Familial hypercholesterolemia (FH) is commonly caused by mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B, and proprotein convertase subtilisin/kexin type 9 genes. The study aim was to investigate patients with FH in Taiwan, using molecular diagnostic methods, and compare the abnormalities in the small mutation and large DNA rearrangement subgroups. In total, 102 unrelated probands with FH were tested for mutations by exon-by-exon sequence analysis (EBESA) and multiple ligation-dependent probe amplification (MLPA). EBESA identified gene apolipoprotein B R3500W in 8 probands and 25 mis-sense, 5 nonsense, and 6 frameshift LDLR mutations in 52 probands; 11 were novel mutations. Of the 42 probands with mutations undetected by EBESA, 8 had abnormal MLPA patterns, including 2 with exon 6 to 18 deletions, 2 with exon 9 deletion, 1 with exon 6 to 8 deletions, 1 with exon 11 deletion, 1 with exon 3 to 5 duplications, and 1 with exon 7 to 12 duplications. Pedigree analysis showed mutation cosegregation with hypercholesterolemia in affected family members. Mean lipid profiles and rate of failure to lower LDL cholesterol <100 mg/dl in response to rosuvastatin/ezetimibe treatment were similar in groups with abnormal MLPA patterns and groups carrying nonsense or frameshift mutations. In conclusion, frequency of large LDLR rearrangement was approximately 8% in Taiwanese patients with FH. The response to statin drugs differed between probands with abnormal MLPA patterns and probands carrying mis-sense or undetected mutations.
Familial hypercholesterolemia (FH) is the most common and severe form of monogenic hypercholesterolemia, which, if untreated, can increase the risk of fatal coronary heart disease by up to 100-fold compared to this risk in the general population. FH is currently commonly diagnosed using exon-by-exon screening methods, such as exon-by-exon sequence analysis (EBESA), to detect mutations in genes for the low-density lipoprotein receptor (LDLR), LDLR binding domain of apolipoprotein B100 ( APOB ), and proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) encoding neural apoptosis-regulated convertase-1. However, EBESA lacks adequate sensitivity to detect certain mutation types, such as large deletions or insertions. Multiplex ligation-dependent probe amplification (MLPA) is an analytical method that detects larger DNA deletions or insertions that would otherwise be overlooked by EBESA. Currently, mutations underlying FH are largely unknown in the Taiwanese, most of whose ancestors migrated from the southeastern coast of China during the previous few centuries and mixed with local aborigines in Taiwan. Accordingly, the objectives of our study were to investigate small mutations of LDLR , APOB , and PCSK9 by EBESA and large DNA rearrangements of LDLR by MLPA in Taiwanese patients with FH and compare abnormalities in the small mutation and large DNA rearrangement subgroups.
Methods
In total, 102 unrelated probands fulfilling the diagnostic criteria for FH of the Simon Broome Familial Hypercholesterolemia Register were included. Patients with hypercholesterolemia due to secondary causes were excluded. Premature coronary artery disease and risk factors for heart disease, including diabetes mellitus, hypertension, family history of premature coronary artery disease in first-degree relatives, and smoking habits, were recorded by a specially trained nurse. Family members with identified mutations were also invited to participate in this investigation. The study protocol was approved by the institutional review board of the hospital, and informed consent was obtained from each patient. Total cholesterol, high-density lipoprotein cholesterol, and triglyceride levels were measured using commercial kits (Boehringer Mannheim, Mannheim, Germany). The concentration of LDL cholesterol was calculated according to the formula of Friedewald et al.
Genomic DNA was extracted from peripheral leukocytes using a QIAamp DNA Blood Minikit (Qiagen, Hilden, Germany). EBESA was performed as described on the 2 strands of the LDLR gene (promoter region, 18 coding exons, and flanking intron regions), APOB gene (part of the ligand binding domains in exon 26 [codons 3,431 to 3,584] and exon 29 [codons 4,310 to 4,396]), and PCSK9 gene (12 exons and flanking intron regions; primer information and sequences available upon request). Standard DNA sequencing reactions were performed using fluorescence-labeled dideoxy chain terminations with the Big Dye Terminator ABI Prism Kit and the ABI PRISM 3700 DNA Analyzer (Applied Biosystems, Foster City, California) according to the manufacturer’s instructions. Sequence variants were identified using ABI PRISM SeqScape 2.2 and confirmed using a polymerase chain reaction amplification protocol followed by restriction endonuclease digestion.
