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Common mutations of familial hypercholesterolemia patients in Taiwan: Characteristics and implications of migrations from southeast China
Gene 498 (2012) 100–106
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Short Communication
Common mutations of familial hypercholesterolemia patients in Taiwan: Characteristics and implications of migrations from southeast China Kuan-Rau Chiou a, c, 1, Min-Ji Charng b, c,⁎, 1 a b c
Division of Cardiology, Department of Medicine, Kaohsiung Veterans General Hospital, Taiwan, ROC Division of Cardiology, Department of Medicine, Taipei Veterans General Hospital, Taiwan, ROC School of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC
a r t i c l e
i n f o
Article history: Accepted 29 January 2012 Available online 14 February 2012 Keywords: Chinese Familial hypercholesterolemia Haplotype Mutation Taiwanese
a b s t r a c t Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in low-density lipoprotein receptor (LDLR), apolipoprotein B-100 (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. This study investigated FH patients carrying common mutations in Taiwan and compared them to FH southeastern Asians. Causal FH mutations were identified by exon-by-exon sequencing with/without multiplex ligation-dependent probe amplification among 208 Taiwanese with clinically diagnosed FH. Haplotype analyses among probands and family members were undertaken using TaqMan® Assays. Totally, LDLR mutations were found in 118 probands, consisting of 61 different loci, and APOB 10579C>T mutations in 12 probands. Three mutations (64delG, 1661C>T, and 2099A>G) were novel. LDLR 986G>A (13.1%), 1747C>T (10.8%), and APOB 10579C>T (9.2%) were common mutations with no differences in phenotypes. LDLR 1747C>T associated with one haplotype (CAAGCCCCATGG/(dTA)n-112nt); LDLR 986G>A with two. APOB 10579C>T associated with the same LDLR binding-domain pattern in Taiwanese and southeastern Asians. We concluded that LDLR 986G>A, 1747C>T and APOB 10579C>T are common mutations, with combined frequency of approximately 33%. The presence of different haplotypes associated with FH common mutations in Taiwan indicates multiple historical migrations, probable multiple recurrent origins from southern China, and haplotype homologies reflect the presence of common ancestors in southern China. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Familial hypercholesterolemia (FH, OMIM #143890) is one of the most common inherited disorders of plasma lipoprotein metabolism, characterized clinically by an increased level of circulating lowdensity lipoprotein (LDL)-cholesterol that leads to lipid accumulation in tendons and arteries, premature atherosclerosis and increased risk of coronary heart disease (Ueda, 2005). FH is known to be associated with mutations in the LDL receptor (LDLR) gene, the apolipoprotein B-100 gene (APOB), and proprotein convertase subtilysin kexin 9 gene (PCSK9) (Abifadel et al., 2003; Brown and Goldstein, 1986; Soria et al., 1989). With the exception of a small number of founder populations where one or two mutations predominate, most geographically based-surveys of FH patients show a large number of Abbreviations: FH, familial hypercholesterolemia; LDLR, low-density lipoprotein receptor; APOB, apolipoprotein B; PCSK9, proprotein convertase subtilisin/kexin type 9; PCR, polymerase chain reaction; MLPA, multiple ligation-dependent probe amplification. ⁎ Corresponding author at: Division of Cardiology, Department of Medicine, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei, 112, Taiwan, ROC. Tel.: + 886 2 2875 7507; fax: + 886 2 2875 6849. E-mail address: [email protected] (M.-J. Charng). 1 K.-R. Chiou and M. -J. Charng contributed equally to this work. 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.01.092
mutations in a given population (Austin et al., 2004). Haplotype analysis of FH Caucasians improved the knowledge and understanding of the origin, spread by migrations and estimation of the ages of pathogenic mutation (Castillo et al., 2002; Miserez and Muller, 2000; Tejedor et al., 2010; Traeger-Synodinos et al., 1998), but no data has been reported concerning the Asian population. Taiwanese, the major population group in Taiwan, are the descendants of early settlers from the southeast coast (Fuchien and Kwangton province) of China during the past 400 years or more in recent history (Lin et al., 2001). Recently, anthropological studies using genetic markers by human leukocyte antigen and microsatellites revealed that Taiwanese, with Singapore Chinese and ThaiChinese, formed a southern Asian cluster with neighboring groups of Thais and Vietnamese, which is separate from the northern Asian cluster consisting of Koreans and Japanese (Chu et al., 1998; Lin et al., 2001; Yang et al., 2009). Although there have been reports of FH from the southern Chinese (Chang et al., 2003; Charng et al., 2006; Chiou and Charng, 2010; Chiu et al., 2005; Khoo et al., 2000; Mak et al., 1998a; Sun et al., 1994; Tai et al., 1998), the relationships need to be further investigated. Accordingly, we performed molecular genetic testing to investigate the frequency of mutations among unrelated FH patients in Taiwan, analyzed haplotype patterns among patients carrying common mutations, and further compared
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the patterns with those of previous FH studies from the southeastern Asians. 2. Materials and methods 2.1. Study populations Patients with LDL-cholesterol levels >190 mg/dl and positive family history of hypercholesterolemia were recruited in a genetic screening program for FH in Taiwan from Sep, 2008 to Mar, 2011. All were clinically diagnosed as having FH by the Simon Broome Familial Hypercholesterolemia Register diagnostic criteria (Scientific Steering Committee on behalf of the Simon Broome Register Group, 1991). Totally, a series of 208 apparently unrelated Taiwanese residing throughout northern, central and southern Taiwan were enrolled, including 102 who had been surveyed in a previous study (Chiou and Charng, 2010). Patients with hypercholesterolemia due to secondary causes were excluded. Premature coronary artery disease and the risk factors for heart disease, including hypertension, diabetes mellitus, smoking habits and family history of premature coronary artery disease in first-degree relatives, were recorded. Family members of the probands with the identified mutations were also invited to participate in this investigation. The study complied with the Declaration of Helsinki of the World Medical Association, was approved by the institutional review board of the hospital, and informed consent was obtained from each patient. 2.2. DNA analyses Genomic DNA was isolated from the leukocytes of peripheral blood of patients. First, exon-by-exon sequence analysis was performed as previously described on both strands of all 3 FH-causing genes—LDLR, APOB, and PCSK9T (Chiou and Charng, 2010). Next, if exon-by-exon sequence
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failed to detect mutations, DNA samples were subsequently analyzed for large deletions or insertions using the multiplex ligation-dependent probe amplification (MLPA) assay with the SALSA P062-B LDLR MLPA kit obtained from MRC-Holland (Amsterdam, the Netherlands) according to manufacturer instructions (http://www.mrc-holland.com). The nomenclature and classification of the found mutations are based on the findings in the updated FH mutation databases (http://www.ucl.ac.uk/ ldlr/Current/index.php?select_db=LDLR, accessed 11 October 2011). The effects of novel nucleotide variants were subjected to online computer program using Poly Phen-2 to predict possible impact of an amino acid substitution on the structure and function of the newly discovered mutation (http://genetics.bwh.harvard.edu/pph2/). 2.3. Genotyping LDLR and APOB polymorphisms To gain more insight into FH Taiwanese, the haplotypes of the common mutations in the study cohort were compared with those of FH Caucasians and southeastern Asians. The particular polymorphisms were selected according to both previous FH studies having haplotype analysis (Castillo et al., 2002; Chang et al., 2003; Mak et al., 1998a; Miserez and Muller, 2000; Tai et al., 1998; Zuliani and Hobbs, 1990) and the haplotype website (www.hapmap.com). Probands and their family members carrying the most common mutations were genotyped for their haplotype pattern. LDLR was genotyped at 13 polymorphic markers within or flanking the LDLR gene: rs2228671 [C/T], rs2304183 [C/T], rs12983082 [A/C], rs12710260 [C/G], rs5929 [C/T], rs4508523 [C/T], rs688 [C/T], rs2738450 [A/C], rs2738452 [A/G], rs5925 [C/T], rs5927 [A/G], rs5742911 [A/G], and hypervariable region of TA dinucleotide repeat marker at the 3′ end. APOB was genotyped at 17 polymorphic sites within or flanking the APOB gene: rs1367117 [A/C/G/T], rs13306198 [C/T], rs1469513 [C/T], rs679899 [A/C/G/T], rs3828293 [A/G], rs3791981 [A/ G], rs11676704 [G/T], rs12713956 [C/T], rs2854725 [A/C], rs693 [A/C/G/
Fig. 1. Haplotype markers and positions for the LDLR gene (Panel A) and APOB gene (Panel B).
