymorphisms and both criteria still resulted in LOD scores more negative than the critical value 
-2, considered to be the exclusion criteria in monogenic diseases (Tables 3 and 4). At all other recombination fractions (=0.05, 0.10, and 0.20), the total LOD scores remained below zero by both diagnostic criteria (data not shown).
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Adoption of an intermediate or recessive mode of inheritance created no significant change in the results obtained. At the recombination fraction =0.00, under a recessive mode of inheritance the LPL markers in all families produced the two-point LOD scores of -11.15 (AC repeat), -2.19 (HindIII), and -5.00 (PvuII) by criterion 1. With criterion 2, the total LOD scores obtained remained more negative than -3. Neither did any of the individual families produce significantly positive LOD scores by criteria 1 and 2. With regard to the intermediate mode of inheritance with the LPL polymorphisms, the total LOD scores of all families obtained by criterion 1 were -7.26, -0.73, and -2.43, and with criterion 2, -7.76, -2.16, and -2.57, respectively. Thus, the LOD score for the HindIII polymorphism and FCHL remained inconclusive (-0.73) by criterion 1 and the intermediate mode of inheritance. This was due to the uninformativeness of this biallelic marker with low heterozygosity (0.38) combined with the lower penetrances of 0.45 (for subjects >20 years old) and 0.35 (for subjects <20 years old) for heterozygous individuals for the disease locus used in the intermediate mode of inheritance, because none of the individual families produced significant evidence for linkage between this marker and FCHL in any of the individual families 1 to 14. The LOD scores of the families 1 to 14 at the recombination fraction 0.00 were 0.36, -0.07, -0.19, 0.18, 0.01, 0.33, 0.12, -0.01, -0.01, -0.52, -0.00, -0.02, -0.07, and -0.82. For the HL polymorphisms, the LOD scores of all families by the recessive pattern of inheritance remained more negative than -3 (criterion 1) and more negative than -2 (criterion 2). For the intermediate mode of inheritance, the two-point LOD scores of all families for the HL polymorphisms were also negative (all more negative than -2) by criterion 1. The results were very similar by criterion 2 (-1.85, -2.00, and -2.09). The LOD scores of the individual families 1 to 14 producing this slightly inconclusive total LOD score of -1.85 at =0.00 were -0.14, -0.58, -0.02, -0.18, 0.03, -0.95, -0.25, 0.07, 0.00, 0.14, -0.01, 0.01, -0.01, and 0.00. Thus, none of the families showed positive evidence for linkage, uninformative LOD scores simply reflecting the uninformativeness of this biallelic marker. Neither did the LOD scores of individual families show any evidence for linkage between the other HL polymorphisms and FCHL (criteria 1 and 2) with both intermediate and recessive modes of inheritance. The results of two-point linkage analysis between FCHL and the HSL AC-repeat polymorphism were more negative than -6 (criterion 1) and more negative than -3 (criterion 2) with the intermediate and recessive modes of inheritance (=0.00). Furthermore, the results of two-point linkage analysis in individual families revealed no evidence for linkage between the HSL gene and FCHL.
The possible heterogeneity of FCHL was tested with every marker of the LPL, HL, and HSL genes with all three modes of inheritance and both diagnostic criteria by use of the program HOMOG. No evidence for locus heterogeneity emerged from these analyses.
To improve the informativeness in the segregation analyses, we constructed the chromosomal haplotypes using three intragenic markers for both LPL and HL (Figure), which revealed no recombination events in the 14 FCHL families analyzed. As expected from data obtained with individual markers, no cosegregation of the haplotype and the disease phenotype could be observed in any of the families (Figure). In two-point linkage analyses between FCHL and the haplotypes of LPL and HL markers, the LOD scores were lower than in pairwise analyses with individual markers using the dominant model (Table 5). At the recombination fraction 0.00, the LOD score was more negative than -16 with LPL haplotypes and more negative than -11 with HL haplotypes by both criteria (=0.00). Again, the results of the individual families showed that none of the families provided evidence for linkage, although the results of the HL haplotypes in families 1 and 12 remained uninformative because of uninformativeness of the haplotypes in these families (Table 5 and Figure). Because the defects in LPL are known to produce mixed hyperlipidemia, we also investigated within families whether members with type IIa hyperlipidemia inherited LPL alleles different from those with type IIb or IV hyperlipidemia. This was seen only in families 4 (haplotype C), 9 (haplotype C), and 11 (haplotype B). However, in families 4 and 9, the same haplotype was detected in healthy family members as well, and in family 11 there were only two affected subjects.
