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REVIEW ARTICLE

Unraveling the complex genetics of familial combined hyperlipidemia

Elina Suviolahti Department of Human Genetics, David Geffen School of Medicine at UCLA, University of California, Los AngelesUSA
,
Heidi E. Lilja Department of Surgery, Meilahti Hospital, University of Helsinki, Helsinki, Finland
&
Päivi Pajukanta Department of Human Genetics, David Geffen School of Medicine at UCLA, University of California, Los AngelesUSA
Pages 337-351 | Published online: 08 Jul 2009

Abstract

Familial combined hyperlipidemia (FCHL) constitutes a substantial risk factor for atherosclerosis since it is observed in about 20% of coronary heart disease (CHD) patients under 60 years. FCHL, characterized by elevated levels of total cholesterol (TC) and triglycerides (TGs), or both, is also one of the most common familial hyperlipidemias with a prevalence of 1%–6% in Western populations. Numerous studies have been performed to identify genes contributing to FCHL. The recent linkage and association studies and their replications are beginning to elucidate the genetic variations underlying the susceptibility to FCHL. Three chromosomal regions on 1q21–23, 11p and 16q22–24.1 have been replicated in different study samples, offering targets for gene hunting. In addition, several candidate gene studies have replicated the influence of the lipoprotein lipase (LPL) gene and apolipoprotein A1/C3/A4/A5 (APOA1/C3/A4/A5) gene cluster in FCHL. Recently, the linked region on chromosome 1q21 was successfully fine‐mapped and the upstream transcription factor 1 (USF1) gene identified as the underlying gene for FCHL. This finding has now been replicated in independent FCHL samples. However, the total number of variants, the risk related to each variant and their relative contributions to the disease susceptibility are not known yet.

Abbreviations
APOA1=

apolipoprotein A1

APOA2=

apolipoprotein A2

APOA5=

apolipoprotein A5

APOB=

apolipoprotein B

APOC3=

apolipoprotein C3

APOE=

apolipoprotein E

APOA1/C3/A4/A5=

apolipoprotein A1/C3/A4/A5

CHD=

coronary heart disease

FCHL=

familial combined hyperlipidemia

HDL‐C=

high‐density lipoprotein cholesterol

HNF4A=

hepatic nuclear factor 4 alpha

LD=

linkage disequilibrium

LDL‐C=

low‐density lipoprotein cholesterol

LEPR=

leptin receptor

LIPC=

hepatic lipase

LPL=

lipoprotein lipase

SNP=

single nucleotide polymorphism

TC=

total cholesterol

TG=

triglycerides

TNFRSF1B=

tumor necrosis factor receptor superfamily, member 1B

TXNIP=

thioredoxin interacting protein

T2DM=

type 2 diabetes mellitus

USF1=

upstream transcription factor 1

Introduction

Familial combined hyperlipidemia (FCHL), first described among young survivors of myocardial infarction in 1973 Citation1–3, is the most common familial dyslipidemia predisposing to coronary heart disease (CHD). FCHL is a common disease with an estimated population prevalence of 1%–6% Citation3,4. Elevated levels of serum total cholesterol (TC), triglycerides (TGs), or both characterize the FCHL disorder. FCHL also features low levels of high‐density lipoprotein cholesterol (HDL‐C), elevated levels of serum apolipoprotein B (apoB), and glucose intolerance as component traits Citation2,3,Citation5.

It has been evident for 30 years that FCHL has a strong genetic component Citation2,3,Citation6. The genetic component in FCHL has been suggested by familial aggregation of dyslipidemia Citation2 and in fact, FCHL was originally suggested to be inherited as an autosomal dominant disorder due to the vertical transmission pattern Citation1. A more complex polygenic background is, however, likely, as suggested by metabolic Citation7 and segregation studies Citation8,9. Furthermore, whole‐genome searches and candidate gene studies performed in FCHL families originating from different populations have identified several putative loci for FCHL Citation10–16. The recent findings and replications of the linkage and association studies are beginning to identify the DNA sequence variations contributing to FCHL. Fine‐mapping of one of the linked regions recently resulted in the characterization of the first positionally cloned gene for FCHL, upstream transcription factor 1 (USF1) Citation17. Since USF1 is a transcription factor known to regulate the expression of a number of genes participating in glucose and lipid metabolism, it provides an excellent candidate for FCHL. A recent study also indicates that common variants and haplotypes in the hepatic nuclear factor 4 alpha (HNF4A) gene are associated with high serum lipid levels and the metabolic syndrome in FCHL families Citation18. Interestingly, cooperative effects of USF1 and HNF4A have been suggested to control the regulation of multiple genes, including apolipoprotein A2 (APOA2) and apolipoprotein C3 (APOC3) Citation19–21. Interactions between DNA sequence variants are likely to contribute to the complex pathogenic mechanism(s) of common cardiovascular traits, raising the possibility that variants in USF1, HNF4A and apolipoproteins may interactively confer susceptibility to FCHL. To summarize, these accumulating data indicate that multiple variants contribute to the susceptibility to FCHL.