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T], rs676210 [A/C/G/T], rs13306196 [A/G], rs1801701 [A/C/G/T], rs2678379 [A/G], rs1042031 [A/C/G/T], rs1042034 [A/C/G/T], and hypervariable region of 15 bp repeat marker at the 3′ end. Fig. 1 maps the position of the haplotype markers of LDLR and APOB genes, respectively. Genotyping was performed using commercially available Pre-made TaqMan® Genotyping assays (Applied Biosystems Inc.). TaqMan® PCR was performed according to the manufacturer's standard PCR protocol. Briefly, 20 ng genomic DNA was mixed with the supplied 2X TaqMan Universal PCR Master Mix No AmpErase UNG and 20X TaqMan Assay Mix to a final volume of 5 μl in a 384-well plate. Each sample underwent 40 amplification cycles on GeneAmp® PCR System 9700 (Applied Biosystems Inc.). Fluorescent signals of the two probes were analyzed for end-point fluorescent data on ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems Inc.). Genotype was determined automatically by Sequence Detection Software (Applied Biosystems, Foster City, CA). The number of hypervariable TA dinucleotide (dTA) repeat marker at the 3′ end of the LDLR gene was genotyped using fluorescent-labeled primers, PCR amplification and capillary electrophoresis on an ABI PRISM™ DNA Analyzer (Applied Biosystems, Foster City, CA) and analyzed using the GeneScan software version 2.2. A 15-base pairs (bp) repeat marker in the hypervariable repeat region (HVR) downstream from the 3′ end of the APOB gene was genotyped by PCR amplification and capillary electrophoresis on an ABI PRISM™ DNA Analyzer and analyzed by ABI PRISM™ SeqScape software (Applied Biosystems, Foster City, CA). An investigator without knowledge of the clinical data performed the genotyping. 2.4. Statistical analyses Continuous variables were expressed as medians with range and compared using the nonparametric Kruskal–Wallis H test. Categorical variables were expressed as numbers or percentages, and compared using Fisher's exact test. Data were collected and analyzed using SPSS version 14.0 (SPSS Inc., Chicago, IL, USA). A P value of less than 0.05 was considered to be statistically significant.
the mutations within the LDLR gene, consisting of 61 different loci (53 point mutations and 8 gene rearrangements). Greater numbers of point mutations were found in exon 4 (11/53), exon 9 (7/53), and exon 7 (5/53) of LDLR. Most of the mutations were distributed in the epidermal growth factor (EGF) precursor homology domain (exons 7– 14) (31/53) and the ligand binding domain (exons 2–6) (18/53) of LDLR. The most common mutations in this series were LDLR 986G>A (Cys329Tyr): 17 cases (13.1%), LDLR 1747C>T (His583Tyr): 14 cases (10.8%), and APOB 10579C>T (Arg3500Trp): 12 cases (9.2%). Three mutations (64delG, 1661C>T, and 2099A>G) were novel and cosegregated with hypercholesterolemia in affected family members. The 64delG mutation, G deletion at position 64, caused a frame-shift which introduced an abnormal downstream amino acids and premature termination at codon 184. The 1661C>T (Ser554Leu) and 2009A>G (Asp700Gly) mutations were predicted by Poly Phen software; the former was predicted as possibly damaging with a score of 0.774, and the latter as probably damaging with a score of 0.999. 3.2. Phenotypes of the probands carrying the most common mutations The clinical features and plasma lipid levels of the patients carrying the most common mutations are shown in Table 1. Four probands were compound heterozygotes exhibiting a homozygous phenotype, carrying LDLR 986G>A with additional 268G>A, 1291G>A, 1432G>A, and 769C>T/1765G>A mutations, respectively. All 4 probands received maximum statin dose and failed to achieve lower LDL-cholesterol below 100 mg/dl. Later, one underwent liver transplantation and one received apheresis. Table 1 shows that the three most common mutation groups among 39 heterozygotes had no gross differences in clinical features, baseline lipid profile levels, or the rates of failure to lower LDLcholesterol below 100 mg/dl in response to rosuvastatin/ezetimibe treatment. 3.3. Haplotype analyses of LDLR 986G>A, 1747C>T and APOB 10579C>T
3. Results 3.1. Identification of mutations The causal mutations of FH were identified in 130 patients, including LDLR mutations in 118 and APOB 10579C>T (a CGG-to-TGG change in codon 3500) in 12. Fig. 2 shows distribution and patient numbers of
The first and second degree relatives were available from 19 of 43 probands with common mutations to analyze their haplotype patterns; the lack of DNA from the other 24 probands family member only presented genotype patterns. Tables 2 and 3 demonstrate the haplotype patterns of LDLR 986G>A, 1747C>T and APOB 10579C>T, respectively. As shown in Table 2, seven family members carrying
Fig. 2. Diagrams of LDLR gene showing the mutations and patient numbers identified in the study. Exons are shown as vertical boxes and introns as the lines connecting them. Bold solid lines represent deletions, double lines represent duplications.
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Table 1 Clinical characteristics of probands with common mutations. Compound heterozygotesa (n = 4)
Age, years Men Body mass index (kg/m2) Total cholesterol (mg/dl) Low-density lipoprotein cholesterol (mg/dl) High-density lipoprotein cholesterol (mg/dl) Triglycerides (mg/dl) Tendon xanthomas Premature coronary artery disease Failure to achieve goal (100 mg/dl) Rosuvastatin 20 mg Rosuvastatin 20 mg + ezetimibe 10 mg
20 (12–45) 1 (75) 22.9 (19.5–24.6) 477 (410–694) 411 (321–570) 47 (36–70) 102 (60–148) 3 (75) 2 (50)
Heterozygotes (n = 39) Probands carrying 986G>A (n = 13)
Probands carrying 1747C>T (n = 14)
Probands carrying 10579C>T (n = 12)
Differences among 3 groups
50 6 22.1 343 263 51 86 4 5
45 (6–69) 6 (42.9) 22.9 (19.5–26.5) 332 (282–453) 233 (204–354) 58 (45–95) 100 (37–203) 4 (28.6) 3 (21.4)
57 2 21.0 327 235 56 125 5 5
0.380 0.244 0.693 0.368 0.172 0.370 0.784 0.537 0.615
4 (100) 4 (100)
(11–69) (46.2) (16.4–25.9) (314–445) (204–370) (31–95) (45–237) (30.8) (38.5)
8 (61.5) 6 (46.2)
8 (57.1) 6 (42.9)
(23–69) (16.7) (18.7–23.8) (300–394) (200–318) (37–116) (46–269) (41.7) (41.7)
8 (66.7) 7 (58.3)
0.884 0.715
Values are medians (range) or number (%) of patients. a Four compound heterozygotes carrying 986G>A also had 268G>A, 1291G>A, 1432G>A, and 769C>T/1765G>A, respectively.
4. Discussion
LDLR 986G>A were associated with CGAGCCCCATGG/(dTA)n-112nt and two were associated with CGAGTCTAGCGA/(dTA)n-106nt. Examples of the inheritance of the haplotype and the co-segregation within family of each proband with LDLR 986G>A are illustrated in Figs. 3A and B. The lack of blood samples from family members among the other 8 probands with LDLR 986G>A could not give unambiguous results (Supplementary Table 1). However, the same haplotype as CGAGCCCCATGG/(dTA)n-112nt can be deduced from 7 probands' genotypes, but one had a minor change in the numbers of dTA repeat (108/106nt). Five family members carrying LDLR 1747C>T were associated with CAAGCCCCATGG/(dTA)n-112nt (Table 2). Fig. 3C shows the haplotype in one pedigree carrying LDLR 1747C>T. Although the lack of blood samples from family members among the other 9 probands precluded unambiguous haplotype, it could be deduced that all may have the same haplotype (Supplementary Table 1). Table 3 shows that three family members carrying 10579C>T were associated with ACCGAATTAGAGCACG/3′HVR-34 repeats and two were associated with GCTAAATTAGAGCACG/3′HVR-34 repeats. Figs. 3D and E show the two haplotype patterns in families. In the other 7 probands, lacking blood samples from other family members, their haplotype could be deduced as ACCGAATTAGAGCACG in 5 cases, and GCTAAATTAGAGCACG in 2 cases, but the 3′HRV repeats were 30, 32 or 34 (Supplementary Table 2).
