© 1997 American Heart Association, Inc.
Articles |
Familial Hypercholesterolemia in the Finnish North Karelia
A Molecular, Clinical, and Genealogical Study
From the Departments of Medicine (A.F.V., K.K.) and History (K-M.P.), University of Helsinki, Helsinki, Finland; the Central Hospital of North Karelia (H.T.), Joensuu, Finland; the North Karelian Data Base (P.R.), Joensuu, Finland, and the Department of Endocrinology (T.K.), University of Lund, Malmö, Sweden.
Correspondence to Kimmo Kontula, MD, Professor of Molecular Medicine, Department of Medicine, University of Helsinki, Haartmaninkatu 4, FIN-00290 Helsinki Finland.
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Abstract A specific mutation termed FH-North Karelia [FH-NK] accounts for almost 90% of familial hypercholesterolemia [FH] cases in the Finnish North Karelia, with a population of about 180 000. Extensive search for its presence in the entire North Karelia province revealed 340 carriers of this mutation. Other mutations of the LDL receptor [LDLR] gene accounted for 67 cases of heterozygous FH. This gives a minimum FH prevalence of 1 in 441 inhabitants in North Karelia, with the highest density of patients in the Polvijärvi commune (1 in 143 inhabitants). Old parish records, confirmation records, and tax records were used to track a common ancestor for most of the present-day North Karelian FH-NK patients in the village of Puso, located within an area where the FH prevalence today is the highest. DNA analysis indicated that 2% of the subjects aged 1 to 25 years would have been diagnosed as false-negative and 7% as false-positive FH patients on the basis of LDL cholesterol [LDL-C] determinations alone. Common genetic variations of apolipoprotein E [apoE], XbaI, polymorphism of apolipoprotein B [apoB], and PvuII polymorphism of the intact LDLR allele contributed little to serum lipid variation in established carriers of the FH-NK allele, although apoE2/4 genotype and the presence of the PvuII restriction site tended to be associated with relatively low LDL-C levels. Coronary heart disease (CHD) was present in 65 (30%) out of the 179 FH gene carriers aged
25 years, and 19
individuals had a previous history of acute myocardial infarction
(AMI). The average age (mean±SD) at onset of CHD was 42±7 years for
males and 48±11 years for females (P<.05). In stepwise
logistic regression analysis carried out in carriers of the
FH-NK allele, age, gender, smoking, and apoE allele E2 all
emerged as independent determinants of risk of CHD or AMI. It may be
concluded that the relatively high prevalence of FH patients in North
Karelia province provides a unique founder population in which genetic
and nongenetic factors modifying the course of FH can be effectively
investigated.
Key Words: LDL receptor • diagnosis • phenotype • genealogy
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Introduction |
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FH is among the most common autosomal dominant disorders, characterized by a life-long elevation of serum LDL-C levels, tendon xanthomatosis, and development of premature atherosclerosis.1 By 1992 more than 150 mutations of the LDLR gene causing FH were reported.2 In most populations, FH has proven to be extremely heterogenous at the DNA level. However, there are few exceptions of inbred populations in which one or a few mutant LDLR genes account for the majority of FH cases.3–7
In Finland, four different mutations—two deletions and two point mutations—of the LDLR gene were shown to account for approximately 75% of all heterozygous FH cases of the population.8–10 The enrichment of these founder genes among the Finns can be explained by a variety of reasons, including the unique geographical position of the country between Eastern and Western cultures as well as an isolation brought about by linguistic barriers.11 Even within the Finnish population, the North Karelia province, with its population of about 180 000 inhabitants, its high prevalence of CHD, and its position close to the eastern border of the country, occupies an exceptional subregion of the country with regard to the molecular genetics of FH. One specific mutation, termed FH-NK and characterized by a deletion of seven nucleotides from exon 6 of the LDLR gene, was shown to be the underlying disorder in approximately 90% of FH patients in this area of the country.9 The FH-NK mutation causes a translational frameshift, is associated with a receptor-negative phenotype of FH, and causes a typical xanthomatic form of heterozygous FH.9 The clinical significance of this mutation was substantiated by demonstration of its presence in 9% of young patients with CHD in North Karelia.12
The present study was conducted to delineate the impact of FH as a determinant of CHD risk in a high-incidence area, to assess the effectiveness of molecular genetic techniques in diagnostics of FH in relatively young individuals, and to identify genetic and other factors modifying the clinical manifestations of a genetically homogenous form of heterozygous FH-NK. North Karelia is an area especially well suited for these purposes because of its well-organized public health services, the presence of one central hospital providing consultations on severe lipoprotein disorders for the whole province, and well-maintained parish records allowing efficient family studies back to the 17th century.