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The total number of affected sib-pairs in these 14 families was 38 by criterion 1 and 32 by criterion 2. The results of the sib-pair analyses supported the data obtained in linkage analyses. With the LPL gene, the two probability values (criteria 1 and 2) of the 2 test for estimated shared versus not-shared alleles between the affected sibs were P=.50 (0.50; 15.5, 20.5) and P=.50 (0.50; 12.8, 19.9) for the AC repeat, P=.15 (0.62; 13.7, 8.3) and P=.50 (0.50; 9.3, 10.9) for HindIII, and P=.50 (0.50; 10.6, 13.3) and P=.50 (0.50; 8.4, 12.1) for PvuII, respectively (mean identical-by-descent for a sib-pair and the calculated numbers of alleles shared versus not shared between the affected sibs are given in parentheses after the probability values). The three HL polymorphisms yielded values of P=.50 (0.50; 9.0, 13.2) and P=.50 (0.50; 9.3, 11.4) for the variable nucleotide in codon 133, P=.28 (0.56; 9.6, 10.0) and P=.50 (0.50; 9.3, 11.3) for the variable nucleotide in codon 457, and P=.11 (0.60; 18.1, 14.7) and P=.33 (0.54; 15.0, 13.9) for the AC repeat with the diagnostic criteria 1 and 2. The AC-repeat polymorphism of the HSL gene exhibited values of P=.50 (0.50; 18.0, 17.5) and P=.32 (0.54; 16.8, 13.7). These data indicate that there is no excess allele-sharing observable in disease chromosomes with any of the analyzed intragenic polymorphisms.
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Discussion |
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Studies aiming to solve the complex metabolic and genetic background of FCHL have been ongoing for >20 years. The most interesting findings have concerned LPL and HSL. HSL has a key role in the regulation of lipolysis in adipocytes by catalyzing the breakdown of triglycerides to glycerol and free fatty acids.55 A recent study21 suggested that in FCHL there is a lipolytic defect due to impaired function of HSL. To the best of our knowledge, the present study represents the first genetic approach to clarify the potential role of the HSL gene in carefully documented FCHL families with both angiographically confirmed premature CHD and FCHL-like dyslipidemia. Our criterion 1 corresponds to the lipid criteria used for the 10 Swedish FCHL subjects in the study by Reynisdottir and coworkers.21 However, neither linkage nor excess allele-sharing was found between the 14 Finnish FCHL families and the HSL gene in the present study. How to interpret this result? One possibility is that there are earlier abnormalities in the lipolytic cascade at the level of adenyl cyclase or cAMP that are more important than a defect in HSL. Also, altered protein kinase function might cause the decreased activity. Conversely, the defect in lipid hydrolysis catalyzed by HSL21 could be a secondary phenomenon in FCHL and reflect a defect in triglyceride synthesis via the acylation-stimulating protein pathway.56 The possibility also remains that the HSL gene is one of the important loci for FCHL in the Swedish population but not in these Finnish pedigrees.
One third of the subjects with FCHL have decreased LPL activity,16 suggesting that the subjects with LPL deficiency could contribute to the FCHL phenotype and that the LPL mutations influencing catalytic activity could be related to the FCHL phenotype. Earlier studies have suggested that mutations in the coding sequence or in the promoter of the LPL gene do not represent a common cause of FCHL.17 19 57 Our study also suggests that LPL is not one of the major genes influencing the expression of FCHL in our study population. In any case, because of its important function in catalyzing the hydrolysis of triglyceride-rich lipoproteins, LPL could still represent a minor or modifying gene behind FCHL. The results of a recent association study would suggest this by revealing that the frequency of the Asn291Ser mutation in the LPL gene was more than two times higher in 169 unrelated FCHL patients than in 215 male control subjects (11.8% versus 4.6%).18 Nevertheless, when the major genetic factors and the gene-environment interactions in FCHL are not known, it is difficult to clarify the true significance of a particular LPL mutation with a low frequency in FCHL subjects when the mutation can also be found in the control population. To be able to evaluate the possible role of LPL in FCHL, the major loci involved in this complex disorder should be identified first.