FH mutations in LDLR are highly heterogeneous among Taiwanese; 986G>A (Cys329Tyr) and 1747C>T (His583Tyr) are the two most common mutations in LDLR genes. Only APOB 10579C>T (Arg3500Trp) mutation, not APOB 10580G>A (Arg3500Gln) was found in the cohort study. The three most common mutations, LDLR 986G>A, LDLR 1747C>T, and APOB 10579C>T, with a combined frequency of 33.1%, have phenotypes indistinguishable among the heterozygotes. These common mutations were very rarely found in Caucasians (Fisher et al., 1999; Zakharova et al., 2005), but often reported among southern Han Chinese in other Asian countries (Chang et al., 2003; Charng et al., 2006; Chiu et al., 2005; Khoo et al., 2000; Mak et al., 1998a; Punzalan et al., 2005; Sun et al., 1994; Tai et al., 1998). Mild clinical phenotypes were shown to be based on the level of lipid profiles and the presence of tendon xanthoma among the three common heterozygotes. LDLR 986G>A and 1747C>T were classified as class 2B transport-defective and class 5 recycling defective LDLR, respectively, by in vitro functional studies (Chang et al., 2003; Sun et al., 1994; Van Hoof et al., 2005). Ethnic backgrounds and environmental factors such as diet and exercise may also play an important role in the phenotypic expression. Haplotype findings, consisting of two patterns in LDLR 986G>A, one in LDLR 1747C>T and two in APOB 10580G>A in the cohort, provide
Table 2 Haplotype associated with LDLR common mutations. Proband no.
Proband 117a Proband 147a Proband 151a Proband 197a Proband 257a Proband 265a Proband 292a Proband 183a Proband 251a Proband 99b Proband 108b Proband 157b Proband 273a Proband 302b
Haplotype rs 2228671
rs 2304183
rs 12983082
rs 12710260
rs 5929
rs 4508523
rs 688
rs 2738450
rs 2738452
rs 5925
rs 5927
rs 5742911
3′UTR
C C C C C C C C C C C C C C
G G G G G G G G G A A A A A
A A A A A A A A A A A A A A
G G G G G G G G G G G G G G
C C C C C C C T T C C C C C
C C C C C C C C C C C C C C
C C C C C C C T T C C C C C
C C C C C C C A A C C C C C
A A A A A A A G G A A A A A
T T T T T T T C C T T T T T
G G G G G G G G G G G G G G
G G G G G G G A A G G G G G
112 112 112 112 112 112 112 106 106 112 112 112 112 112
Unequivocal haplotype patterns of probands and recruited family members carrying LDLR common mutations. a Probands 117, 147, 151, 183, 197, 251, 257, 265, and 292 carried 986G>A mutation. b Probands 99, 108, 157, 273, and 302 carried 1747C>T mutation.
Unequivocal haplotype patterns of probands and recruited family members carrying APOB 10579C>T mutation. The markers of rs693, rs676210, rs1801701, rs1042031, and rs1042034, are the same sites as those of XbaI, MaeI, 3500W MspI, EcoRI, and Eco57, which were studied by Tai et al., 1998, respectively.
G G G G G C C C C C A A A A A C C C C C G G G G G A A A A A G G G G G A A A A A T T T T T T T T T T A A A A A A A A A A G G G A A C C C T T C C C C C A A A G G 111 123 261 229 298 Proband Proband Proband Proband Proband
rs 1042034 rs 1042031 rs 2678379 rs 1801701 rs 13306196 rs 676210 rs 693 rs 2854725 rs 12713956 rs 11676704 rs 3791981 rs 3828293 rs 679899 rs 1469513 rs 13306198 rs 1367117
Haplotype Proband no.
Table 3 Haplotype associated with APOB 10579C>T mutation.
34 34 34 34 34
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insights into their origins and the presence of common ancestors from southern Chinese. FH patients carrying LDLR 986G>A was first reported in Hong Kong and speculated to be a possible common mutation in Chinese (Mak et al., 1998b). Later, 2 patients carrying LDLR 986G>A with different haplotype patterns was identified in Taiwan, including one pattern that was the same as Mak et al.'s findings (Chang et al., 2003). The mutation was also found in Malaysia, but there was no data of haplotype analysis (Khoo et al., 2000). Here, we demonstrated that there were 2 distinct haplotypes for LDLR 986G>A: haplotype CGAGCCCCATGG/(dTA)n-112nt was more common and probably present in 13 probands; CGAGTCTAGCGA/(dTA)n-106nt was less frequent and present only in 2 probands. The polymorphic loci and haplotype pattern in our study (rs-2228671–rs12710260–rs5929–rs688–rs5925– rs5927–rs5742911–dTA) were the same as those in Chang et al's (SfaNI–SmaI–AciI–HincII–AvaII–MspI–NcoI–dTA). The possibilities of the origin of these two haplotypes may result from recurrent 986G>A mutation occurred independently on the two haplotypes, or 986G>A mutation occurred once on a haplotype and subsequently spread through multiple recombination events. We favor that 986G>A mutation arose independently on the two haplotypes. There existed 5 different polymorphic markers in the two LDLR 986G>A haplotypes. Although Alu repeats exist frequently in the intronic sequence of LDLR and lead to frequent recombination events, multiple recombination events leading to 5 different markers is less likely and may create many more haplotype patterns rather than two as found in this study. Sun et al. first identified a Chinese carrying LDLR 1747C>T (Sun et al., 1994), but no polymorphic haplotype analysis was carried out. The family lived in Jiangsu province of China. To the best of our knowledge, this is the first time a haplotype pattern for probands carrying LDLR 1747C>T was analyzed. Five probands clearly showed the haplotype as CAAGCCCCATGG/(dTA)n-112nt, and the other 9 probands probably had the same haplotype. Thus, the common ancestor of LDLR 1747C>T may originate from the indigenous population (Yueh) living on the southeast coast of China, including Jiangsu, Fujian and Guangdong provinces. APOB 10580G>A (a CGG-to-CAG change in APOB codon 3500) is a common mutation of APOB in Caucasians. This mutation is predicted to have emerged from common ancestors who lived in Central Europe north of the Alps and the Pyrenees. APOB 10580G>A was not detected in the Finnish population, thus the mutation might have emerged after the divergence of the Finnish and other Caucasoid populations 10,000–20,000 years ago (Miserez and Muller, 2000). In contrast, APOB 10579C>T is very rarely reported in Caucasians, more often in southern Asians. Studies showed that 10579C>T haplotype patterns were different between Caucasians and Asians (Choong et al., 1997; Fisher et al., 1999; Tai et al., 1998). In previous studies in southeastern Asians, all cases were reported carrying the same diallelic markers (XbaI−/MspI+/EcoRI+) in LDLR binding domains (exons 26–29) (Choong et al., 1997; Tai et al., 1998). In agreement with these studies, our 12 APOB 10579C>T probands had the same diallelic markers in the LDLR binding domains, rs693/rs1801701/rs1042031, are the same sites as those of XbaI/MspI/EcoRI except a variety of repeated numbers at 3′ HVR (including 30, 32 and 34). In addition, two distinct haplotypes, at different sites in non-LDLR binding domains, were found in the study: ACCG-AATTAGAGCACG and GCTAAATTAGAGCACG. This could result from 1) APOB 10579C>T mutation occurring once on a haplotype and subsequently spreading by recombination events within the APOB gene later, or 2) mutations occurring independently on the two haplotypes. Interestingly, APOB 10579C>T has not yet been detected in northern Asian populations, including northern Chinese, Koreans and Japanese (Miyake et al., 2009). Considering that the same haplotype at LDLR binding domains of APOB is found among the descendants of southern Asians, mostly southern Chinese, it would be worthwhile continuing efforts to investigate the regional founder effects and gain insights into the evolution of APOB 10579C>T mutation in southern China.