The SALSA P062-B LDLR MLPA kit was obtained from MRC-Holland (Amsterdam, The Netherlands). The LDLR MLPA kit contained 37 sets of probes, 19 of which were LDLR specific and the others were control standards. Reactions were carried out in 200-μl tubes in a model 9700 thermocycler (PE-Applied Biosystems, Foster City, California). The genomic DNA (approximately 200 to 250 ng) from each subject was diluted in 5 μl of distilled water and denatured at 98°C for 5 minutes. MLPA buffer and probe mix (1.5 μl of each) were then added and the mixture was heated at 95°C for 1 minute and incubated at 60°C for 16 hours to allow probes to anneal to target genomic DNA. Annealed probes were ligated at 54°C for 15 minutes followed by inactivation at 98°C for 5 minutes. Exactly 7.5 μl of ligation reaction mixture was removed for multiplex amplification using a pair of common primers, 1 of which was labeled with fluorescent dye 5-carboxyfluorescein. The addition of Taq polymerase to the reaction at 60°C was followed by 33 cycles of 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute, and a final extension step of 72°C for 20 minutes. Two microliters of reaction solution was used for fragment analysis on the 3130 capillary sequencer, with LIZ-500 size standards (PE-Applied Biosystems). The procedure was performed according to the manufacturer’s instructions.
MLPA data analysis was performed using GeneMapper 3.5 (PE-Applied Biosystems). Electropherograms of fragments from MLPA analysis of LDLR from normal subjects show a profile composed of 37 peaks (range 130 to 445 nt). In addition, the multiplex contains 5 control fragments generating an amplification product <120 nt; 4 DNA quantity fragments were ligation independent and were included to demonstrate that sufficient template DNA was present for the entire multiplex amplification reaction. Successful ligation was indicated by a peak (representing the fifth DNA quantity fragment, of 92 nt) of comparable size to that of other chromosome-specific probes in the multiplex. Relative areas under the curve for peaks in each sample were determined. The relative peak area under the curve for each probe was calculated using approximately 4 to 7 adjacent peaks as internal controls. The fraction of each peak was then divided by the median peak fraction of the corresponding fragment from 15 normal control samples. In the 15 control subjects, these calculations provided values close to 1.0, which corresponded to the normalized mean peak area and SDs for a subject with 2 copies of the target sequence. Copy number results >1.25 or <0.75 were flagged. Calculations were performed on samples processed within an assay run.
DNA was amplified using a long polymerase chain reaction technique and the sequence or position of the deletion break in 1 proband with homozygous FH was identified. Family members of probands carrying abnormal MLPA were also recruited for pedigree analysis to ascertain whether these abnormalities cosegregated with hypercholesterolemia in each family.
All probands, after being on the diet recommended by the National Cholesterol Education Program Adult Treatment Panel III therapeutic lifestyle changes for 4 weeks, followed a 4-step titration protocol (each step lasting 4 weeks) until the target goal (LDL cholesterol <100 mg/dl) was reached: (1) starting dose of rosuvastatin 10 mg/day, (2) rosuvastatin 20 mg/day, (3) coadministration with ezetimibe 10 mg/day, and (4) rosuvastatin 40 mg/day combined with ezetimibe 10 mg/day. Probands were divided into 5 subgroups according to molecular diagnosis: (1) heterozygous for mis-sense mutations, (2) heterozygous for nonsense or frameshift mutations, (3) heterozygous for abnormal MLPA patterns, (4) heterozygous for APOB R3500W, and (5) no mutation found by EBESA or MLPA analysis. Baseline clinical characteristics, lipid profile, and percent statin doses failing to achieve the LDL cholesterol goal (100 mg/dl) were compared among subgroups.
Continuous variables are expressed as medians with interquartile ranges and compared using nonparametric Mann–Whitney U-test. Categorical variables were expressed as numbers or percentages and compared using Fisher’s exact test. Data were collected and analyzed using SPSS 12.0 (SPSS, Inc., Chicago, Illinois). A p value <0.05 was considered statistically significant.