HL participates in the conversion of IDL to LDL, in chylomicron remnant catabolism, and in HDL metabolism in humans.58 59 Nevertheless, there is no consensus regarding the precise physiological function of HL. Recently, a preliminary study showed that the missense mutation Val73Met of the HL gene was overrepresented in 25 German FCHL subjects compared with controls.20 The sample size in this interesting study is too small to justify any definite conclusions. According to our data, however, the HL gene is not the major locus causing FCHL in the Finnish population.
According to the diagnostic FCHL family criterion, different lipid phenotypes (IIa, IIb, IV) as well as elevated apo B levels should be present within the family,1 4 which makes the definite determination of the affection status of individual family members relatively complicated and causes difficulties in distinguishing between FCHL and elevated apo B levels segregating in families. Our results obtained by two different diagnostic criteria were rather similar. This reflects the fact that the affection status for the majority of the cases in generations I and II remained the same with both criteria. In generation III, however, the percentage of affected family members was higher by criterion 1 than criterion 2 (50.00% versus 29.03%). This difference can represent the age-dependent penetrance of elevated apo B levels segregating in families, suggesting that apo B status should be defined independently in FCHL families.
Although in FCHL, the disease clearly shows familial aggregation, it is much more difficult than in simple monogenic traits to define the mode of inheritance without knowing the precise number of genes involved and the extent of the environmental influence. The Finnish population, which has remained genetically isolated for linguistic and geographic reasons for hundreds of years, may be advantageous in this situation.60 Because of this isolation, disease-causing mutations can be expected to originate from only a few ancestors, often only one,61 which we have also shown to hold for monogenic diseases.62 63 In the majority of disease chromosomes of currently living individuals, there tends to be the same distinctive haplotype on a relatively wide DNA region around the disease gene.64 65 66 Consequently, selected predisposing loci, enriched in this population, should possibly be easier to identify in Finland by a sparser marker map in the random genome search than in more mixed populations.
We wanted to further enrich the genetic form of FCHL by using strict and well-characterized criteria for the FCHL families and by collecting extended FCHL families, many of them having affected members in three generations. By deliberately combining the advantage of the Finnish genetic isolation with the strict and well-characterized FCHL criteria in extended FCHL families, we have obviously selected an extreme form of FCHL phenotype with high genetic influence. Using this study material, we hope to identify at least one major gene influencing the expression of the complex FCHL phenotype. In the statistical analyses of the genotypes, we used pairwise linkage analysis with three different modes of inheritance. Furthermore, to avoid the disadvantageous fact that linkage analysis is sensitive to the particular inheritance pattern selected for the disease, affected sib-pair analysis was also performed, because this method assumes no pattern for the inheritance of the disease.
There was no evidence for either linkage in pairwise linkage analysis or excess allele-sharing in affected sib-pair analysis between FCHL and the intragenic markers of the LPL, HL, or HSL genes. The only two LOD scores, both with intermediate mode of inheritance for the HindIII polymorphism of the LPL gene (criterion 1) and the variable nucleotide in codon 133 of the HL gene (criterion 2), that remained inconclusive (-0.73, -1.85) were simply due to uninformativeness of these biallelic markers with low heterozygosity and did not result from widely deviating LOD scores in the individual families. The intermediate mode of inheritance may be sensitive to producing inconclusive results with uninformative markers because of low penetrance of heterozygotes for the disease locus used in this model. The negative findings of the nonparametric sib-pair analysis also support this explanation. Furthermore, no LPL or HL haplotype was shared by the affected family members. Because the number of diabetic and obese family members was higher in the affected than in the unaffected group (P=.03 and P=.001), and taking into account the most likely genetic heterogeneity in FCHL,6 we also allowed for heterogeneity in our calculations to find out whether a subgroup of families would exist, for example due to this glucose tolerance or obesity parameters, that were linked to any of the lipolytic enzyme genes we studied. However, no significant LOD scores were found in these analyses either. Thus, our data would suggest that it is unlikely that the LPL, HL, or HSL genes are major genetic factors in FCHL, although the possible genetic heterogeneity has to be taken into account in evaluation of the significance of our results for FCHL in other, more mixed populations. Neither can we exclude a modifying or minor genetic effect of these loci, but the search for minor and modifying loci is justified only after the major loci have been identified. In the future, we aim to continue with other interesting candidate genes, for example with the apo A-I/C-III/A-IV gene complex and the putative locus for small, dense LDL in chromosome 19.67 Thereafter, we aim to initiate a genome-wide random search to identify major genes involved in FCHL in Finnish families.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by the Academy of Finland and the Finnish Heart Foundation. We wish to thank Drs Jurg Ott and Joseph Terwillinger for their computer programs and helpful comments concerning linkage and sib-pair analysis and Mari Sipilä and Liisa Ikävalko for sophisticated technical help. The primary genotype data on which the present report is based are available to other researchers upon request (P.P.).