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Fig. 3. Five pedigrees of families with common mutations. (A and B), Two families with members carrying 986G>A having two different haplotype patterns. (C), One family with members carrying 1747C>T (D and E), Two pedigrees of families with members carrying APOB 10579C>T having two different haplotype patterns. Below each symbol, pedigree number and lipoprotein levels are presented. Lipid profiles are presented in the following order: total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides. Proband in each family is indicated with an arrow. Square symbols indicate males; circles, females. The half-filled symbols indicate heterozygosity. No data was available for individuals with an asterisk.
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5. Conclusions LDLR 986G>A, LDLR 1747C>T and APOB 10579C>T, being the most common mutations in this series, have a combined frequency of approximately 33% among the Taiwanese population. The presence of different haplotype patterns associated with these common mutations reflects multiple waves of migration from the southeast coast of China into Taiwan during the last centuries, and haplotype homologies reflect the presence of common ancestors in southern China. A larger screening program to clarify the epidemiological features of FH and survey the regional founder effects in the southeast coast of China would be worthwhile. Supplementary materials related to this article can be found online at doi:10.1016/j.gene.2012.01.092. Acknowledgments We acknowledge the valuable technical assistance provided by Ming-Wei Lin, PhD, and Yen-Hui Ho, MSc and Hsing-Yi Liu, MSc. This study was supported by grants from the National Science Council (NSC 96-2314-B-075-055-MY3, NSC 98-2314-B-075B-005), Taipei Veterans General Hospital (V99C1-029), and Kaohsiung Veterans General Hospital, Taiwan (VGHKS 99-014). References Abifadel, M., et al., 2003. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat. Genet. 34, 154–156. Austin, M.A., Hutter, C.M., Zimmern, R.L., Humphries, S.E., 2004. Genetic causes of monogenic heterozygous familial hypercholesterolemia: a HuGE prevalence review. Am. J. Epidemiol. 160, 407–420. Brown, M.S., Goldstein, J.L., 1986. A receptor-mediated pathway for cholesterol homeostasis. Science 232, 34–47. Castillo, S., et al., 2002. The apolipoprotein B R3500Q gene mutation in Spanish subjects with a clinical diagnosis of familial hypercholesterolemia. Atherosclerosis 165, 127–135. Chang, J.H., et al., 2003. Identification and characterization of LDL receptor gene mutations in hyperlipidemic Chinese. J. Lipid Res. 44, 1850–1858. Charng, M.J., Chiou, K.R., Chang, H.M., Cheng, H.M., Ye, Z.X., Lin, S.J., 2006. Identification and characterization of novel low-density lipoprotein receptor mutations of familial hypercholesterolaemia patients in Taiwan. Eur. J. Clin. Invest. 36, 866–874. Chiou, K.R., Charng, M.J., 2010. Detection of mutations and large rearrangements of the low-density lipoprotein receptor gene in Taiwanese patients with familial hypercholesterolemia. Am. J. Cardiol. 105, 1752–1758. Chiu, C.Y., Wu, Y.C., Jenq, S.F., Jap, T.S., 2005. Mutations in low-density lipoprotein receptor gene as a cause of hypercholesterolemia in Taiwan. Metabolism 54, 1082–1086. Choong, M.L., Koay, E.S., Khoo, K.L., Khaw, M.C., Sethi, S.K., 1997. Denaturing gradientgel electrophoresis screening of familial defective apolipoprotein B-100 in a mixed
Asian cohort: two cases of arginine3500→tryptophan mutation associated with a unique haplotype. Clin. Chem. 43, 916–923. Chu, J.Y., et al., 1998. Genetic relationship of populations in China. Proc. Natl. Acad. Sci. U. S. A. 95, 11763–11768. Fisher, E., et al., 1999. Mutations in the apolipoprotein (apo) B-100 receptor-binding region: detection of apo B-100 (Arg3500→Trp) associated with two new haplotypes and evidence that apo B-100 (Glu3405→Gln) diminishes receptor-mediated uptake of LDL. Clin. Chem. 45, 1026–1038. Khoo, K.L., et al., 2000. Low-density lipoprotein receptor gene mutations in a Southeast Asian population with familial hypercholesterolemia. Clin. Genet. 58, 98–105. Lin, M., et al., 2001. The origin of Minnan and Hakka, the so-called “Taiwanese”, inferred by HLA study. Tissue Antigens 57, 192–199. Mak, Y.T., et al., 1998a. Mutations in the low-density lipoprotein receptor gene in Chinese familial hypercholesterolemia patients. Arterioscler. Thromb. Vasc. Biol. 18, 1600–1605. Mak, Y.T., et al., 1998b. Possible common mutations in the low density lipoprotein receptor gene in Chinese. Hum. Mutat. Suppl. 1, S310–S313. Miserez, A.R., Muller, P.Y., 2000. Familial defective apolipoprotein B-100: a mutation emerged in the mesolithic ancestors of Celtic peoples? Atherosclerosis 148, 433–436. Miyake, Y., Yamamura, T., Sakai, N., Miyata, T., Kokubo, Y., Yamamoto, A., 2009. Update of Japanese common LDLR gene mutations and their phenotypes: Mild type mutation L547V might predominate in the Japanese population. Atherosclerosis 203, 153–160. Punzalan, F.E., et al., 2005. A Low density lipoprotein–receptor (LDL-R) gene mutations among Filipinos with familial hypercholesterolemia. J. Atheroscler. Thromb. 12, 276–283. Scientific Steering Committee on behalf of the Simon Broome Register Group, 1991. Risk of fatal coronary heart disease in familial hypercholesterolaemia. Br. Med. J. 303, 893–896. Soria, L.F., Ludwig, E.H., Clarke, H.R., Vega, G.L., Grundy, S.M., McCarthy, B.J., 1989. Association between a specific apolipoprotein B mutation and familial defective apolipoprotein B-100. Proc. Natl. Acad. Sci. U. S. A. 86, 587–591. Sun, X.M., et al., 1994. Familial hypercholesterolemia in China. Identification of mutations in the LDL-receptor gene that result in a receptor-negative phenotype. Arterioscler. Thromb. 14, 85–94. Tai, D.Y., Pan, J.P., Lee-Chen, G.J., 1998. Identification and haplotype analysis of apolipoprotein B-100 Arg3500→Trp mutation in hyperlipidemic Chinese. Clin. Chem. 44, 1659–1665. Tejedor, M.T., et al., 2010. Haplotype analyses, mechanism and evolution of common double mutants in the human LDL receptor gene. Mol. Genet. Genomics 283, 565–574. Traeger-Synodinos, J., et al., 1998. Analysis of low density lipoprotein receptor gene mutations and microsatellite haplotypes in Greek FH heterozygous children: six independent ancestors account for 60% of probands. Hum. Genet. 102, 343–347. Ueda, M., 2005. Familial hypercholesterolemia. Mol. Genet. Metab. 86, 423–426. Van Hoof, D., Rodenburg, K.W., Van der Horst, D.J., 2005. Intracellular fate of LDL receptor family members depends on the cooperation between their ligand-binding and EGF domains. J. Cell Sci. 118, 1309–1320. Yang, K.L., Chen, S.P., Shyr, M.H., Lin, P.Y., 2009. High-resolution human leukocyte antigen (HLA) haplotypes and linkage disequilibrium of HLA-B and -C and HLADRB1 and -DQB1 alleles in a Taiwanese population. Hum. Immunol. 70, 269–276. Zakharova, F.M., et al., 2005. Familial hypercholesterolemia in St-Petersburg: the known and novel mutations found in the low density lipoprotein receptor gene in Russia. BMC Med. Genet. 6, 6. Zuliani, G., Hobbs, H.H., 1990. Dinucleotide repeat polymorphism at the 3′ end of the LDL receptor gene. Nucleic Acids Res. 18, 4300.