Both rare and common variants have been suggested to contribute for example to low plasma levels of HDL‐C in the general population Citation22,23. However, the extent to which each group influences this susceptibility is not known. It is thus likely that multiple DNA sequence variants, both common and rare, underlie the genetic susceptibility to FCHL. In addition to the human DNA sequence and variation data produced by the Human Genome Project and the HapMap Project, recent advances in genotyping technologies and statistical approaches should enable an accelerated investigation of the sequence variations at the genomic level to identify all the variants involved in the disease susceptibility of FCHL.

Key messages

  • Familial combined hyperlipidemia (FCHL) is a common complex disorder with both genetic and environmental factors affecting the disease susceptibility.

  • Most likely the genetic susceptibility to FCHL is determined by multiple DNA sequence variants and their interactions.

  • This review focuses on the recent findings that are beginning to elucidate the FCHL susceptibility genes, including the upstream transcription factor 1 (USF1) and lipoprotein lipase (LPL) genes, as well as the apolipoprotein A1/C3/A4/A5 (APOA1/C3/A4/A5) gene cluster.

Genes conferring the susceptibility to FCHL

USF1 identified as the gene underlying the linkage signal on chromosome 1q21

Previously a locus for FCHL was identified on human chromosome 1q21–q23 in FCHL families originating from the genetically relatively isolated population of Finland Citation10. Since then, this finding has been replicated in several FCHL samples, originating from other, more heterogeneous populations Citation15,Citation24–26. Linkage has been observed to FCHL, as well as to several FCHL component traits, including TG, TC and apoB levels Citation10,Citation15,Citation25,26. On 1q21 no significant evidence of genetic heterogeneity was observed in the Finns Citation17. However, in Mexican, German and Chinese samples the proportion of families contributing to linkage ranged from 22% to 71% Citation24,Citation26. Interestingly, the same markers in the 1q21 region have also been linked to type 2 diabetes mellitus (T2DM) in numerous studies Citation27–33. Most recently, 1q23–31 was also shown to be linked to the metabolic syndrome Citation34. The evidence for linkage obtained for 1q21 has varied in these FCHL and T2DM studies, most likely reflecting the underlying genetic heterogeneity, as well as population‐based and diagnostic differences. Interestingly, many of the critical metabolic features of FCHL, e.g. hypertriglyceridemia and insulin resistance, also represent trait components of T2DM. Taken together, these linkage findings suggest that one or more genes in this particular chromosomal region predispose to both FCHL and T2DM, two clinical phenotypes with overlapping component traits and shared diagnostic features.

The upstream transcription factor 1 (USF1) was recently linked and associated with FCHL in 60 extended Finnish FCHL families with 721 genotyped individuals (P = 0.00002) Citation17. The evidence for association was strongest among males for high serum TGs (P = 0.0000009) and extended to a ∼46‐kb region, containing also the adjacent F11 receptor (F11R) gene Citation17 (). An association was also observed for apoB, TC and the LDL peak particle size Citation17. The known functions of F11R, related to regulation of tight junction assembly in epithelia, pathogenesis of viral infections, and transendothelial migration of certain T cell types Citation35–37, make it a less likely candidate gene for FCHL than USF1. USF1 is a ubiquitously expressed transcription factor of the basic helix‐loop‐helix leucine zipper family. It forms homo‐ and heterodimers (with USF2) and recognizes a CACGTG motif termed E box, resulting in activation of the gene transcription and enhanced expression in response to various stimuli such as glucose and dietary carbohydrates Citation38. USF1 regulates the expression of several genes participating in glucose and lipid metabolism, such as apolipoprotein C3 (APOC3), apolipoprotein A2 (APOA2), apolipoprotein A5 (APOA5), apolipoprotein E (APOE), hormone sensitive lipase (LIPE), hepatic lipase (LIPC), glucokinase (GCK), islet‐specific glucose‐6‐phosphatase catalytic‐subunit‐related protein (IGRP), insulin (INS), glucagon receptor (GCGR), ATP‐binding cassette transporter A1 (ABCA1), fatty acid synthase (FAS), acetyl‐CoA carboxylase alpha (ACACA) and plasminogen activator inhibitor‐1 (PAI1) Citation20,21,Citation38–51. A more complete list of the USF1 target genes is available in a recent paper Citation52.