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Methods |
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Patients
Subsequent to the identification of the FH-NK allele as accounting for the majority of FH cases in North Karelia area,9 all public health centers in the province were advised of the finding and encouraged to admit patients suspected of having FH as well as their first-degree relatives for further studies on lipid levels and for DNA examinations in the Central Hospital of North Karelia, Joensuu. One of the authors (H.T.) personally examined all subjects attending the lipid outpatient clinic. Clinical examination was supplemented by the collection of family histories to the level of second-degree relationship. A total of 966 subjects attending the lipid outpatient clinic of the Central Hospital of North Karelia, Joensuu town, were examined. Of the whole cohort, 569 subjects fulfilled the clinical criteria of FH (see below) or had a family history of FH. DNA samples became available for 541 (95%) of this group. The remaining subjects (n=397) had hypercholesterolemia, but, on subsequent examination, did not fulfill the clinical diagnostic criteria of FH. DNA samples were available of all the individuals of this latter group as well and were screened for the presence of the two common Finnish types of LDLR mutations (see below).
The clinical diagnostic criteria for adult FH patients included: (1) serum total cholesterol [TC] level over 8 mmol/L; (2) the presence of tendon xanthomas in the proband and/or in his/her first-degree relative; and (3) the presence of hypercholesterolemia (>8 mmol/L) in at least one of the first-degree relatives of the proband. Throughout the study, clinical and laboratory examinations were carried out to rule out the presence of secondary causes of hyperlipidemia, such as hypothyroidism, hepatic disease, excess alcohol intake, or renal failure. Data on places of birth and site of current residence for all the FH-NK carriers were also collected. The present study was carried out at the North Karelia Central Hospital, Joensuu, Finland, from January 1992 to August 1996 and was approved by the ethical review committee of the Department of Medicine, University of Helsinki, and special permission to obtain clinical data from the FH-NK patients was received from the Ministry of Health.
Genealogical Study
We randomly picked 18 probands from apparently unrelated
families living in different parts of North Karelia (Polvijärvi,
Kontiolahti, and Juuka). To discover a putative common ancestor of the
North Karelia mutation, we traced their family trees and any available
health records back to the end of the 17th century. The information
was based on several sources. First, detailed questionnaires on family
data were collected from each proband. Second, Lutheran parish
records were available from the 18th century for all the probands
and, for some subjects, from the end of the 17th century. Parish
records and records on confirmations that included the list of
communicants were used as a main source of information. These documents
contained data on dates of birth, marriage and death, and, in many
cases, records of the cause of death were recorded from the
year 1749 forward. Third, parish records of the Greek Church
starting in the first quarter of the 19th century were also utilized.
Fourth, tax records giving information on household structure were
available from the end of the 17th century.
Tracing family histories, medical information, and causes of death back
from one generation to the preceding one for each of the families was
carried out essentially as described by Bertolini et
al.13 The strategy employed for the genealogical
study is based on the assumption that the carrier of the mutant gene
was the partner who either died prematurely (men 
55 y, women 65 y),
or had siblings who died prematurely.
Risk factors for CHD and AMI
Clinical data of 179 carriers of the FH-NK allele aged
25 y or more were available to classify them as those with or
without CHD. CHD was considered to be present if the patient had a
typical effort-induced angina pectoris, if there was a previous
diagnostic finding on a bicycle ergometer test, thallium
scanning, or coronary angiogram, if the patient had a
documented history of AMI, or if a previous coronary
angioplasty or bypass operation had been carried out. AMI was diagnosed
by a characteristic clinical history, combined with
diagnostic changes in electrocardiographic
recordings or clinical enzyme assays.
Data on cigarette smoking was collected from clinical records. The
subjects were classified as smokers (current or former smokers) or
nonsmokers (subjects who had never smoked and subjects whose smoking
status was unknown). Hypertension was considered to be present if
the subject used drugs for established hypertension, if
systolic blood pressure was 160 mm Hg, or if
diastolic blood pressure was 95 mm Hg. We
considered the patient as diabetic if he/she used antidiabetic drugs
(oral hypoglycemic drugs or insulin).