Results
Table 1 lists baseline clinical characteristics of the study cohort. Typical clinical manifestations such as tendon xanthoma and/or xanthelasma were identified in 29 probands (28.4%). Probands with detected gene mutations had higher levels of LDL cholesterol, lower levels of triglycerides, and a higher incidence of premature coronary artery disease.
| Variable | All Patients | Patients With LDLR APOB or PCSK9 Mutation | Patients Without LDLR APOB or PCSK9 Mutation | Difference Between 2 Groups |
|---|---|---|---|---|
| Age (years) | 44 (34–54) | 43 (33–54) | 46 (42–53) | 0.617 |
| Men | 40 (39.2%) | 29 (28.4%) | 11 (10.8%) | 0.315 |
| Body mass index (kg/m 2 ) | 23.3 (20.8–25.7) | 23.3 (20.7–26.0) | 23.3 (22.0–25.2) | 0.607 |
| Total cholesterol (mg/dl) | 344 (302–379) | 352 (301–387) | 328 (302–346) | 0.200 |
| Low-density lipoprotein cholesterol (mg/dl) | 255 (214–285) | 264 (219–299) | 224 (210–250) | 0.038 |
| High-density lipoprotein cholesterol (mg/dl) | 57 (45–65) | 56 (44–64) | 58 (45–68) | 0.447 |
| Triglycerides (mg/dl) | 136 (83–167) | 116 (78–135) | 177 (109–236) | <0.001 |
| Tendon xanthomas | 29 (28.4%) | 24 (23.5%) | 5 (4.9%) | 0.037 |
| Premature coronary artery disease | 31 (30.4%) | 25 (24.5%) | 6 (5.9%) | 0.030 |
EBESA detected gene mutations in 60 probands (58.8% of sample): 52 (51.0%) had LDLR small mutations, including 8 probands with multiple point mutation sites and 8 (7.8%) had APOB R3500W mutation. Table 2 presents a summary of LDLR point mutations, including 25 different LDLR mis-sense mutations, 5 nonsense mutations, and 6 frameshift mutations. C13S, D151E, F179C, N295S, P318L, V441I, 562delT, 656delGCCCCG, 1,174insT, W422X, and K582X were novel mutations. All LDLR mutations had cosegregated with hypercholesterolemia in affected family members. In addition, all LDLR mutations were absent from the genome of 100 healthy control subjects. PCSK 9 mutations were not detected in our series. Six PCSK9 polymorphisms and 12 APOB polymorphisms were identified in the proband genomes ( Table 3 ).
| Group | Designation ⁎ | Exon Number | Nucleotide Change † | Amino Acid Change | Proband Number ‡ | Novelty |
|---|---|---|---|---|---|---|
| Mis-sense mutations | C13S | 02 | G → C at 101 | Cys → Ser at 13 | 138 | novel |
| D69N | 03 | G → A at 268 | Asp → Asn at 69 | 147, 158, 174, 189 | ||
| R94H | 04 | G → A at 344 | Arg → His at 94 | 149 | ||
| D151E | 04 | C → G at 516 | Asp → Glu at 151 | 103, 194 | novel | |
| C176Y | 04 | G → A at 590 | Cys → Tyr at 176 | 195 | ||
| F179C | 04 | T → G at 599 | Phe → Cys at 179 | 122 | novel | |
| R236W | 05 | C → T at 769 | Arg → Trp at 236 | 117, 193 | ||
| N295 S | 07 | A → G at 947 | Asn → Ser at 295 | 119 | novel | |
| C308Y | 07 | G → A at 986 | Cys → Tyr at 308 | 112, 117, 132, 144, 147, 151, 156, 183 | ||
| P318L | 07 | T → C at 1,016 | Pro → Leu at 318 | 96, 140, 154 | novel | |
| R385W | 09 | C → T at 1,216 | Arg → Trp at 385 | 190 | ||
| R395W | 09 | C → T at 1,246 | Arg → Trp at 395 | 104 | ||
| R395L | 09 | G → T at 1,247 | Arg → Leu at 395 | 162 | ||
| A410T | 09 | G → A at 1,291 | Ala → Thr at 410 | 151 | ||
| I420T | 09 | T → C at 1,322 | Ile → Thr at 420 | 100, 184, 188 | ||