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Short Communication
Common mutations of familial hypercholesterolemia patients in Taiwan: Characteristics and implications of migrations from southeast China Kuan-Rau Chiou a, c, 1, Min-Ji Charng b, c,⁎, 1 a b c
Division of Cardiology, Department of Medicine, Kaohsiung Veterans General Hospital, Taiwan, ROC Division of Cardiology, Department of Medicine, Taipei Veterans General Hospital, Taiwan, ROC School of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC
a r t i c l e
i n f o
Article history: Accepted 29 January 2012 Available online 14 February 2012 Keywords: Chinese Familial hypercholesterolemia Haplotype Mutation Taiwanese
a b s t r a c t Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in low-density lipoprotein receptor (LDLR), apolipoprotein B-100 (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. This study investigated FH patients carrying common mutations in Taiwan and compared them to FH southeastern Asians. Causal FH mutations were identified by exon-by-exon sequencing with/without multiplex ligation-dependent probe amplification among 208 Taiwanese with clinically diagnosed FH. Haplotype analyses among probands and family members were undertaken using TaqMan® Assays. Totally, LDLR mutations were found in 118 probands, consisting of 61 different loci, and APOB 10579C>T mutations in 12 probands. Three mutations (64delG, 1661C>T, and 2099A>G) were novel. LDLR 986G>A (13.1%), 1747C>T (10.8%), and APOB 10579C>T (9.2%) were common mutations with no differences in phenotypes. LDLR 1747C>T associated with one haplotype (CAAGCCCCATGG/(dTA)n-112nt); LDLR 986G>A with two. APOB 10579C>T associated with the same LDLR binding-domain pattern in Taiwanese and southeastern Asians. We concluded that LDLR 986G>A, 1747C>T and APOB 10579C>T are common mutations, with combined frequency of approximately 33%. The presence of different haplotypes associated with FH common mutations in Taiwan indicates multiple historical migrations, probable multiple recurrent origins from southern China, and haplotype homologies reflect the presence of common ancestors in southern China. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Familial hypercholesterolemia (FH, OMIM #143890) is one of the most common inherited disorders of plasma lipoprotein metabolism, characterized clinically by an increased level of circulating lowdensity lipoprotein (LDL)-cholesterol that leads to lipid accumulation in tendons and arteries, premature atherosclerosis and increased risk of coronary heart disease (Ueda, 2005). FH is known to be associated with mutations in the LDL receptor (LDLR) gene, the apolipoprotein B-100 gene (APOB), and proprotein convertase subtilysin kexin 9 gene (PCSK9) (Abifadel et al., 2003; Brown and Goldstein, 1986; Soria et al., 1989). With the exception of a small number of founder populations where one or two mutations predominate, most geographically based-surveys of FH patients show a large number of Abbreviations: FH, familial hypercholesterolemia; LDLR, low-density lipoprotein receptor; APOB, apolipoprotein B; PCSK9, proprotein convertase subtilisin/kexin type 9; PCR, polymerase chain reaction; MLPA, multiple ligation-dependent probe amplification. ⁎ Corresponding author at: Division of Cardiology, Department of Medicine, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei, 112, Taiwan, ROC. Tel.: + 886 2 2875 7507; fax: + 886 2 2875 6849. E-mail address: [email protected] (M.-J. Charng). 1 K.-R. Chiou and M. -J. Charng contributed equally to this work. 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.01.092
mutations in a given population (Austin et al., 2004). Haplotype analysis of FH Caucasians improved the knowledge and understanding of the origin, spread by migrations and estimation of the ages of pathogenic mutation (Castillo et al., 2002; Miserez and Muller, 2000; Tejedor et al., 2010; Traeger-Synodinos et al., 1998), but no data has been reported concerning the Asian population. Taiwanese, the major population group in Taiwan, are the descendants of early settlers from the southeast coast (Fuchien and Kwangton province) of China during the past 400 years or more in recent history (Lin et al., 2001). Recently, anthropological studies using genetic markers by human leukocyte antigen and microsatellites revealed that Taiwanese, with Singapore Chinese and ThaiChinese, formed a southern Asian cluster with neighboring groups of Thais and Vietnamese, which is separate from the northern Asian cluster consisting of Koreans and Japanese (Chu et al., 1998; Lin et al., 2001; Yang et al., 2009). Although there have been reports of FH from the southern Chinese (Chang et al., 2003; Charng et al., 2006; Chiou and Charng, 2010; Chiu et al., 2005; Khoo et al., 2000; Mak et al., 1998a; Sun et al., 1994; Tai et al., 1998), the relationships need to be further investigated. Accordingly, we performed molecular genetic testing to investigate the frequency of mutations among unrelated FH patients in Taiwan, analyzed haplotype patterns among patients carrying common mutations, and further compared
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the patterns with those of previous FH studies from the southeastern Asians. 2. Materials and methods 2.1. Study populations Patients with LDL-cholesterol levels >190 mg/dl and positive family history of hypercholesterolemia were recruited in a genetic screening program for FH in Taiwan from Sep, 2008 to Mar, 2011. All were clinically diagnosed as having FH by the Simon Broome Familial Hypercholesterolemia Register diagnostic criteria (Scientific Steering Committee on behalf of the Simon Broome Register Group, 1991). Totally, a series of 208 apparently unrelated Taiwanese residing throughout northern, central and southern Taiwan were enrolled, including 102 who had been surveyed in a previous study (Chiou and Charng, 2010). Patients with hypercholesterolemia due to secondary causes were excluded. Premature coronary artery disease and the risk factors for heart disease, including hypertension, diabetes mellitus, smoking habits and family history of premature coronary artery disease in first-degree relatives, were recorded. Family members of the probands with the identified mutations were also invited to participate in this investigation. The study complied with the Declaration of Helsinki of the World Medical Association, was approved by the institutional review board of the hospital, and informed consent was obtained from each patient. 2.2. DNA analyses Genomic DNA was isolated from the leukocytes of peripheral blood of patients. First, exon-by-exon sequence analysis was performed as previously described on both strands of all 3 FH-causing genes—LDLR, APOB, and PCSK9T (Chiou and Charng, 2010). Next, if exon-by-exon sequence
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failed to detect mutations, DNA samples were subsequently analyzed for large deletions or insertions using the multiplex ligation-dependent probe amplification (MLPA) assay with the SALSA P062-B LDLR MLPA kit obtained from MRC-Holland (Amsterdam, the Netherlands) according to manufacturer instructions (http://www.mrc-holland.com). The nomenclature and classification of the found mutations are based on the findings in the updated FH mutation databases (http://www.ucl.ac.uk/ ldlr/Current/index.php?select_db=LDLR, accessed 11 October 2011). The effects of novel nucleotide variants were subjected to online computer program using Poly Phen-2 to predict possible impact of an amino acid substitution on the structure and function of the newly discovered mutation (http://genetics.bwh.harvard.edu/pph2/). 2.3. Genotyping LDLR and APOB polymorphisms To gain more insight into FH Taiwanese, the haplotypes of the common mutations in the study cohort were compared with those of FH Caucasians and southeastern Asians. The particular polymorphisms were selected according to both previous FH studies having haplotype analysis (Castillo et al., 2002; Chang et al., 2003; Mak et al., 1998a; Miserez and Muller, 2000; Tai et al., 1998; Zuliani and Hobbs, 1990) and the haplotype website (www.hapmap.com). Probands and their family members carrying the most common mutations were genotyped for their haplotype pattern. LDLR was genotyped at 13 polymorphic markers within or flanking the LDLR gene: rs2228671 [C/T], rs2304183 [C/T], rs12983082 [A/C], rs12710260 [C/G], rs5929 [C/T], rs4508523 [C/T], rs688 [C/T], rs2738450 [A/C], rs2738452 [A/G], rs5925 [C/T], rs5927 [A/G], rs5742911 [A/G], and hypervariable region of TA dinucleotide repeat marker at the 3′ end. APOB was genotyped at 17 polymorphic sites within or flanking the APOB gene: rs1367117 [A/C/G/T], rs13306198 [C/T], rs1469513 [C/T], rs679899 [A/C/G/T], rs3828293 [A/G], rs3791981 [A/ G], rs11676704 [G/T], rs12713956 [C/T], rs2854725 [A/C], rs693 [A/C/G/
Fig. 1. Haplotype markers and positions for the LDLR gene (Panel A) and APOB gene (Panel B).