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Footnotes |
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Reprint requests to Päivi Pajukanta, MD, National Public Health Institute, Department of Human Molecular Genetics, Mannerheimintie 166, FIN-00300 Helsinki, Finland.
Received May 10, 1996; accepted September 24, 1996.
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Goldstein JL, Schrott HG, Hazzard WR, Bierman EL, Motulsky AG. Hyperlipidemia in coronary heart disease, II: genetic analysis of lipid levels in 176 families and delineation of a new inherited disorder, combined hyperlipidemia. J Clin Invest. 1973;52:1544-1568.
2. Nikkilä EA, Aro A. Family study of serum lipids and lipoproteins in coronary heart disease. Lancet. 1973;1:954-959.[Medline] [Order article via Infotrieve]
3.
Genest JJ Jr, Martin-Munley SS, McNamara JR, Ordovas JM, Jenner J, Myers RH, Silberman SR, Wilson PWF, Salem DN, Schaefer EJ. Familial lipoprotein disorders in patients with premature coronary artery disease. Circulation. 1992;85:2025-2033.
4. Brunzell JD, Albers JJ, Chait A, Grundy SM, Groszek E, McDonald GB. Plasma lipoproteins in familial combined hyperlipidemia and monogenic familial hypertriglyceridemia. J Lipid Res. 1983;24:147-155.[Abstract]
5.
Cullen P, Farren B, Scott J, Farrall M. Complex segregation analysis provides evidence for a major gene acting on serum triglyceride levels in 55 British families with familial combined hyperlipidemia. Arterioscler Thromb. 1994;14:1233-1249.
6.
Jarvik GP, Brunzell JD, Austin MA, Krauss RM, Motulsky A, Wijsman E. Genetic predictors of FCHL in four large pedigrees: influence of apoB level major locus predicted genotype and LDL subclass phenotype. Arterioscler Thromb. 1994;14:1687-1694.
7. Cortner JA, Coates PM, Gallagher PR. Prevalence and expression of familial combined hyperlipidemia in childhood. J Pediatr. 1990;116:514-519.[Medline] [Order article via Infotrieve]
8. Cortner JA, Coates PM, Liacouras CA, Jarvik GP. Familial combined hyperlipidemia in children: clinical expression, metabolic defects, and management. J Pediatr. 1993;123:177-184.[Medline] [Order article via Infotrieve]
9. Chait A, Albers JJ, Brunzell JD. Very low density lipoprotein overproduction in genetic forms of hypertriglyceridaemia. Eur J Clin Invest. 1980;10:17-22.[Medline] [Order article via Infotrieve]
10. Kissebah AH, Alfarsi S, Adams PW. Integrated regulation of very low density lipoprotein triglyceride and apolipoprotein-B kinetics in man: normolipemic subjects, familial hypertriglyceridemia and familial combined hyperlipidemia. Metabolism. 1981;30:856-868.[Medline] [Order article via Infotrieve]
11. Castro Cabezas M, de Bruin TWA, Kock LAW, Kortlandt W, van Linde-Sibenius-Trip M, Jansen H, Erkelens DW. Simvastatin improves chylomicron remnant removal in familial combined hyperlipidemia without changing chylomicron conversion. Metabolism. 1993;42:497-503.[Medline] [Order article via Infotrieve]
12. Castro Cabezas M, de Bruin TWA, de Valk HW, Shoulders CC, Jansen H, Erkelens DW. Impaired fatty acid metabolism in familial combined hyperlipidemia: a mechanism associating hepatic apolipoprotein B overproduction and insulin resistance. J Clin Invest. 1993;92:160-168.