Figure 1. Overview of the associatedUSF1 region in Finnish and Mexican familial combined hyperlipidemia (FCHL) families. The associated region was restricted by ∼70% (from 46 kb to 14 kb) using two different populations, the Finns and Mexicans Citation17,Citation26. The rs numbers of the single nucleotide polymorphisms (SNPs) are as follows: f11rs1 (rs836), f11rs4 (hCV1459766), f11rs5 (rs4339888), usf1s1 (rs3737787), usf1s2 (rs2073658), usf1s8 (rs2516838).

Figure 1. Overview of the associatedUSF1 region in Finnish and Mexican familial combined hyperlipidemia (FCHL) families. The associated region was restricted by ∼70% (from 46 kb to 14 kb) using two different populations, the Finns and Mexicans Citation17,Citation26. The rs numbers of the single nucleotide polymorphisms (SNPs) are as follows: f11rs1 (rs836), f11rs4 (hCV1459766), f11rs5 (rs4339888), usf1s1 (rs3737787), usf1s2 (rs2073658), usf1s8 (rs2516838).

In the original study, genetic data were supported by preliminary functional data, as the USF1 risk haplotype had an effect on the expression profiles of fat biopsies Citation17. Expression profiles of fat biopsies of FCHL cases seemed to differ depending on their carrier status for the associated USF1 haplotype Citation17. A total of 25 genes were significantly upregulated and 73 genes downregulated in the susceptibility haplotype carriers Citation17. The upregulated genes belonged to functional classes mainly related to fat metabolism, and the downregulated genes included several functional classes related to immune response. Interestingly, the upregulated genes included the stearoyl‐CoA desaturase (SCD) gene Citation17. The SCD activity has been shown to be associated with plasma TG levels Citation53. The downregulated genes also included several known important atherosclerosis‐related genes, such as APOE, phospholipid transfer protein (PLTP), macrophage scavenger receptor 1 (MSP1), arachidonate 5‐lipoxygenase (ALOX5), and complement component 3a receptor 1 (C3AR1).

A recent study further demonstrated differential expression of known USF1 target genes, APOE, ABCA1 and angiotensinogen (AGT), between USF1 risk allele carriers and non‐carriers using a larger number of fat biopsies of FCHL and low HDL‐C‐affected patients (n = 19) Citation52. No differences in the characteristically low USF1 transcript levels were observed between carriers of the USF1 risk versus non‐risk alleles in the original Citation17 or in this subsequent study Citation52. However, it is possible that even a subtle difference may have significant effects on the expression of USF1 target genes.

Hoffstedt et al. studied the effect of USF1 variants on lipolysis in adipocytes Citation54. Lipolysis is a critical aspect of adipocyte function and manifests large differences between individuals potentially due to genetic differences. In this study including fat biopsies from 196 normolipidemic obese women, the usf1s2 (rs2073658) variant was associated with the maximum lipolytic action in response to stimulation by noradrenaline, as well as by β1‐, β2‐ and β3‐ adrenergic receptor agonists (dobutamine, terbutaline, CGP12177 and forskolin). Importantly, the carriers of the protective allele of the usf1s2 variant had a significantly increased maximum lipolytic activity in response to these drugs. This association between the USF1 variant and the maximum lipolytic activity was observed in normolipidemic women without any disease such as FCHL, CHD or T2DM. The result may imply that the defect caused by USF1 is present already before the clinical manifestation of the disease. Defect in USF1 may ultimately lead to disease when other genetic or environmental factors accumulate in the same individual.