Laboratory Methods
DNA was isolated from 5 to 20 mL venous blood samples using
standard techniques. Presence of the FH-NK and FH-Helsinki [FH-HKI]
mutations of the LDLR gene was assayed using the duplex polymerase
chain reaction [PCR] method described previously by our
laboratory.12 The common allelic variation of
apoE was determined by a solid-phase minisequencing
technique.14 The XbaI (codon 2488)
polymorphism of the apoB gene and the AvaII
polymorphism in exon 13 of the LDLR gene were assayed by techniques
combining amplification of the genomic areas involved by PCR, followed
by digestion of the PCR products with the respective restriction
enzyme and analysis on polyacrylamide gel
electrophoresis. Assay for the PvuII polymorphism in
intron 15 of the LDLR gene was carried out by Southern blot
techniques.15 Alleles were named using the
nomenclature X-/X-, X-/X+ or X+/X+ for the XbaI
restriction site, and A+ or A- and P+ or P- for the AvaII
and PvuII restriction sites of the normal LDLR allele,
respectively.
Lipid analyses on fasting serum samples were always carried out before any hypolipidemic drug treatment. TC16 and triglyceride (TG)17 levels were determined by enzymatic methods using commercial kits obtained from Boehringer-Mannheim. The concentration of serum HDL cholesterol (HDL-C) was measured enzymatically after precipitation of LDL and VLDL fractions with dextran sulfate and MgCl2,18 and serum LDL-C level was calculated using the formula of Friedewald et al.19
Statistical Analysis
Age- and sex-specific comparisons of lipid levels were carried
out using the Mann-Whitney's nonparametric test or
2 test using Yates correction when necessary.
To examine whether serum LDL-C values were significantly different during puberty from those present in other age groups, we first calculated regression equations between serum LDL-C levels and ages, separately for the carriers of the FH-NK allele and their noncarrier siblings, with the pubertal individuals (aged 12 to 18 y) omitted from the calculations. The resulting regression equations were as follows: gene carriers: LDL-C=0.024*AGE+2.59; and noncarriers: LDL-C=0.049*AGE+6.42, respectively. Using the paired t test, we compared the actual LDL-C values from regression equation estimates depending on the carrier status of the individual. Regression method has to be used, because the age-covariance equations for LDL-C were significant, and they were also significantly different for FH-NK individuals and nonaffected individuals.
The correctness of the clinical diagnosis of FH-NK or non-FH in young
affected subjects and their nonaffected siblings was compared to the
DNA diagnosis of the same individuals. To assess the clinical diagnosis
on the basis of serum lipid measurements, we used the maximum
likelihood method to divide the apparently bimodal LDL-C distribution
in the whole study cohort to two different normal distributions
representing the FH-NK allele carriers and their
healthy siblings. The maximum likelihood function was given by:
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Results |
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Occurrence of the FH-NK Allele in the Province of North Karelia and Its Subregions
A total of 966 subjects were examined at the lipid outpatient clinic of the Central Hospital of North Karelia, and DNA samples were available from 938 of these individuals to be screened by the duplex PCR assay. Of these 938 individuals, 340 were carriers of the FH-NK allele according to the present study and our initial study,9 and 67 were diagnosed to have heterozygous FH due to mutant genes other than the FH-NK; the remaining subjects were their relatives (n=134) or had hypercholesterolemia due to other genetic or nongenetic factors (n=397). It was not possible to totally exclude the diagnosis of heterozygous FH in the 397 subjects who all were found to be negative for the FH-NK and FH-HKI allele. These data provide a minimum prevalence of FH of 407/179 526 (1 in 441 inhabitants) in North Karelia. The FH-NK allele was found to account for 340/407 (84%) of these FH cases, while the FH-HKI allele was present in 18 (4%) individuals. Other yet undetermined mutant alleles were inferred to be present in 49 subjects. We did not find a single living subject with a homozygous form of FH.
There were striking differences in the occurrence of FH in the
different communes of North Karelia. A marked accumulation of FH,
mostly explained by the occurrence of the FH-North Karelia allele,
was seen in the commune Polvijärvi with a minimum prevalence of
approximately 7 in 1000 inhabitants, and its neighboring communes (Fig 1
). This accumulation was even more
obvious when the origin of the patients was located on the basis of
their birth places (data not shown). Of the living 340 carriers of the
FH-NK allele, 156 (46%) were males and 184 (54%) females. In the
age group of 50 y or more (n=82), females (67%) predominated
males (33%), and there were only two heterozygous FH patients older
than 75 y (aged 76 and 78 y), both females.