13. Wojciechowski AP, Farrall M, Cullen P, Wilson TME, Bayliss JD, Farren B, Griffin BA, Caslake MJ, Packard CJ, Shepherd J, Thakker R, Scott J. Familial combined hyperlipidaemia linked to the apolipoprotein AI-CIII-AIV gene cluster on chromosome 11q23-q24. Nature. 1991;349:161-164.[Medline] [Order article via Infotrieve]
14. Wijsman EM, Motulsky AG, Guo SW, Yang M, Austin MA, Brunzell JD, Deeb S. Evidence against linkage of familial combined hyperlipidemia to the AI-CIII-AIV gene complex. Circulation. 1992;86(suppl I):410. Abstract.
15.
Babirak SP, Iverius PH, Fujimoto WY, Brunzell JD. Detection and characterization of the heterozygous state for lipoprotein lipase deficiency. Arteriosclerosis. 1989;9:326-334.
16. Babirak SP, Brown G, Brunzell JD. Familial combined hyperlipidemia and abnormal lipoprotein lipase. Arterioscler Thromb. 1992;12:1176-1183.[Abstract]
17.
Gagne E, Genest J, Zhang H, Clark CA, Hayden MR. Analysis of DNA changes in the LPL gene in patients with familial combined hyperlipidemia. Arterioscler Thromb. 1994;14:1250-1257.
18.
Reymer PWA, Groenemeyer BE, Gagne E, Miao L, Appelman EEG, Seidel JC, Kromhout D, Bijvoet SM, van de Oever K, Bruin T, Hayden MR, Kastelein JJP. A frequently occurring mutation in the lipoprotein lipase gene (Asn291Ser) contributes to the expression of familial combined hyperlipidemia. Hum Mol Genet. 1995;4:1543-1549.
19.
Yang W-S, Nevin DN, Peng R, Brunzell JD, Deeb SS. A mutation in the promoter of the lipoprotein lipase (LPL) gene in a patient with familial combined hyperlipidemia and low LPL activity. Proc Natl Acad Sci USA. 1995;92:4462-4466.
20. Gehrisch S, Tesche R, Kostka H, Julius U, Jaross W. Point mutations in the hepatic triglyceride lipase (HTGL) gene in familial combined hyperlipidemia (FCHL). Circulation. 1995;92(suppl I):493. Abstract.
21. Reynisdottir S, Eriksson M, Angelin B, Arner P. Impaired activation of adipocyte lipolysis in familial combined hyperlipidemia. J Clin Invest. 1995;95:2161-2169.
22.
Syvänne M, Vuorinen-Markkola H, Hilden H, Taskinen M-R. Gemfibrozil reduces postprandial lipemia in non–insulin-dependent diabetes mellitus. Arterioscler Thromb. 1993;13:286-295.
23.
Taskinen M-R, Kuusi T, Helve E, Nikkilä E, Yki-Järvinen H. Insulin therapy induces antiatherogenic changes of serum lipoproteins in non–insulin-dependent diabetes. Arteriosclerosis. 1988;18:168-177.
24. Lehtimäki T, Moilanen T, Viikari J, Åkerblom HK, Ehnholm C, Rönnemaa T, Marniemi J, Dahlen G, Nikkari T. Apolipoprotein E phenotypes in Finnish youths: a cross-sectional and 6-year follow-up study. J Lipid Res. 1990;31:487-495.[Abstract]
25. Cuthbert JA, East CA, Bilheimer DW. Detection of familial hypercholesterolemia by assaying functional low-density-lipoprotein receptors on lymphocytes. N Engl J Med. 1986;314:879-883.[Abstract]
26.
Vartiainen E, Puska P, Jousilahti P, Korhonen HJ, Tuomilehto J, Nissinen A. Twenty-year trends in coronary risk factors in North Karelia and in other areas of Finland. Int J Epidemiol. 1994;23:495-504.