Since the original finding in Finnish FCHL families, the association between the DNA sequence variations in USF1 and FCHL has been replicated in several study samples Citation26,Citation55–57 (). The first replication study including 24 extended multigenerational Mexican FCHL families, replicated the association, and moreover the associated region was restricted to 14 kb Citation26 versus more than 46 kb in the Finns Citation17 (). These data provides a shorter region with fewer variants available for functional analyses. No gender differences were observed in Mexican FCHL families Citation26. The most significantly associated single nucleotide polymorphisms (SNPs), usf1s1 (rs3737787) and usf1s2 (rs2073658), or SNPs in linkage disequilibrium (LD) with usf1s1/s2, were investigated in three other study samples ascertained for CHD or for family history of CHD Citation55–57. In extended Utah pedigrees with family members suffering early death due to CHD, early strokes, or early onset hypertension, the USF1 SNPs and haplotype were associated with FCHL, TG and low‐density lipoprotein cholesterol (LDL‐C) levels Citation57. In the European Atherosclerosis Research Study II, Putt et al. examined the lipid levels before and after meal and after oral glucose tolerance test Citation56. They found differences in the correlation between body mass index (BMI), fasting LDL‐C and glucose levels according to the USF1 genotypes. It is worth noting that as in Finns Citation17 the common allele of usf1s1 (rs3737787), usf1s2 (rs2073658) or SNPs in LD with usf1s1/s2 represents the associated allele in all of these studies, replicating the original study. It can be concluded that these SNPs seem to capture the disease‐associated signal, although their direct relationship to the functional defect contributing to FCHL pathogenesis is not known yet.

Table I. Summary of the genetic studies on the USF1 gene, the first gene identified for familial combined hyperlipidemia (FCHL) using positional cloning approach.

Most recently, Komulainen et al. investigated the role of USF1 variants and haplotypes as a risk factor for cardiovascular disease events at the population level Citation55 (). Importantly, they observed that female carriers of a USF1 risk allele had a two‐fold risk of a cardiovascular event and an increased risk of all‐cause mortality during the follow‐up period in a Finnish prospective population cohort consisting of 14,000 individuals, followed up for cardiovascular events in a period of 7–10 years Citation55.

Two different studies have focused on the contribution of USF1 SNPs to T2DM Citation58,59 (). In the first study by Ng et al., a significant association of USF1 polymorphisms with T2DM and the metabolic syndrome‐related traits was observed in Chinese T2DM families, although no single SNP explained the previous linkage to the 1q21 Citation58. In the Chinese case‐control sample, the population‐based hospital cases of T2DM were, however, not associated with usf1s1 Citation58. In the second study, Gibson et al. did not find differences in allele frequencies between French diabetics and healthy controls Citation59. To summarize, the results for USF1 with T2DM and the metabolic syndrome have included both positive and negative associations Citation58,59. These data imply that there are also other regional genes on 1q21 that contribute to the linkage signals of these two disorders.

Interestingly, a gene for combined hyperlipidemia (Hyplip1) in mouse was mapped to a region on chromosome 3 in the HcB‐19/Dem mouse that was orthologous to human chromosome 1q21 Citation60. The underlying gene, thioredoxin interacting protein (TXNIP), was recently identified as a gene for combined hyperlipidemia in mouse Citation61. TXNIP provided thus a strong positional candidate for human FCHL Citation62. However, several recent studies show that variations in the TXNIP gene do not confer the susceptibility to FCHL Citation17,Citation63,64. Regarding the possibility whether the identified USF1 risk haplotype would have a long‐range effect on the expression of TXNIP, the TXNIP expression profiles of fat biopsies from affected Finnish FCHL family members having the USF1 risk haplotype were compared with affected FCHL family members, homozygous for the putative protective haplotype. No haplotype‐dependent difference in TXNIP expression was detected Citation17. To summarize, it seems unlikely that TXNIP accounts for the observed evidence of linkage between FCHL and the 1q21 region.

Variants in the apolipoprotein gene cluster APOA1/C3/A4/A5 are implicated in FCHL

Multiple studies predict the importance of the APOA1/C3/A4 gene cluster as a modifier gene complex in the development of FCHL Citation65–73 (), although not all studies have shown the connection Citation74–76. The most investigated polymorphisms of this gene cluster are three restriction enzyme polymorphisms, XmnI and MspI residing upstream to the apolipoprotein A1 (APOA1) gene and the SstI site in the 3′ untranslated region of exon 4 of the APOC3 gene Citation77. The positive studies include a Dutch study, in which the minor alleles of these polymorphisms were associated with elevated plasma TC, TG, LDL‐C, apoB, and apoC3 levels in Dutch FCHL families Citation67. Furthermore, a suggestive evidence for linkage between the MspI minor allele and plasma LDL‐C levels was detected Citation67. Based on the results, Dallinga‐Thie et al. suggested that the APOAI/C3/A4 gene cluster is not the primary cause of FCHL, but rather it has a specific modifying effect on plasma TG and LDL‐C levels in this lipid disorder Citation67. The negative studies include a Finnish study in which the MspI polymorphism was associated with serum TC and apoB levels in spouses, but no evidence of direct involvement of the APOAI/C3/A4 loci or haplotypes in the expression of FCHL in the Finnish FCHL families was found Citation76.