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Genealogical Studies
Eighteen apparently unrelated families, with the probands coming
from the communes Polvijärvi, Juuka and Kontiolahti, were
randomly selected for our genealogical studies. The ancestors of most
families were observed to have lived in a geographical area which was
within the area where the prevalence of the FH-NK mutation today is the
highest. After studying the life histories of about 1300 individuals,
we were able to construct the family tree shown in Fig 2. Fourteen of eighteen families were
historically linked together (Fig 2), and, in twelve of them, premature
deaths were documented. The mean (±SE) age of death for the inferred
or DNA-verified carriers of the FH-NK allele (n=74) was 59±2 y, a
figure significantly (P=.01) lower than the corresponding
age (66±2 y) for their deceased spouses (n=59). In these fourteen
families, the mean age (±SE) of death of the heterozygous carriers of
the FH-NK allele was 58±2 y for men and 62±3 y for women. The
available records suggested a cardiac cause of death, with a
documentation of preceding chest disease, chest pain, edema or heart
disease, in 12 out of those 25 presumed carriers of the FH-NK
allele who had written health documents available. The postulated
ancestor family lived in the village of Puso in the late 17th century
and in the beginning of the 18th century; the male and female ancestors
were born in 1686 and 1690, respectively. This village is located
within the present-day Kontiolahti commune with a high prevalence
of the FH-NK allele (Fig 1
).
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= a presumed
homozygous patient who died in 1966 (see
text).
There were four probands that we could not link to the offspring of the
suspected ancestor from the village Puso. However, the ancestors of
three of these probands were traced back to the nearby Juuka area,
suggesting the possibility for a relatedness to the other families,
while one seemed to have his roots in a more distant place, the
Tohmajärvi area (Fig 1).
The family histories disclosed only one putative homozygous FH-NK
patient, deceased in 1966. This individual (black symbol in Fig 2) was
a female subject who was examined for skin xanthomatosis at the age of
9 y and who died suddenly at the age of 10 y. An autopsy
revealed widespread atheroslerotic changes, including severe CHD.
Serum Lipid Levels in the FH Patients and Their Non-FH
Siblings
Mean serum lipid values in 326 heterozygous DNA-documented FH-NK
patients, grouped according to their age class and sex, are summarized
in Table 1. There was a slight increase
of serum LDL-C and TG levels in the older age groups, while serum HDL-C
levels remained relatively stable. Male and female patients did not
appear to differ significantly from each other, with the exception of
serum HDL-C levels, which in men were higher in the youngest age group
and lower after the age of 20 y in comparison to the corresponding
levels in women (Table 1).
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Scatterograms demonstrating individual serum lipid levels in
heterozygous DNA-verified FH patients and their available siblings,
shown to be noncarriers of the FH-NK allele by DNA
analysis, are depicted in Fig 3.
Although serum TC and LDL-C levels were essentially nonoverlapping in
affected and nonaffected subjects from the youngest age on, occasional
exceptions to this rule were noticed (Fig 3). Mean serum HDL-C levels
were significantly lower in the DNA-verified subjects aged 1 to 40
y than in their non-FH siblings (data not shown). During teenage years
from 12 to 18 y, serum LDL-C levels were significantly
(P=.0025) lower in noncarrier siblings but not in
heterozygous FH-NK patients (P=.087), compared to expected
levels at that age derived from the other age groups by regression
method (Fig 3).
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)
and their healthy siblings (
). Solid lines indicate the mean
values and dotted lines the 95% confidence intervals for the lipid
levels (separate lines for the FH and non-FH subjects are
indicated).
Young members of families with a known carrier status of the FH-NK
allele were recruited in particular for the present study, to
evaluate the accuracy of FH diagnostics based on lipid
measurements in subjects aged 1 to 25 y. Using a maximum
likelihood method taking into account individual serum LDL-C levels and
utilizing DNA analysis as a standard in 208 subjects, we found
that two (2%) out of the 121 true FH patients were diagnosed as
false-negatives and six (7%) out of the 87 non-FH subjects were
diagnosed as false-positives. The situation is illustrated in Fig 4 that shows the actual bimodal frequency
distribution of serum LDL-C levels in the whole cohort and the
separately calculated normal curves for mutation carriers and
noncarriers.
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= actual distribution of the LDL
cholesterol levels measured, ——–= sum of all subjects
(bimodal curve), - - - = unaffected subjects (left curve), and
... ... . = affected subjects (right
curve).
Occurrence of CHD and Its Risk Factors in FH Patients
We conducted a systematic study on CHD risk factors in carriers of
the FH-NK allele who were at least 25 years of age; data was
collected from 179 subjects (73 males and 106 females). Fifty-five (26
men, 29 women) had convincing evidence for CHD, and 19 (14 men, 5
women) out of these 55 individuals were survivors of AMI. The mean
(±SD) age of onset of symptomatic CHD was 42±7 y for men
and 48±11 year for women (P<.05), and the corresponding
ages at the time of first AMI were 47±12 y and 59±13 y
(P=.08), respectively (Fig 5).