27. Porkka KVK, Viikari J, Rönnemaa T, Marniemi J, Åkerblom HK. Age and gender specific serum lipid percentiles of Finnish children and young adults: the Cardiovascular Risk in Young Finns study. Acta Paediatr. 1994;83:838-848.[Medline] [Order article via Infotrieve]
28. SAS Institute Inc. SAS/STAT User's Guide. Release 6.10 ed. Cary, NC: SAS Institute Inc; 1990.
29. Norusis MJ. SPSS for Windows: Base System Users Guide, Release 5.0. Chicago, Ill: SPSS Inc; 1992.
30. Tomfohrde J, Wood S, Schertzer M, Wagner MJ, Wells DE, Parrish J, Sadler LA, Blanton SH, Daiger SP, Wang Z, Wilkie PJ, Weber JL. Human chromosome 8 linkage map based on short tandem repeat polymorphisms: effect of genotyping errors. Genomics. 1992;14:144-152.[Medline] [Order article via Infotrieve]
31. Wood S, Mitchell HK, Schertzer M. Isolation and analysis of dinucleotide repeat polymorphisms from a flow-sorted chromosome 8 library. Cytogenet Cell Genet. 1991;58:1932. Abstract.
32.
Heinzmann C, Ladias J, Antonarakis S, Kirchgessner T, Schotz M, Lusis AJ. RFLP for the human lipoprotein lipase (LPL) gene: HINDIII. Nucleic Acids Res. 1987;15:6763.
33.
Oka K, Tkalcevic GT, Stocks J, Galton DJ, Brown WV. Nucleotide sequence of PvuII polymorphic site at the human lipoprotein lipase locus. Nucleic Acids Res. 1989;17:6752.
34.
Bhattacharaya S, Ameis D, Cullen P, Narcisi TM, Bayliss J, Greten H, Schotz MC, Scott J. VNTR polymorphism in the hepatic lipase gene (LIPC). Nucleic Acids Res. 1991;19:5088.
35. Hegele RA, Tu L, Conelly PW. Human hepatic lipase mutations and polymorphisms. Hum Mutat. 1992;1:320-324.[Medline] [Order article via Infotrieve]
36. Mori A, Tagagi A, Ikeda Y, Fukuoka T, Yamamoto A. Polymorphisms on human hepatic lipase gene: haplotypes of hepatic lipase gene in a Japanese population. Atherosclerosis. 1994;109:69. Abstract.
37.
Levitt RC, Liu Z, Nouri N, Meyers DA, Brandriff, Mohrenweiser HM. Mapping of the gene for hormone sensitive lipase (LIPE) to chromosome 19q13.1
q13.2. Cytogenet Cell Genet. 1995;69:211-214.[Medline]
[Order article via Infotrieve]
38.
Vandenplas S, Wud I, Grobler-Rabie A, Brebner K, Ricketts M, Wallis G, Besler A, Boyd C, Mathew C. Blot hybridization analysis of genomic DNA. J Med Genet. 1984;21:164-172.
39.
Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of betaglobin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985;230:1350-1354.
40. Syvänen A-C, Sajantila A, Lukka M. Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing. Am J Hum Genet. 1993;52:46-59.[Medline] [Order article via Infotrieve]
41. Mansfield DC, Brown AF, Green DK, Carothers AD, Morris SW, Evans HJ, Wright AF. Automation of genetic linkage analysis using fluorescent microsatellite markers. Genomics. 1994;24:225-233.[Medline] [Order article via Infotrieve]
42. Lathrop GM, Lalouel J-M, Julier CA, Ott J. Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci USA. 1984;81: 3443-3446.
43. Ott J. Analysis of Human Genetic Linkage. 2nd ed. Baltimore, Md: Johns Hopkins University Press; 1991.
44. Terwillinger JD, Ott J. Handbook of Human Genetic Linkage. Baltimore, Md: Johns Hopkins University Press; 1994.
45. Lathrop GM, Lalouel J-M, White RL. Construction of human genetic linkage maps: likelihood calculations for multilocus analysis. Genet Epidemiol. 1986;3:39-52.[Medline] [Order article via Infotrieve]
46. Cottingham RW Jr, Idury RM, Schaffer AA. Faster sequential genetic linkage computations. Am J Hum Genet. 1993;53:252-263.[Medline] [Order article via Infotrieve]
47. Schaffer AA, Gupta SK, Shriram K, Cottingham RW Jr. Avoiding recomputation in linkage analysis. Hum Hered. 1994;44:225-237.[Medline] [Order article via Infotrieve]
48. Lander E, Kruglyak L. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet. 1995;11:241-247.[Medline] [Order article via Infotrieve]