Table II. Genes associated with familial combined hyperlipidemia (FCHL) in previous studies.

Recently, APOA5 gene was added as part of the APOA1/C3/A4 gene cluster Citation78. Variants of this gene cluster have been linked to high TGs in both the general population Citation78–87 and in FCHL Citation88–90. In the Dutch FCHL families, APOA1/C3/A4/A5 showed an association with TG levels and LDL particle size, and the strongest evidence of association was obtained with SNPs in APOA1 and APOA5Citation89. Ribalta and colleagues also suggested a potential implication of APOA5 in the hypertriglyceridemia present in FCHL, because in hyperlipidemic patients with FCHL, the carriers of the minor allele of a SNP in APOA5 had significantly increased plasma TG levels when compared with the carriers of the common allele Citation90. In British FCHL families, alleles of the APOA1/C3/A4/A5 gene cluster were overtransmitted to subjects with FCHL, and the transmission of the common APOA1/C3/A4/A5 haplotype to the affected subjects was reduced Citation88. Several studies also imply that APOA5 is a potential risk factor for cardiovascular disease Citation82,Citation91,92.

APOA5 is suggested to reduce plasma TGs by inhibiting lipidation of apoB and thus reducing the hepatic very low‐density lipoprotein (VLDL) production rate, as well as by stimulating LPL‐mediated clearance of TG‐rich lipoproteins Citation93. Overexpression of APOA5 has been shown to lower TGs in mice and ApoA5 knockout mice have severe hypertriglyceridemia Citation78,Citation94. Recently, a study by Nowak et al. implied that APOA5 is regulated by insulin Citation46. Interestingly, insulin induces a dose‐dependent downregulation of APOA5 expression by reducing the binding of upstream stimulatory factors USF1 and USF2 to E‐Box (5′‐CACGTG‐3′) of the APOA5 promoter. It was also suggested, that the inhibitory effect of insulin on the APOA5 transcription involves a phosphorylation mechanism of USF that modulates their binding to the APOA5 promoter and results in APOA5 transrepression Citation46. The downregulation of APOA5 by insulin could explain the association between hypertriglyceridemia and hyperinsulinemia. Interestingly, APOA5 is also a highly responsive target gene of the peroxisome proliferator‐activated receptor alpha and may act as a major mediator for fibrates in reduction of plasma TGs Citation95.

Lipoprotein lipase and hepatic lipase genes in FCHL

The variants in the LPL gene have been of interest in FCHL because LPL catalyzes the hydrolysis of TGs of VLDL and chylomicrons and thus delivers fatty acids to tissues (). Nevin et al. identified variations in the coding region of the LPL gene in 6 of 20 FCHL patients, the variations including Asp9Asn, Val108Val and Ser447stop Citation96. Reymer et al. found a common Asn291Ser variant of the LPL gene in FCHL patients and observed an association with FCHL and HDL‐C levels, as well as with catalytic activity of LPLCitation97. Since these studies, the Asn291Ser and Asp9Asn variants have been associated with reduced HDL‐C, elevated TG, apoB and/or insulin levels in FCHL patients in several different studies Citation98–103. In addition to coding variants in LPL, variations in the promoter region have been identified in FCHL patients Citation101,102,Citation104,105. Yang et al. reported a heterozygous carrier of a promoter variant (−39C/T) in LPL when screening 20 FCHL probands for mutations in the putative LPL promoter region Citation105. The variant resulted in diminished transcriptional activity of the LPL gene, potentially by abolishing the transcription factor Oct‐1 binding site. The −93G/T and −53C/G variants were also reported to affect promoter activity Citation104. However, not all studies have supported the role of LPL in FCHL Citation74,Citation106,107. In addition to FCHL, variations in LPL have been associated with CHD Citation108. Evidence for linkage to the hepatic lipase (LIPC) locus (P<0.003) and to the lecithin:cholesterol acyltransferase (LCAT) locus (P<0.0006) has also been observed Citation13 and a coding variant in LIPC was associated with elevated TC and apoB levels in the Dutch FCHL families Citation109. However, the linkage to LIPC locus was not confirmed in the Finnish FCHL families Citation106.