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Not unexpectedly, the mean age of the FH-NK patients with clinical CHD,
with or without past history of AMI, were higher than the corresponding
age in FH-NK carriers without CHD (Tables 2 and 3).
Hypertension and diabetes were more common in patients with CHD than in
those without it, and there were more smokers among those who had CHD
compared to those with no clinical symptoms of CHD (Table 2). FH
patients with and without CHD, whether manifesting with a previous AMI
or not, could not be distinguished from each other by their
pretreatment HDL-C levels (Tables 2 and 3). There was a trend towards
elevated serum LDL-C levels in individuals with some clinical
manifestation of CHD or AMI, and serum TG levels were significantly
higher in patients with CHD than in those without it (Tables 2 and 3).
A combination of elevated serum TG concentration (2.0
mmol/L) and low HDL-C concentration (1.0
mmol/L) was present in 8 (15%) out of 55 CHD patients and 7
(6%) out of the 124 non-CHD subjects (2=2.9,
P=.09).
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Occurrence of any manifestation of CHD as a function of various risk
factors was also analyzed separately in men and women (Table 4). Males with CHD had slightly higher
serum TG levels than those without it, but other lipid
parameters did not show significant differences in groups
with or without CHD irrespective of sex. Among women, there were more
hypertensive and diabetic individuals in sufferers of CHD than in
non-CHD subjects (Table 4).
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Common Polymorphisms of the Lipid-Regulatory Genes
A number of commonly occurring variations of gene loci previously
proposed to show association with risk of CHD were examined in those
carriers of the FH-NK allele aged 25 y or more and whose
clinical records permitted analysis of occurrence of CHD
(n=179, see above). In the case of LDLR, the heterozygous FH subjects
possessed one functional allele only. Our previous studies had
shown that the mutant FH-NK allele was negative (P-) for the
polymorphic PvuII site,15 and
every patient was found to have the genotype A+/A- or A+/A+
for the AvaII site showing that FH-NK mutation occurred in
the A+ allele, thus permitting an unequivocal typing of the intact
LDLR allele. We did not find any significant association of the
various polymorphisms examined, including the common
polymorphism of apoE, XbaI polymorphism of apoB
gene, and PvuII and AvaII polymorphisms of
the LDLR gene with occurrence of any manifestations of CHD (Table 2).
When survivors of AMI alone were compared to those without its past
history, we found a lower prevalence of the LDLR P+ allele in the
former group, but the numbers of subjects under comparison were rather
small (Table 3).
The relationship of the common polymorphisms of lipid regulatory genes to serum lipid levels was examined in extended material of 288 carriers of the FH-NK allele aged 1 to 73 y. In this material, apoE allele E4 was apparently not associated with elevated serum LDL-C levels. Thus, the mean LDL-C levels in apoE genotypes E2E2+E2E3, E3E3, and E3E4+E4E4 were 7.74±0.28 mmol/L, 8.05±0.15 mmol/L and 8.10±0.21 mmol/L, respectively (P=NS), and males and females did not differ significantly from each other. The mean LDL-C level in the genotype E2/4, though present in only seven individuals, was significantly lower (6.47±0.71 mmol/L) than the corresponding levels in other apoE genotypes (7.99±0.10 mmol/L, P<.05). There was no significant association between the apoB XbaI polymorphism and serum LDL-C levels (data not shown). Both male and female carriers of the FH-NK allele possessing the P- type functional LDLR allele tended to have higher LDL-C levels (7.84±0.18 and 8.12±0.17 mmol/L, respectively) than those with the P+ allele (7.36±0.41 and 7.92±0.35 mmol/L, respectively), but this difference did not reach statistical significance. There were no significant associations between any of the common polymorphisms studied and serum HDL-C or TG levels (data not shown).
Multivariate Analysis of the Genetic and
Other Risk Factors of CHD
To evaluate the possible independent association of genetic and
other variables to the risk of CHD as a whole or AMI in particular,
stepwise logistic regression analyses were carried out, both
separately for each sex and combined. When men and women were
analyzed together, male gender, age, and smoking appeared as
significant risk factors of CHD (Table 5). A similar discriminant
analysis of risk factors of AMI alone indicated that male
gender, age, smoking, and apoE allele E2 all played significant
roles (Table 6). When similar
analyses were carried out separately for men and women, only
age emerged as a significant risk factor of CHD, while the impact of
other risk factors did not reach statistical significance (data not
shown).