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T], rs676210 [A/C/G/T], rs13306196 [A/G], rs1801701 [A/C/G/T], rs2678379 [A/G], rs1042031 [A/C/G/T], rs1042034 [A/C/G/T], and hypervariable region of 15 bp repeat marker at the 3′ end. Fig. 1 maps the position of the haplotype markers of LDLR and APOB genes, respectively. Genotyping was performed using commercially available Pre-made TaqMan® Genotyping assays (Applied Biosystems Inc.). TaqMan® PCR was performed according to the manufacturer's standard PCR protocol. Briefly, 20 ng genomic DNA was mixed with the supplied 2X TaqMan Universal PCR Master Mix No AmpErase UNG and 20X TaqMan Assay Mix to a final volume of 5 μl in a 384-well plate. Each sample underwent 40 amplification cycles on GeneAmp® PCR System 9700 (Applied Biosystems Inc.). Fluorescent signals of the two probes were analyzed for end-point fluorescent data on ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems Inc.). Genotype was determined automatically by Sequence Detection Software (Applied Biosystems, Foster City, CA). The number of hypervariable TA dinucleotide (dTA) repeat marker at the 3′ end of the LDLR gene was genotyped using fluorescent-labeled primers, PCR amplification and capillary electrophoresis on an ABI PRISM™ DNA Analyzer (Applied Biosystems, Foster City, CA) and analyzed using the GeneScan software version 2.2. A 15-base pairs (bp) repeat marker in the hypervariable repeat region (HVR) downstream from the 3′ end of the APOB gene was genotyped by PCR amplification and capillary electrophoresis on an ABI PRISM™ DNA Analyzer and analyzed by ABI PRISM™ SeqScape software (Applied Biosystems, Foster City, CA). An investigator without knowledge of the clinical data performed the genotyping. 2.4. Statistical analyses Continuous variables were expressed as medians with range and compared using the nonparametric Kruskal–Wallis H test. Categorical variables were expressed as numbers or percentages, and compared using Fisher's exact test. Data were collected and analyzed using SPSS version 14.0 (SPSS Inc., Chicago, IL, USA). A P value of less than 0.05 was considered to be statistically significant.
the mutations within the LDLR gene, consisting of 61 different loci (53 point mutations and 8 gene rearrangements). Greater numbers of point mutations were found in exon 4 (11/53), exon 9 (7/53), and exon 7 (5/53) of LDLR. Most of the mutations were distributed in the epidermal growth factor (EGF) precursor homology domain (exons 7– 14) (31/53) and the ligand binding domain (exons 2–6) (18/53) of LDLR. The most common mutations in this series were LDLR 986G>A (Cys329Tyr): 17 cases (13.1%), LDLR 1747C>T (His583Tyr): 14 cases (10.8%), and APOB 10579C>T (Arg3500Trp): 12 cases (9.2%). Three mutations (64delG, 1661C>T, and 2099A>G) were novel and cosegregated with hypercholesterolemia in affected family members. The 64delG mutation, G deletion at position 64, caused a frame-shift which introduced an abnormal downstream amino acids and premature termination at codon 184. The 1661C>T (Ser554Leu) and 2009A>G (Asp700Gly) mutations were predicted by Poly Phen software; the former was predicted as possibly damaging with a score of 0.774, and the latter as probably damaging with a score of 0.999. 3.2. Phenotypes of the probands carrying the most common mutations The clinical features and plasma lipid levels of the patients carrying the most common mutations are shown in Table 1. Four probands were compound heterozygotes exhibiting a homozygous phenotype, carrying LDLR 986G>A with additional 268G>A, 1291G>A, 1432G>A, and 769C>T/1765G>A mutations, respectively. All 4 probands received maximum statin dose and failed to achieve lower LDL-cholesterol below 100 mg/dl. Later, one underwent liver transplantation and one received apheresis. Table 1 shows that the three most common mutation groups among 39 heterozygotes had no gross differences in clinical features, baseline lipid profile levels, or the rates of failure to lower LDLcholesterol below 100 mg/dl in response to rosuvastatin/ezetimibe treatment. 3.3. Haplotype analyses of LDLR 986G>A, 1747C>T and APOB 10579C>T
3. Results 3.1. Identification of mutations The causal mutations of FH were identified in 130 patients, including LDLR mutations in 118 and APOB 10579C>T (a CGG-to-TGG change in codon 3500) in 12. Fig. 2 shows distribution and patient numbers of
The first and second degree relatives were available from 19 of 43 probands with common mutations to analyze their haplotype patterns; the lack of DNA from the other 24 probands family member only presented genotype patterns. Tables 2 and 3 demonstrate the haplotype patterns of LDLR 986G>A, 1747C>T and APOB 10579C>T, respectively. As shown in Table 2, seven family members carrying
Fig. 2. Diagrams of LDLR gene showing the mutations and patient numbers identified in the study. Exons are shown as vertical boxes and introns as the lines connecting them. Bold solid lines represent deletions, double lines represent duplications.
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Table 1 Clinical characteristics of probands with common mutations. Compound heterozygotesa (n = 4)
Age, years Men Body mass index (kg/m2) Total cholesterol (mg/dl) Low-density lipoprotein cholesterol (mg/dl) High-density lipoprotein cholesterol (mg/dl) Triglycerides (mg/dl) Tendon xanthomas Premature coronary artery disease Failure to achieve goal (100 mg/dl) Rosuvastatin 20 mg Rosuvastatin 20 mg + ezetimibe 10 mg
20 (12–45) 1 (75) 22.9 (19.5–24.6) 477 (410–694) 411 (321–570) 47 (36–70) 102 (60–148) 3 (75) 2 (50)
Heterozygotes (n = 39) Probands carrying 986G>A (n = 13)
Probands carrying 1747C>T (n = 14)
Probands carrying 10579C>T (n = 12)
Differences among 3 groups
50 6 22.1 343 263 51 86 4 5
45 (6–69) 6 (42.9) 22.9 (19.5–26.5) 332 (282–453) 233 (204–354) 58 (45–95) 100 (37–203) 4 (28.6) 3 (21.4)
57 2 21.0 327 235 56 125 5 5
0.380 0.244 0.693 0.368 0.172 0.370 0.784 0.537 0.615
4 (100) 4 (100)
(11–69) (46.2) (16.4–25.9) (314–445) (204–370) (31–95) (45–237) (30.8) (38.5)
8 (61.5) 6 (46.2)
8 (57.1) 6 (42.9)
(23–69) (16.7) (18.7–23.8) (300–394) (200–318) (37–116) (46–269) (41.7) (41.7)
8 (66.7) 7 (58.3)
0.884 0.715
Values are medians (range) or number (%) of patients. a Four compound heterozygotes carrying 986G>A also had 268G>A, 1291G>A, 1432G>A, and 769C>T/1765G>A, respectively.
4. Discussion
LDLR 986G>A were associated with CGAGCCCCATGG/(dTA)n-112nt and two were associated with CGAGTCTAGCGA/(dTA)n-106nt. Examples of the inheritance of the haplotype and the co-segregation within family of each proband with LDLR 986G>A are illustrated in Figs. 3A and B. The lack of blood samples from family members among the other 8 probands with LDLR 986G>A could not give unambiguous results (Supplementary Table 1). However, the same haplotype as CGAGCCCCATGG/(dTA)n-112nt can be deduced from 7 probands' genotypes, but one had a minor change in the numbers of dTA repeat (108/106nt). Five family members carrying LDLR 1747C>T were associated with CAAGCCCCATGG/(dTA)n-112nt (Table 2). Fig. 3C shows the haplotype in one pedigree carrying LDLR 1747C>T. Although the lack of blood samples from family members among the other 9 probands precluded unambiguous haplotype, it could be deduced that all may have the same haplotype (Supplementary Table 1). Table 3 shows that three family members carrying 10579C>T were associated with ACCGAATTAGAGCACG/3′HVR-34 repeats and two were associated with GCTAAATTAGAGCACG/3′HVR-34 repeats. Figs. 3D and E show the two haplotype patterns in families. In the other 7 probands, lacking blood samples from other family members, their haplotype could be deduced as ACCGAATTAGAGCACG in 5 cases, and GCTAAATTAGAGCACG in 2 cases, but the 3′HRV repeats were 30, 32 or 34 (Supplementary Table 2).