Variants in HNF4A gene associated with lipid levels in FCHL

A region on chromosome 20q12–q13 has been linked to T2DM and obesity in numerous studies Citation110–114. Recently, several independent groups have identified associations between SNPs in the HNF4A gene residing in this 20q12–q13.1 region and T2DM Citation115–119. Previously mutations in HNF4A have been demonstrated to cause maturity onset diabetes of the young type I (MODY1) Citation120. Interestingly, the same chromosomal region on 20q12–q13 has also been linked to TGs and low HDL‐C in FCHL families Citation11,Citation121,122. Subsequently, evidence for linkage has been found with high TGs in other populations Citation123,124. There is a clear phenotypic overlap between FCHL and T2DM, patients with T2DM exhibiting often hypertriglyceridemia and patients with FCHL glucose intolerance and/or insulin resistance Citation5,Citation125. Both diseases also predispose to CHD.

Considering this clear phenotypic overlap between T2DM and FCHL, and the fact that both disorders have been linked to the same chromosomal region on 20q Citation11,Citation110,111,Citation113,114,Citation121,122, it is possible that HNF4A contributes to linkage signals in both diseases. Recently common HNF4A variants and their haplotypes were investigated for association in Finnish and Mexican FCHL families, comprising 1020 subjects Citation18 (). The common HNF4A variants and haplotypes were associated with elevated serum lipid levels, the metabolic syndrome as well as with elevated glucose parameters in the Finnish FCHL families Citation18. Importantly, both the Finnish and Mexican FCHL families shared two common lipid‐associated HNF4A haplotypes Citation18. This is the first study demonstrating that common HNF4A variants and their haplotypes are associated with high plasma lipid levels and the metabolic syndrome.

HNF4A is a transcription factor, regulating several genes in lipid and glucose metabolism. Interestingly, it has been shown that cooperative binding of USF1 and HNF4A drives the transcription of the human APOA2 gene Citation20. In addition, sequences from sites bound by HNF4A and USF1 were demonstrated to show significant overlap in HepG2 cells Citation19. As interactions between DNA sequence variants are suggested to be critical in the etiology of complex traits such as FCHL, it is possible that DNA sequence variants in USF1 and HNF4A interactively influence the susceptibility to FCHL.

TNFRSF1B and LEPR as candidate genes for QTL affecting apoB levels on chromosome 1

A genome‐wide scan in 18 Dutch FCHL families revealed a quantitative trait locus (QTL) affecting apoB levels on chromosome 1 short arm Citation15. The candidate genes associated with FCHL in this region include tumor necrosis factor receptor superfamily, member 1B (TNFRSF1B) Citation126 and leptin receptor (LEPR)Citation127. TNFRSF1B, located on 1p36.2, was associated with susceptibility to FCHL in the Dutch family sample Citation126. For further support, the levels of soluble extracellular domain of TNF‐R p75 were lower in the hyperlipidemic than in the normolipidemic relatives of FCHL patients Citation128. A polymorphism in the coding region of the LEPR gene (Gln223Arg), located on 1p31 was associated with an increased risk of FCHL and a difference in HDL‐C levels Citation127. To determine whether one or both of these genes explain the linkage signal, or if there are additional regional genes involved, requires further investigation.

Other candidate genes investigated for FCHL include manganese superoxide dismutase locus on chromosome 6, which showed suggestive evidence of linkage in Dutch FCHL families with FCHL, as well as with related traits such as TC, apoB and apoC3 levels (P<0.02) Citation13. Biochemical and genetic associations of plasma apoA2 levels with FCHL have also been suggested Citation129. The role of APOA2 in FCHL has been discussed recently Citation130,131.

Gene expression profiling provides novel candidate genes for FCHL

Gene expression is the major determinant of the phenotype and function of a living organism. The profile of expressed genes in a particular cell is highly dynamic and changes rapidly in response to cellular events and external stimuli. For long, methods have been available to measure the gene expression of a limited number of genes at a time. Recently, DNA microarray technology has provided a tool to monitor the expression pattern of a whole genome simultaneously on a single chip Citation132. This technology provides a useful tool to profile gene expression at the genomic level, and to determine sets of genes expressed or turned off together. Consequently, these techniques allow for identification of the genes and metabolic pathways that are differentially expressed in healthy and diseased individuals and, thus, provide an alternative approach to identify novel genes and pathways involved in FCHL.