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Discussion |
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FH-NK: A Founder Mutation Enriched in Finnish North Karelia
Our data show that the FH-NK allele, accounting for approximately 85% of heterozygous FH cases among Finnish North Karelians, represents a founder LDLR gene allele in this population and occurs in at least one in 500 inhabitants of the whole province. Although not a rarity, it occurs less frequently among FH patients in other Finnish provinces,9 but its presence has not been reported outside Finland. Even considering other founder mutations that have been screened in large FH patient cohorts, such as the promoter mutation among French Canadians3 and the two FH-Afrikaner mutations,5 the FH-NK mutation is an LDLR mutation allele showing the highest relative frequency in a given geographical area.
Origin of the FH-NK Allele
Our results are in agreement with our initial hypothesis that the
mutation may have occurred once in the geographical area where its
prevalence today is the highest. We have been able to pinpoint a common
ancestor couple for most of the present-day carriers of the FH-NK
allele (Fig 2). It should, however, be emphasized that the
records employed to track the mutation may contain some
inaccuracies. Thus, there were registration gaps in specific communes
during certain periods; methods ensuring paternity were not available,
and, in cases of illegitimacy, the biological father was missing.
However, the evidence from the pedigree data (Fig 2) that indicated not
only a convergence of most probands into a single ancestor couple but
also early deaths (males 55 y, females 65 y) significantly more
often in inferred carriers of the FH-NK allele (33/69 or 48%) than
in their spouses (14/59 or 24%; P<.01), as well as the
localization of the ancestor couple within the high-incidence area
convinces us that we have correctly followed the mutation back to the
17th century.
In 1617, the North Karelia province, which formerly belonging to Russia, became part of Finland, which, in turn, was part of Sweden at that time. During the 17th century, new Lutheran inhabitants coming from Savo (located southwest of North Karelia) and Kainuu (located north of North Karelia) pushed the earlier inhabitants belonging to the Greek Church toward the East and Russia. We presume that one of the original settlers coming from the West was a carrier of the FH-NK allele, or that the mutation took place immediately after this immigration. In 1749, there were about 21 000 inhabitants in North Karelia, and, by 1805, the population had increased up to 56 000. However, the population density was still very low. People lived in large families, and multiple family households were quite common in the 18th century.20–24 Gene flow between these large breeding units called "superisolates"25 was very low in the community. Considering these circumstances, it may not be unexpected that a mutant gene not affecting fertility could be maintained in the population and within the same geographical area for centuries.
There are but few previous systematic genealogical studies of FH. Three Italian families carrying the same mutant LDLR allele (FH-Pavia) were traced back to a common ancestor living in the 17th century.13 In these families, the mean ages at death of the presumed heterozygous FH heterozygotes were 48 y for males and 63 y for females,13 which compare rather well with the mean ages of 58 y (males) and 62 y (females) recorded by us for the former carriers of the FH-NK allele. Common mutations causing FH among the South African Afrikaners may be of Dutch origin.26 Interestingly, Torrington et al27 found the original founder surname in 57 of the present-day Afrikaans-speaking FH index cases. In our study, surname analysis proved to be of no value in the search for origin of the FH-NK mutation. Fumeron et al28 have demonstrated a probable French origin for the large promoter deletion among the French Canadian FH patients, and Jomphe et al29 have shown that most ancestors of FH patients carrying this deletion lived in a specific region 120 miles east of Quebec.
Lipoprotein Levels and Diagnostic Aspects
With the exception of the age class 60 y or more, we did not
see significant differences in the mean serum LDL-C levels between male
and female FH patients (Table 1), which is compatible with the data
from earlier studies carried out in FH cohorts without mutation
typing.30–32 In most adult age classes, serum
HDL-C levels were higher in females than in males (Table 1). This is in
accordance with the data of Gagné et al,33
who showed a similar sex difference of HDL-C levels in the heterozygous
FH patients living in the Quebec area. Studies in healthy Finnish
children aged 3 to 24 y revealed the lowest serum
cholesterol levels around the age 15
y.34 We found a similar nadir of serum total and
LDL-C levels in teenaged healthy siblings of the FH-NK individuals and
a remarkably similar trend in the FH individuals of the same age (Fig 3).