FH mutations in LDLR are highly heterogeneous among Taiwanese; 986G>A (Cys329Tyr) and 1747C>T (His583Tyr) are the two most common mutations in LDLR genes. Only APOB 10579C>T (Arg3500Trp) mutation, not APOB 10580G>A (Arg3500Gln) was found in the cohort study. The three most common mutations, LDLR 986G>A, LDLR 1747C>T, and APOB 10579C>T, with a combined frequency of 33.1%, have phenotypes indistinguishable among the heterozygotes. These common mutations were very rarely found in Caucasians (Fisher et al., 1999; Zakharova et al., 2005), but often reported among southern Han Chinese in other Asian countries (Chang et al., 2003; Charng et al., 2006; Chiu et al., 2005; Khoo et al., 2000; Mak et al., 1998a; Punzalan et al., 2005; Sun et al., 1994; Tai et al., 1998). Mild clinical phenotypes were shown to be based on the level of lipid profiles and the presence of tendon xanthoma among the three common heterozygotes. LDLR 986G>A and 1747C>T were classified as class 2B transport-defective and class 5 recycling defective LDLR, respectively, by in vitro functional studies (Chang et al., 2003; Sun et al., 1994; Van Hoof et al., 2005). Ethnic backgrounds and environmental factors such as diet and exercise may also play an important role in the phenotypic expression. Haplotype findings, consisting of two patterns in LDLR 986G>A, one in LDLR 1747C>T and two in APOB 10580G>A in the cohort, provide
Table 2 Haplotype associated with LDLR common mutations. Proband no.
Proband 117a Proband 147a Proband 151a Proband 197a Proband 257a Proband 265a Proband 292a Proband 183a Proband 251a Proband 99b Proband 108b Proband 157b Proband 273a Proband 302b
Haplotype rs 2228671
rs 2304183
rs 12983082
rs 12710260
rs 5929
rs 4508523
rs 688
rs 2738450
rs 2738452
rs 5925
rs 5927
rs 5742911
3′UTR
C C C C C C C C C C C C C C
G G G G G G G G G A A A A A
A A A A A A A A A A A A A A
G G G G G G G G G G G G G G
C C C C C C C T T C C C C C
C C C C C C C C C C C C C C
C C C C C C C T T C C C C C
C C C C C C C A A C C C C C
A A A A A A A G G A A A A A
T T T T T T T C C T T T T T
G G G G G G G G G G G G G G
G G G G G G G A A G G G G G
112 112 112 112 112 112 112 106 106 112 112 112 112 112
Unequivocal haplotype patterns of probands and recruited family members carrying LDLR common mutations. a Probands 117, 147, 151, 183, 197, 251, 257, 265, and 292 carried 986G>A mutation. b Probands 99, 108, 157, 273, and 302 carried 1747C>T mutation.
Unequivocal haplotype patterns of probands and recruited family members carrying APOB 10579C>T mutation. The markers of rs693, rs676210, rs1801701, rs1042031, and rs1042034, are the same sites as those of XbaI, MaeI, 3500W MspI, EcoRI, and Eco57, which were studied by Tai et al., 1998, respectively.
G G G G G C C C C C A A A A A C C C C C G G G G G A A A A A G G G G G A A A A A T T T T T T T T T T A A A A A A A A A A G G G A A C C C T T C C C C C A A A G G 111 123 261 229 298 Proband Proband Proband Proband Proband
rs 1042034 rs 1042031 rs 2678379 rs 1801701 rs 13306196 rs 676210 rs 693 rs 2854725 rs 12713956 rs 11676704 rs 3791981 rs 3828293 rs 679899 rs 1469513 rs 13306198 rs 1367117
Haplotype Proband no.
Table 3 Haplotype associated with APOB 10579C>T mutation.
34 34 34 34 34
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insights into their origins and the presence of common ancestors from southern Chinese. FH patients carrying LDLR 986G>A was first reported in Hong Kong and speculated to be a possible common mutation in Chinese (Mak et al., 1998b). Later, 2 patients carrying LDLR 986G>A with different haplotype patterns was identified in Taiwan, including one pattern that was the same as Mak et al.'s findings (Chang et al., 2003). The mutation was also found in Malaysia, but there was no data of haplotype analysis (Khoo et al., 2000). Here, we demonstrated that there were 2 distinct haplotypes for LDLR 986G>A: haplotype CGAGCCCCATGG/(dTA)n-112nt was more common and probably present in 13 probands; CGAGTCTAGCGA/(dTA)n-106nt was less frequent and present only in 2 probands. The polymorphic loci and haplotype pattern in our study (rs-2228671–rs12710260–rs5929–rs688–rs5925– rs5927–rs5742911–dTA) were the same as those in Chang et al's (SfaNI–SmaI–AciI–HincII–AvaII–MspI–NcoI–dTA). The possibilities of the origin of these two haplotypes may result from recurrent 986G>A mutation occurred independently on the two haplotypes, or 986G>A mutation occurred once on a haplotype and subsequently spread through multiple recombination events. We favor that 986G>A mutation arose independently on the two haplotypes. There existed 5 different polymorphic markers in the two LDLR 986G>A haplotypes. Although Alu repeats exist frequently in the intronic sequence of LDLR and lead to frequent recombination events, multiple recombination events leading to 5 different markers is less likely and may create many more haplotype patterns rather than two as found in this study. Sun et al. first identified a Chinese carrying LDLR 1747C>T (Sun et al., 1994), but no polymorphic haplotype analysis was carried out. The family lived in Jiangsu province of China. To the best of our knowledge, this is the first time a haplotype pattern for probands carrying LDLR 1747C>T was analyzed. Five probands clearly showed the haplotype as CAAGCCCCATGG/(dTA)n-112nt, and the other 9 probands probably had the same haplotype. Thus, the common ancestor of LDLR 1747C>T may originate from the indigenous population (Yueh) living on the southeast coast of China, including Jiangsu, Fujian and Guangdong provinces. APOB 10580G>A (a CGG-to-CAG change in APOB codon 3500) is a common mutation of APOB in Caucasians. This mutation is predicted to have emerged from common ancestors who lived in Central Europe north of the Alps and the Pyrenees. APOB 10580G>A was not detected in the Finnish population, thus the mutation might have emerged after the divergence of the Finnish and other Caucasoid populations 10,000–20,000 years ago (Miserez and Muller, 2000). In contrast, APOB 10579C>T is very rarely reported in Caucasians, more often in southern Asians. Studies showed that 10579C>T haplotype patterns were different between Caucasians and Asians (Choong et al., 1997; Fisher et al., 1999; Tai et al., 1998). In previous studies in southeastern Asians, all cases were reported carrying the same diallelic markers (XbaI−/MspI+/EcoRI+) in LDLR binding domains (exons 26–29) (Choong et al., 1997; Tai et al., 1998). In agreement with these studies, our 12 APOB 10579C>T probands had the same diallelic markers in the LDLR binding domains, rs693/rs1801701/rs1042031, are the same sites as those of XbaI/MspI/EcoRI except a variety of repeated numbers at 3′ HVR (including 30, 32 and 34). In addition, two distinct haplotypes, at different sites in non-LDLR binding domains, were found in the study: ACCG-AATTAGAGCACG and GCTAAATTAGAGCACG. This could result from 1) APOB 10579C>T mutation occurring once on a haplotype and subsequently spreading by recombination events within the APOB gene later, or 2) mutations occurring independently on the two haplotypes. Interestingly, APOB 10579C>T has not yet been detected in northern Asian populations, including northern Chinese, Koreans and Japanese (Miyake et al., 2009). Considering that the same haplotype at LDLR binding domains of APOB is found among the descendants of southern Asians, mostly southern Chinese, it would be worthwhile continuing efforts to investigate the regional founder effects and gain insights into the evolution of APOB 10579C>T mutation in southern China.