Three studies have been performed to identify the differential gene expression between FCHL patients and healthy controls Citation133–135. One of the studies investigated lymphoblastic cell lines Citation133, and two others focused on adipose tissue Citation134,135. The first study by Erlings et al. detected 25 differentially expressed genes out of 588 genes in subcutaneous adipose tissue obtained from 5 unrelated FCHL and 4 control individuals Citation134. Many of the differentially expressed genes had been implicated in the activation of the adipocyte cell cycle. Expression of tumor necrosis factor, alpha (TNFA) was upregulated in FCHL patients, which is of particular interest since TNFRSF1B variants and decreased levels of soluble extracellular domain of TNF‐R p75 have been observed to be associated with FCHL previously Citation126,Citation128.

Morello et al. studied the gene expression in immortalized lymphoblastic cells obtained from FCHL cases and their normolipidemic spouses and relatives Citation133. Of the 7647 genes expressed in these samples, 166 genes were differentially expressed between cases and controls. Categorizing the differentially expressed genes according to biological processes that the genes were involved in revealed that almost half of the genes were taking part in metabolism. Interestingly, the early growth response 1 gene (EGR‐1) encoding a transcription factor was upregulated in lymphoblasts Citation133, as well as in the adipocytes derived from FCHL patients in the previous study Citation134. Of the 166 genes differentially expressed in the lymphoblastic cell lines, surrounding sequences of 16 genes contained the EGR‐1 consensus sequence (5′‐CGCCCCCGC‐3′) Citation133.

Meex et al. Citation135 measured the expression levels of 640 genes in 5 unrelated FCHL patients and 10 control individuals. Initially, 27 genes were identified differentially expressed between FCHL cases and controls. After validation CD36/FAT was shown to be upregulated in the original sample of FCHL cases using quantitative reverse transcription polymerase chain reaction (RT‐PCR), as well as in five additional pairs of FCHL cases and controls. CD36/FAT is a multiligand class B scavenger receptor that has been implicated in the transmembrane transport of long‐chain fatty acids and in the regulation of angiogenesis. Interestingly, Meex et al. found a significant correlation between the CD36/FAT expression and esterification of fatty acids into phospholipids and TGs, suggesting that this gene has a functional role in lipid metabolism Citation135.

To summarize the candidate gene studies and the evidence obtained in replication studies (), it seems evident that DNA sequence variants at least in USF1, LPL and the APOA1/C3/A4/A5 gene cluster confer the susceptibility to FCHL. However, none of the candidate genes investigated so far account solely for a major genetic component in FCHL, further confirming the current understanding that the complex FCHL phenotype is an end result of genetic and environmental interplay of multiple factors ().

Figure 2. Multiple genetic and environmental factors confer the susceptibility to familial combined hyperlipidemia(FCHL).

Figure 2. Multiple genetic and environmental factors confer the susceptibility to familial combined hyperlipidemia(FCHL).

Chromosomal loci identified for FCHL

To identify novel loci for FCHL, genome‐wide scans have been performed in the Finnish Citation11,Citation122, Dutch Citation14,15 and British families with FCHL Citation16. Loci on chromosomes 1q21, 2p, 2q31, 8q, 10p11.2, 10q11.2–10qter, 16q, 20q and 21q21 were identified in the Finnish FCHL families Citation10,11,Citation122, and loci on chromosomes 1p, 2p, 11p, 16q and 19q in the Dutch FCHL families Citation14,15. A combined genome scan of the Dutch and Finnish families with FCHL identified three loci for HDL‐C on 2p, 9p and 16q Citation12. In addition, a recent genome scan in the British FCHL families replicated the 11p locus and identified two novel candidate loci on 6q and 8p Citation16.

Three of these chromosomal regions, 1q21–23, 11p and 16q22–24.1, have been replicated in several FCHL study samples. The 1q21 locus has been observed in the Finnish and Dutch genome scans, as well as in independent studies of Chinese, German, US Caucasian and Mexican families with FCHL Citation10,11,Citation15,Citation24–26. Another replicated locus is the 11p region, which has been detected in the Dutch and British FCHL families Citation14,Citation16. This region showed evidence of linkage to FCHL, as well as to the TC and TG traits in the British study sample Citation16. No evidence of linkage for apoB levels was observed for this 11p region Citation14, implying that this locus may not directly contribute to the observed elevation of apoB‐containing particles in FCHL. Genetic heterogeneity was demonstrated for the 11p region, as 49% of the British FCHL families were linked to this region Citation16.