Comparison of serum TG levels in young DNA-documented carriers and
noncarriers of the FH-NK allele revealed only minor differences in
these two groups. In contrast, in age classes from 1 to 40 y, mean
serum HDL-C levels were lower in FH subjects than in their non-FH
siblings (Fig 3). These data are compatible with several earlier
studies showing diminished serum HDL-C levels in FH patients in
comparison to nonaffected controls33,35,36 or
relatives.37 In fact, when present, reduction
of serum HDL-C level may impart an elevated risk of coronary
artery disease in FH.35,37
The lipid phenotype-based diagnosis of heterozygous FH was compared to molecular genetic assessment of the disorder in 121 carriers of the FH-NK allele and in 87 of their nonaffected siblings. The phenotype analysis, carried out by a maximum likelihood method, was targeted to younger aged group for whom other clinical manifestations of FH are still negligible. The level of misdiagnosis by lipid measurements (8 out of the 208 subjects examined, or 4%) is relatively low and in accordance with our previous estimate in a group of families with the FH-HKI allele.38 Two facts may have, however, rendered this percentage lower than experienced in normal clinical routine. First, we were operating with one mutation category only, whereas, in most clinical situations, FH patients carry different types of mutations that may cause subtle variations in serum lipid levels. Second, healthy siblings came from families with known FH members. Dietary habits in these families may have been more strict than average, thus accentuating differences between pretreatment serum lipid levels in FH patients and lipid levels in the siblings. Previous experience of the usefulness of DNA-based screening of FH comes from South Africa39 and Canada.40
Risk Factors of CHD
In its heterozygous form, FH shows marked interindividual
phenotypic variability, the reasons for which are only poorly
understood.1,41,42 A portion of this variation in
the phenotypic expression may be due to variation of the causative
mutation per se,43–46 but other genetic and
other factors may be equally or even more important. Although the role
of lipid and nonlipid risk factors as modifiers of CHD risk has been
approached in several earlier studies, this has been carried out in
genetically defined FH cohorts in only few
studies.40,43,47–49 It should be emphasized,
however, that there may be inherent caveats to consider in studies on
genetically homogenous populations: these populations are not only
homogenous for the gene locus under investigation, but also for other
covariates.
When only established mutation carriers older than 25 y were considered, signs of CHD were present in 36% of men and 27% of women (NS). On average, evidence for CHD emerged six years earlier in men than in women, an interval comparable to that of approximately 9 y reported in several earlier studies.33,35,50 Ferriéres et al40 studied the expression of CHD in 263 heterozygous carriers of the same >10 kb deletion of the LDLR gene in French Canadians and found a pattern remarkably similar to that in our cohort: the mean age of onset of CHD was 39 y for affected males and 46 y for affected females. It should be emphasized that onset of CHD in heterozygous FH patients is likely to be delayed in future with the advent of extensive use of cholesterol biosynthesis inhibitors in these subjects.
Of the various nonlipid risk factors widely documented to be related to
risk of CHD, smoking, elevated blood pressure, and diabetes appeared to
be more common in FH patients with documented CHD than in those without
it (Table 2). However, when studied using stepwise discriminant
analysis, of these three factors, only smoking was shown to
have an independent association with the occurrence of CHD (Table 5) as
well as a past history of AMI (Table 6) when the both sexes were
considered together, although our study is relatively small for firm
conclusions. Some previous studies using genetically nondefined FH
subjects have lent support for the role of smoking and elevated blood
pressure as determinants of CHD risk in heterozygous FH, even if not
constantly in both genders,30,35 but these
relationships have not been confirmed in all earlier
surveys.32,51 In the cohort examined by Wiklund
et al,52 smokers were more common in the FH
patients with CHD compared to those without CHD, but the contribution
of smoking disappeared on multiple regression analysis of the
data. In the genetically uniform population of heterozygous FH patients
described by Ferriéres et al,40
hypertension was more common in FH patients with CHD than in those
without CHD, but the difference approached statistical significance in
women only.
Previous comparative studies on serum lipid levels in heterozygous FH
subjects with and without CHD have yielded somewhat
inconsistent results. Thus, some studies support the assumption
that a greater extent of serum LDL-C elevation imparts an increased
risk of CHD in heterozygous FH,35,40,50,51
whereas LDL-C levels in affected and nonaffected FH heterozygotes were
reported to be similar by other
researchers.43,52,53 Moreover, there seem to be
noncongruent data on the possible role of diminished serum HDL-C levels
as a predictor of CHD risk: some studies pinpoint this lipid alteration
as a risk factor;37,50,51,54 other studies
support its role only in men35,40 or
women,33 while additional
surveys43,52,53 do not favor the role of serum
HDL-C level as an adjunct CHD risk factor in FH at all. In our own
study, there was a nonsignificant trend towards elevated serum LDL-C
levels in subjects with CHD in comparison to those without it (Tables 2 to 4). With regard to serum HDL-C and risk of CHD, it appears that the
presence or absence of the mutant FH-NK allele itself overrides the
impact of any reduction of HDL-C concentration. In addition to the
lipid variables listed above, elevated concentrations of serum
lipoprotein(a) have been suggested to be associated with an increased
risk of CHD in heterozygous FH,52,53 but this
finding has been questioned in several subsequent
studies.40,50,55
Although serum lipid measurements in our study were always carried out before any hypolipidemic treatment, all except one of the 179 individuals analyzed for risk of AMI or CHD were subsequently treated with lipid-lowering drugs, which may have modified the time of occurrence of CHD. However, the average duration of drug treatment (7.8 y) in our cohort was relatively short in terms of lifelong CHD prevention, and inhibitors of cholesterol biosynthesis (statins) became available only at a relatively late phase of follow-up. These facts, including the possible bias resulting from more agressive drug treatment in cases with established CHD, preclude any definite conclusions related to the use of hypolipidemic drugs and risk of CHD.