49. Kidd KK, Ott J. Power and sample size in linkage studies. Cytogenet Cell Genet. 1984;37:510-511.
50. Suarez BK, Rice J, Reich T. The generalized sib pair IBD distribution: its use in the detection of linkage. Ann Hum Genet. 1978;42:87-94.[Medline] [Order article via Infotrieve]
51. Weitkamp LR, Stancer HC, Persad E, Flood C, Guttormsen S. Depressive disorders and HLA: a gene on chromosome 6 that can affect behavior. N Engl J Med. 1981;305:1301-1305.[Abstract]
52. Elston RC, Keats BJB. Genetic Analysis Workshop, III: sib pair analyses to determine linkage groups and to order loci. Genet Epidemiol. 1985;2:211-213.
53. Holmans P. Asymptotic properties of affected-sib-pair linkage analysis. Am J Hum Genet. 1993;52:362.[Medline] [Order article via Infotrieve]
54. Grundy SM, Chait A, Brunzell JD. Familial combined hyperlipidemia workshop. Arteriosclerosis. 1987;7:203-207.
55. Strålfors P, Olsson H, Belfrage P. Hormone-sensitive lipase. In: Boyer PD, Krebs EG, eds. The Enzymes. New York, NY: Academic Press; 1987;18:147-177.
56. Teng B, Forse A, Rodriguez A, Kwiterovich PO Jr, Sniderman AD. Adipose tissue triglyceride synthesis in patients with hyperapobetalipoproteinemia. J Physiol Pharmacol. 1988;66:239-242.
57.
Nevin DN, Brunzell JD, Deeb SS. The LPL gene in individuals with familial combined hyperlipidemia and decreased LPL activity. Arterioscler Thromb. 1994;14:869-873.
58. Murase T, Itakura H. Accumulation of intermediate density lipoprotein in plasma after intravenous administration of hepatic triglyceride lipase antibody in rats. Atherosclerosis. 1981;39:293-300.[Medline] [Order article via Infotrieve]
59. Demant T, Carlsson LA, Holmquist L, Karpe F, Nilsson-Ehle P, Packard CJ, Shepherd J. Lipoprotein metabolism in hepatic lipase deficiency: studies on the turnover of apolipoprotein B and on the effect of hepatic lipase on high density lipoprotein. J Lipid Res. 1988;29:1603-1611.[Abstract]
60.
Lander ES, Schork NJ. Genetic dissection of complex traits. Science. 1994;265:2037-2048.
61. Norio R. Diseases of Finland and Scandinavia. In: Rothschild HR, ed. Biocultural Aspects of Diseases. New York, NY: Academic Press; 1981:359-415.
62. Syvänen A-C, Ikonen E, Manninen T, Bengtström M, Söderlund H, Aula P, Peltonen L. Convenient and quantitative determination of the frequency of a mutant allele using solid-phase minisequencing: application to aspartylglucosaminuria in Finland. Genomics. 1992;12:590-595.[Medline] [Order article via Infotrieve]
63. Vesa J, Hellsten E, Verkruyse LA, Camp LA, Rapola J, Santavuori P, Hofmann SL, Peltonen L. Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis. Nature. 1995;376:584-587.[Medline] [Order article via Infotrieve]
64. Hästbacka J, de la Chapelle A, Kaitila I, Sistonen P, Weaver A, Lander E. Linkage disequilibrium mapping in isolated founder populations: diastrophic dysplasia in Finland. Nat Genet. 1992;2:204-211.[Medline] [Order article via Infotrieve]
65. Hellsten E, Vesa J, Speer MC, Mäkelä TP, Järvelä I, Alitalo K, Ott J, Peltonen L. Refined assignment of the infantile neuronal ceroid lipofuscinosis (INCL, CLN1) locus at 1p32: incorporation of linkage disequilibrium in multipoint analysis. Genomics. 1993;16:720-725.[Medline] [Order article via Infotrieve]
66. Peltonen L, Pekkarinen P, Aaltonen J. Messages from an isolate: lessons from the Finnish gene pool. Biol Chem. 1995;376:697-704.
67.
Nishina PM, Johnson JP, Naggert JK, Krauss RM. Linkage of atherogenic lipoprotein phenotype to the low density lipoprotein receptor locus on the short arm of chromosome 19. Proc Natl Acad Sci USA. 1992;89:708-712.
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