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Fig. 3. Five pedigrees of families with common mutations. (A and B), Two families with members carrying 986G>A having two different haplotype patterns. (C), One family with members carrying 1747C>T (D and E), Two pedigrees of families with members carrying APOB 10579C>T having two different haplotype patterns. Below each symbol, pedigree number and lipoprotein levels are presented. Lipid profiles are presented in the following order: total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides. Proband in each family is indicated with an arrow. Square symbols indicate males; circles, females. The half-filled symbols indicate heterozygosity. No data was available for individuals with an asterisk.
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5. Conclusions LDLR 986G>A, LDLR 1747C>T and APOB 10579C>T, being the most common mutations in this series, have a combined frequency of approximately 33% among the Taiwanese population. The presence of different haplotype patterns associated with these common mutations reflects multiple waves of migration from the southeast coast of China into Taiwan during the last centuries, and haplotype homologies reflect the presence of common ancestors in southern China. A larger screening program to clarify the epidemiological features of FH and survey the regional founder effects in the southeast coast of China would be worthwhile. Supplementary materials related to this article can be found online at doi:10.1016/j.gene.2012.01.092. Acknowledgments We acknowledge the valuable technical assistance provided by Ming-Wei Lin, PhD, and Yen-Hui Ho, MSc and Hsing-Yi Liu, MSc. This study was supported by grants from the National Science Council (NSC 96-2314-B-075-055-MY3, NSC 98-2314-B-075B-005), Taipei Veterans General Hospital (V99C1-029), and Kaohsiung Veterans General Hospital, Taiwan (VGHKS 99-014). References Abifadel, M., et al., 2003. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat. Genet. 34, 154–156. Austin, M.A., Hutter, C.M., Zimmern, R.L., Humphries, S.E., 2004. Genetic causes of monogenic heterozygous familial hypercholesterolemia: a HuGE prevalence review. Am. J. Epidemiol. 160, 407–420. Brown, M.S., Goldstein, J.L., 1986. A receptor-mediated pathway for cholesterol homeostasis. Science 232, 34–47. Castillo, S., et al., 2002. The apolipoprotein B R3500Q gene mutation in Spanish subjects with a clinical diagnosis of familial hypercholesterolemia. Atherosclerosis 165, 127–135. Chang, J.H., et al., 2003. Identification and characterization of LDL receptor gene mutations in hyperlipidemic Chinese. J. Lipid Res. 44, 1850–1858. Charng, M.J., Chiou, K.R., Chang, H.M., Cheng, H.M., Ye, Z.X., Lin, S.J., 2006. Identification and characterization of novel low-density lipoprotein receptor mutations of familial hypercholesterolaemia patients in Taiwan. Eur. J. Clin. Invest. 36, 866–874. Chiou, K.R., Charng, M.J., 2010. Detection of mutations and large rearrangements of the low-density lipoprotein receptor gene in Taiwanese patients with familial hypercholesterolemia. Am. J. Cardiol. 105, 1752–1758. Chiu, C.Y., Wu, Y.C., Jenq, S.F., Jap, T.S., 2005. Mutations in low-density lipoprotein receptor gene as a cause of hypercholesterolemia in Taiwan. Metabolism 54, 1082–1086. Choong, M.L., Koay, E.S., Khoo, K.L., Khaw, M.C., Sethi, S.K., 1997. Denaturing gradientgel electrophoresis screening of familial defective apolipoprotein B-100 in a mixed
Asian cohort: two cases of arginine3500→tryptophan mutation associated with a unique haplotype. Clin. Chem. 43, 916–923. Chu, J.Y., et al., 1998. Genetic relationship of populations in China. Proc. Natl. Acad. Sci. U. S. A. 95, 11763–11768. Fisher, E., et al., 1999. Mutations in the apolipoprotein (apo) B-100 receptor-binding region: detection of apo B-100 (Arg3500→Trp) associated with two new haplotypes and evidence that apo B-100 (Glu3405→Gln) diminishes receptor-mediated uptake of LDL. Clin. Chem. 45, 1026–1038. Khoo, K.L., et al., 2000. Low-density lipoprotein receptor gene mutations in a Southeast Asian population with familial hypercholesterolemia. Clin. Genet. 58, 98–105. Lin, M., et al., 2001. The origin of Minnan and Hakka, the so-called “Taiwanese”, inferred by HLA study. Tissue Antigens 57, 192–199. Mak, Y.T., et al., 1998a. Mutations in the low-density lipoprotein receptor gene in Chinese familial hypercholesterolemia patients. Arterioscler. Thromb. Vasc. Biol. 18, 1600–1605. Mak, Y.T., et al., 1998b. Possible common mutations in the low density lipoprotein receptor gene in Chinese. Hum. Mutat. Suppl. 1, S310–S313. Miserez, A.R., Muller, P.Y., 2000. Familial defective apolipoprotein B-100: a mutation emerged in the mesolithic ancestors of Celtic peoples? Atherosclerosis 148, 433–436. Miyake, Y., Yamamura, T., Sakai, N., Miyata, T., Kokubo, Y., Yamamoto, A., 2009. Update of Japanese common LDLR gene mutations and their phenotypes: Mild type mutation L547V might predominate in the Japanese population. Atherosclerosis 203, 153–160. Punzalan, F.E., et al., 2005. A Low density lipoprotein–receptor (LDL-R) gene mutations among Filipinos with familial hypercholesterolemia. J. Atheroscler. Thromb. 12, 276–283. Scientific Steering Committee on behalf of the Simon Broome Register Group, 1991. Risk of fatal coronary heart disease in familial hypercholesterolaemia. Br. Med. J. 303, 893–896. Soria, L.F., Ludwig, E.H., Clarke, H.R., Vega, G.L., Grundy, S.M., McCarthy, B.J., 1989. Association between a specific apolipoprotein B mutation and familial defective apolipoprotein B-100. Proc. Natl. Acad. Sci. U. S. A. 86, 587–591. Sun, X.M., et al., 1994. Familial hypercholesterolemia in China. Identification of mutations in the LDL-receptor gene that result in a receptor-negative phenotype. Arterioscler. Thromb. 14, 85–94. Tai, D.Y., Pan, J.P., Lee-Chen, G.J., 1998. Identification and haplotype analysis of apolipoprotein B-100 Arg3500→Trp mutation in hyperlipidemic Chinese. Clin. Chem. 44, 1659–1665. Tejedor, M.T., et al., 2010. Haplotype analyses, mechanism and evolution of common double mutants in the human LDL receptor gene. Mol. Genet. Genomics 283, 565–574. Traeger-Synodinos, J., et al., 1998. Analysis of low density lipoprotein receptor gene mutations and microsatellite haplotypes in Greek FH heterozygous children: six independent ancestors account for 60% of probands. Hum. Genet. 102, 343–347. Ueda, M., 2005. Familial hypercholesterolemia. Mol. Genet. Metab. 86, 423–426. Van Hoof, D., Rodenburg, K.W., Van der Horst, D.J., 2005. Intracellular fate of LDL receptor family members depends on the cooperation between their ligand-binding and EGF domains. J. Cell Sci. 118, 1309–1320. Yang, K.L., Chen, S.P., Shyr, M.H., Lin, P.Y., 2009. High-resolution human leukocyte antigen (HLA) haplotypes and linkage disequilibrium of HLA-B and -C and HLADRB1 and -DQB1 alleles in a Taiwanese population. Hum. Immunol. 70, 269–276. Zakharova, F.M., et al., 2005. Familial hypercholesterolemia in St-Petersburg: the known and novel mutations found in the low density lipoprotein receptor gene in Russia. BMC Med. Genet. 6, 6. Zuliani, G., Hobbs, H.H., 1990. Dinucleotide repeat polymorphism at the 3′ end of the LDL receptor gene. Nucleic Acids Res. 18, 4300.