Common polymorphism of apoE accounts for a considerable portion of the interindividual variability of serum LDL-C levels56 and, by this mechanism or by some other mechanism, affects the risk of CHD.57 Data on apoE variation in heterozygous FH cannot be interpreted without difficulties. Apolipoprotein phenotype E4 was related to elevated serum and/or LDL-C levels in some surveys,43,58 whereas many other studies did not disclose such a relationship.35,49,59,60 Cholesterol absorption was reportedly higher in the apoE phenotype E4/3 than E3/3, but this was not reflected by any significant differences in serum cholesterol levels.59 Data on apolipoprotein E variation and HDL-C levels seem also to be somewhat variable: while occasional studies suggest that the phenotype E4 is associated with diminished concentrations of serum HDL-C in women,49 the consensus maintains that common polymorphism of apoE does not appreciably affect serum HDL-C concentrations in heterozygous FH.43,47,58–60
The study carried out by Ferriéres et al48
in a genetically uniform cohort of FH patients deserves a special note
in this respect. A careful analysis indicated that the effects
of apoE genotypes on interindividual lipid variability are
dependent on whether FH or non-FH subjects are examined and are
sex-dependent. Of the three alleles, E2 displayed the greatest
influence, showing an LDL-C lowering effect that was strongest in
females.48 Although we were not able to find any
significant differences in mean serum LDL-C, HDL-C and TG levels in the
three common apoE genotypes (E3/2, E3/3 and E4/3), serum LDL-C
levels were the lowest in the seven FH subjects with the E4/2
genotype. Interestingly, a multivariate
stepwise analysis favored the role of allele E2, but not
E4, as an independent risk factor of AMI (Table 6). It is tempting to
speculate that any atherogenic effect of allele E2 exerted on
hypercholesterolemic FH patients could be due to
mechanisms related to VLDL metabolism. First, two earlier
studies have indicated that the E2 phenotype was associated
with increased serum TG levels in heterozygous
FH.35 Second, the highest VLDL
cholesterol levels were recorded in those FH patients
with the phenotype E2/2.48 Third, Hopkins
et al61 were able to provide evidence for an
interaction between FH status and the presence of the E2 allele,
resulting in increased levels of potentially atherogenic ß-VLDL
cholesterol. Collectively, these data, combined with ours,
suggest that heterozygous FH patients carrying at least one E2
allele may be at a particular risk of CHD. Prospective studies are
required in order to clarify whether these subjects should be treated
with a particularly agressive hypolipidemic therapy.
In conclusion, we have found an unique isolate of FH patients in Finnish North Karelia and have tracked the causative mutation to the 17th century. Among the present-day carriers of the FH-NK allele, CHD appeared an average of six years earlier in males than females, and a past history of AMI was more common in males. Although the number of subjects with ischemic heart disease was rather small for definitive conclusions, our data suggest that smoking and the occurrence of apoE allele E2 signify an increased risk of AMI heterozygous FH.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We thank Eija Eklund-Mähönen for help at the lipid outpatient clinic, Kaija Kettunen and Susanna Tverin for expert technical assistance, and Pertti Vuorinen for invaluable cooperation on the genealogical studies. This work was supported by grants from The Medical Council of The Finnish Academy, The Sigrid Juselius Foundation, The University of Helsinki, The Paulo Foundation, The Finnish Heart Foundation, The Finnish Medical Society Duodecim, The Finnish Culture Foundation, The Ida Montin Foundation, and The Science Foundation of Orion Corporation.
Received June 30, 1997; accepted August 27, 1997.
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References |
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A. F. Vuorio, H. Gylling, H. Turtola, K. Kontula, P. Ketonen, and T. A. Miettinen Stanol Ester Margarine Alone and With Simvastatin Lowers Serum Cholesterol in Families With Familial Hypercholesterolemia Caused by the FH-North Karelia Mutation Arterioscler Thromb Vasc Biol, February 1, 2000; 20(2): 500 - 506. [Abstract] [Full Text] [PDF]
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