| V441I | 10 | G → A at 1,384 | Val → Ile at 441 | 185 | novel | |
| G457R | 10 | G → A at 1,432 | Gly → Arg at 457 | 144, 178 | ||
| D471N | 10 | G → A at 1,474 | Asp → Asn at 471 | 145, 146 | ||
| N543S | 11 | A → G at 1,691 | Asn → Ser at 543 | 97 | ||
| H562Y | 12 | C → T at 1,747 | His → Tyr at 562 | 99, 101, 108, 156, 157, 175 | ||
| D568N | 12 | G → A at 1,765 | Asp → Asn at 568 | 117, 193 | ||
| R574W | 12 | C → T at 1,783 | Arg → Trp at 574 | 92 | ||
| A606T | 13 | G → A at 1,879 | Ala → Thr at 585 | 81 | ||
| R612C | 13 | C → T at 1,897 | Arg → Cys at 612 | 124 | ||
| V766M | 16 | G → A at 2,389 | Val → Met at 766 | 142, 155 | ||
| Nonsense or frameshift mutations | 510delC | 04 | del[C] at 510 | frameshift at 150 | 161 | |
| 562delT | 04 | del[T] at 562 | frameshift at 167 | 150 | novel | |
| 656delGCCCCG | 04 | del[GCCCCG] at 656 | frameshift at 198 | 152 | novel | |
| C255X | 06 | C → A at 828 | Cys → stop at 255 | 129 | ||
| R329X | 07 | C → T at 1,048 | Arg → stop at 329 | 87 | ||
| 1,174insT | 08 | ins[T] at 1,174 | frameshift at 371 | 82 | novel | |
| W422X | 09 | G → A at 1,329 | Trp → stop at 422 | 188 | novel | |
| K582X | 12 | A → T at 1,807 | Lys → stop at 582 | 163 | novel | |
| 1,851delAGTATTTTGGAC | 13 | del[AGTATTTTGGAC] at 1,851–1,862 | 597–600delVFWT | 190 | ||
| 631delTA | 13 | del[TA] at 1,953, 1,954 | frameshift at 631 | 182 | ||
| Q718X | 15 | C → T at 2,215 | Gln → stop at 718 | 159 |
⁎ Named according to the amino acid numbering of Yamamoto et al.
† Nomenclature at the DNA level, according to international nomenclature and the Nomenclature Working Group.
‡ Proband 193 was homozygous for FH who carried 2 point mutations (R236W/D568N) in each allele. Probands 117, 144, 147, 151, 156, 188, and 190 were compound heterozygous for FH (R236W/D568N and C308Y; D69N and C038Y; C308Y and G457R; C308Y and A410I; C308Y and H562Y; I420T and W422X; R385W and 597 to 600delVFWT, respectively).
| PCSK9/APOB | Designation | Exon Number | Gene Position | Nucleotide Change ⁎ | Amino Acid Change |
|---|---|---|---|---|---|
| PCSK9 | L22LL | 1 | 66 | ins GCT at 66–67 | ins Leu at 22 |
| A53V | 1 | 158 | C → T at 158 | Ala → Val at 53 | |
| C92R | 2 | 277 | C → T at 277 | Cys → Arg at 92 | |
| V474I | 9 | 1,420 | G → A at 1,420 | Val → Ile at 474 | |
| P576L | 11 | 1,727 | C → T at 1,727 | Pro → Leu at 576 | |
| E670G | 12 | 2,009 | G → A at 2,009 | Glu → Gly at 670 | |
| APOB | Y1395C | 26 | 7,524 | T → C at 7,524 | Tyr → Cys at 1,395 |
| I2286V | 26 | 4,852 | T → C at 4,852 | Ile → Val at 2,286 | |
| K2548I | 26 | 4,065 | T → A at 4,065 | Lys → Ile at 2,548 | |
| M2550L | 26 | 4,060 | T → G at 4,060 | Met → Leu at 2,550 | |
| P2712L | 26 | 3,573 | G → A at 3,573 | Pro → Leu at 2,712 | |
| N2758H | 26 | 3,436 | T → G at 3,436 | Asn → His at 2,758 | |
| T3065R | 26 | 2,514 | C → G at 2,514 | Thr → Arg at 3,065 | |
| E4154K | 29 | 1,152 | C → T at 1,152 | Glu → Lys at 4,154 | |
| I4167T | 29 | 1,112 | A → G at 1,112 | Ile → Thr at 4,167 | |
| K4181E | 29 | 1,152 | C → T at 1,152 | Lys → Glu at 4,181 | |
| T4243R | 29 | 884 | C → G at 884 | Thr → Arg at 4,243 | |
| N4311S | 29 | 13,013 | C → T at 680 | Asn → Ser at 4,311 |
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