Given the known difficulties in replicating and verifying the results of complex traits, international collaboration to replicate findings in independent and combined study samples is of utmost importance to accelerate the gene identification process. This strategy is based on increased statistical power to verify those of the identified regions that have the highest statistical likelihood to harbor causative genes. The power of this strategy has recently been demonstrated with other complex diseases, such as inflammatory bowel disease and asthma Citation136–138, as well as in a combined data analysis of Dutch and Finnish genome‐wide scans for FCHL Citation12. In that study, three regions, 16q24.1, 2p25.1, and 9p23, were identified where the evidence for linkage emerged from the combined study sample Citation12. The region on 16q24.1 resulted in most significant evidence for linkage, a lod score of 3.4, for HDL‐C, 50% of the Finnish and Dutch families being linked to this region. This very same 16q24.1 region was also recently implicated for HDL‐C in Mexican Americans Citation139, as well as in several other independent studies Citation12,Citation26,Citation122,Citation139–141. Different study populations could thus be utilized first to replicate the loci that harbor susceptibility genes for FCHL, and second, to fine‐map these verified regions.

It has not been directly evaluated whether the three replicated regions on 1q21, 11p and 16q explain the dyslipidemia in some or all FCHL families. These types of studies are clearly warranted to estimate the risk related to each variant, their contribution to FCHL and its component traits, as well as to investigate the likely gene‐gene interactions in FCHL.

Future directions and concluding comments

FCHL is a typical complex trait with several genes, environmental factors and their interactions contributing to the disease phenotype (). The possible high genetic complexity and the fact that the current strategies are based on functional characterization of mutations of monogenic diseases make the dissection of the wide allelic spectrum of variants conferring the susceptibility to complex diseases very challenging. These variants can result in minor changes in protein product or reside in promoters or other regulatory regions; or they can cause small changes in the binding affinity of a transcription factor or slightly alter gene expression levels, timing and tissue specificity. In all of these cases, it may be difficult to demonstrate not only the functional consequences of a single variant but also of the allelic combinations of multiple variants. Nutrients or other environmental factors may potentially also modulate differences introduced by genetic variation. It is thus possible that each individual variant causes subtle changes in the disease phenotype. Accumulation of multiple deleterious gene variants and environmental factors in a particular individual will then ultimately lead to the expression of the disease phenotype.

Candidate gene studies, as well as positional cloning have provided novel candidate genes for FCHL of which variants in USF1, the APOA1/C3/A4/A5 gene cluster and LPL have been replicated in several study samples from distinct populations. Many of the variants currently associated with FCHL are common and the risk involved with them at the population level is not known at the moment. Extensive population‐based studies, such as recently conducted for USF1Citation55 and multiple T2DM candidate genes Citation142, estimating the risk related to each variant and their relative contribution to the disease susceptibility, are warranted.

Now in the post‐genomic era, over a hundred different genomes including the human genome have been sequenced, and the HapMap project has described the human sequence variation throughout the human genome Citation143. This vast amount of information together with advancing genotyping technologies have enabled genome‐wide LD and association studies using hundreds of thousands of SNPs. These novel genomic approaches are expected to facilitate the dissection of the molecular basis of human complex diseases and provide novel insights into disease pathogenesis in near future. Utilization of the HapMap data offers short‐cuts to gene identification providing that common variants contribute significantly to common diseases Citation144–146. Moreover, strategies using tag SNPs, derived from the HapMap data, have most power to detect these common causative variants Citation147. Consequently, the novel genome‐wide association approaches are more likely to identify common than rare variants influencing the FCHL susceptibility. Based on the recent candidate gene studies of DNA sequence variants contributing to levels of plasma HDL‐cholesterol in different populations, it is, however, likely that rare DNA sequence variants also contribute to disease susceptibility Citation22,23. As large‐scale sequencing of candidate genes in affected individuals is currently the only approach to identify new rare variants, more efficient and cost‐effective sequencing approaches are warranted.

The Human Genome Project and HapMap Project have provided the basic tools for unraveling the complex genetics of FCHL and other complex traits. However, this challenging task can be accomplished only by combining the accumulating biological information with interdisciplinary knowledge and international collaboration. Skillful combining of molecular medicine and systems biology is needed to overcome the greatest challenge of any complex disease, i.e. the meaningful analysis of the enormous amount of data using tools reflecting accurately the underlying phenotypic and genetic complexity.

Acknowledgements

Many students and collaborators contributed to the work and ideas presented in this review and we would like to thank them. Work in the laboratory of PP is supported by the National Institutes of Health grants HL‐28481, HL‐82762 and HL‐70150, as well as the American Heart Association grant 0430180N.

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