Molecular genetic diagnosis is currently the most specific method for identifying patients with FH. Although numerous LDLR gene mutations have been identified in FH patients, genetic data for the Malaysian population are rare [4, 8]. The present study cohort of 154 patients with clinical features of FH was relatively young (with a mean age of 44.6 years), and already had a high prevalence of CVD (68.2%). The average LDL-C level was classified as high, according to National Cholesterol Education Program (NCEP ATP III) classification [9]. This, together with the high prevalence of CVD, were consistent with the recognized association of elevated LDL-C levels with a high risk of atherosclerotic disease [10].
The presence of gene sequence variants in the promoter region and in exons 2-15 of the LDLR gene was investigated, and LDLR variants were identified in 117 out of 154 patients (76.0%). These comprised 8 previously unreported and 21 previously reported LDLR sequence variations. Eight pathogenic mutations were reported, including one intronic (c.190+4A>T).
One subset was relatively common, such as a variant in exon 10 (p.Arg471Gly), which was found in 17 patients. The donor splice site mutation (c.190+4A>T) was the commonest among Filipino FH [11], while L547V mutation was predominant among Japanese FH [12]. The frequent detection of a deleterious mutation may be the result of consanguinity, recurrent mutational events, genetic drift, or multiple introduction of the mutation into a population [13].
The overall mutation rate of 42.2% (65/154), which was higher than that reported in previous studies of Malaysian FH (26%) [4] or Filipino FH (20%) patients [11], but lower than that reported in European populations (52%) [14].
Only the p.Cys255Ser, p.Asn591Asn, and p.Val653Val variants detected in the current study have previously been reported among Malaysian FH subjects [8]; the remainders were newly identified in this study population. This may be attributed to the high sensitivity of DHPLC for detecting gene sequence variants, and the restricted inclusion criteria.
Five missense mutations that resulted in altered amino acids (p.GluA101Lys, p.Asp139His, p.Glu201Lys, p.Cys255Ser and p.Asp700Glu) were predicted to exert pathogenic effects based on the in silico analyses.
p.Glu101Lys was predicted to be pathogenic by Alamut™software with change from medium size and acidic (Glutamate) to large size and basic (Lysine). This residue occurs in the second disulfide-rich repeat in the binding domain of the receptor protein, and affects processing and intracellular transport of the newly synthesized protein. It was suggested that the normal formation of disulfide bonds in the second repeat may be impeded in the mutant protein [15].
p.Asp700Glu was predicted to be pathogenic by Alamut™software with change in the 3 D structure of the protein. It was reported among Spanish FH subjects [16].
p.Asp139His is a missense mutation, reported for the first time in this study, located in the ligand binding domain and was predicted to be ''probably damaging'' by PolyPhen due defect in the ligand binding site.
p.Glu201Lys and p.Cys255Ser are previously reported mutations among Russian patients [17] and Malaysian patients [8], respectively and were predicted to be ''probably damaging'' by PolyPhen.
It has been reported that a significant proportion (approximately 18.9%) of the gene sequence variants observed in patients with FH have no effect on the protein coding sequence [18]. These variations are often located in the 5' splice-donor site or the 3' splice-acceptor site of an intron, and are predicted to result in either exon skipping or retention of an intron in the mRNA, although this has not always been confirmed experimentally. One such variant, c.190+4A>T, has been reported in this study as well as in the Netherlands [19] and the Philippines [11]. We reported the possibility that it may cause splicing abnormalities through an in-silico analysis. However, the true effect should be experimentally clarified using splicing analysis.
For c.1060+10G>C variant in intron 7, it was reported in French and Greek FH patients [20, 21]. In the present study this variant was reported among FH subjects and control groups with a non significant difference in the occurrence between both groups which may indicate that this variant may be a polymorphism.
c.1359-30C>T is detected in unrelated FH individuals in Denmark [22]. Webb et al. [23] failed to detect any effect of this polymorphism on plasma lipid concentrations in their population. p.Ile398 Ile, p.Pro539Pro, and p.Cys27Cys are previously reported among European [24], Russian [25] and Chinese FH patients [26], respectively, with unknown clinical associations with the disease.
p.Trp666ProfsX45 was identified in four unrelated patients within this study, which could be attributed to a founder effect. The extent or nature of the deletion in the DNA was not clear, and the amplified mutant fragment was therefore cloned and sequenced. The predicted translation products of the alleles carrying this mutation resulted in the creation of premature stop codon downstream of the deletion.
We identified a large genomic rearrangement (c.1706_1845dup;p.Asp616IlefsX96), a duplication of exon 12, in the LDLR gene which have not previously been detected among Malaysian FH patients [8, 27]. It is interesting to note that while large deletion generally represents 85% of large rearrangements in the LDLR gene [5], our finding showed the predominance of a large duplication and the absence of large deletions. Large duplications seem to be much more prevalent than large deletions among the Malaysian population, while large deletions are more common than large duplications in North European Caucasians [28]. Therefore, the prevalence of large duplications/rearrangements in FH patients indicates that MLPA should be included in the diagnostic service for dominant hypercholesterolemia.
The duplicated exon 12 (c. c.1706_1845dup; p.Asp616IlefsX96) is predicted to cause a frameshift protein, with premature termination 95 positions downstream. Due to its nucleotide number (1706-1845 bp) that were duplicated, either deletion or duplication of exon 12 will cause frameshifted protein, (i.e. disruption of reading frame starting from the point of deletion or duplication). The mutation effect is different with that of previously reported deletion of exon 15 [29] which is a common yet mild cause of FH in Finland. Deletion of exon 15 caused only internal truncation of the protein without inducing premature termination, since the reading frame was maintained.
In silico predictions of the eight variants categorized as pathogenic may provide grounds for further experimental studies aimed at revealing mRNA abnormalities, as well as altered protein-protein interactions and loss of function.
mRNA expression studies could be performed for the c.1706_1845dup; p.Asp616IlefsX96 and c.1996_2012del;p.Trp666ProfsX45, as they were also predicted to be targets for nonsense-mediated decay.
There were significant differences in baseline LDL-C levels between the two patients groups; subjects with pathogenic mutations had a mean baseline LDL-C level of 5.2 mmol/l, compared with level of 4.9 mmol/l in patients of the non pathogenic group.
Also those with pathogenic mutations show a more severe phenotype (higher frequencies of CVD, TX, and family history of hyperlipidemia) than those with non pathogenic variants which may increase the likelihood of their pathogenicity. The exact decision about the functional implications should be done by in vitro functional study.
The presentation of TX as a diagnostic sign of FH was interesting in the current study population, as only 40.9% of FH subjects showed TX. This emphasizes the importance of DNA analysis, because no definite diagnosis of FH can be made without the identification of TX or a defined mutation.
Regarding those patients who showed definite clinical manifestations of FH but no identified LDLR sequence variations, the following possibilities exist: (1) they may have defects that are linked to the LDLR gene variant within deep intronic regions, affecting expression or splicing of the gene; (2) they may have exon defects that have not been screened; (3) the defect may lie in another gene involved in LDL-C metabolism, such as the apolipoprotein gene or the proprotein convertase subtilisin/kexin type 9 (PCSK 9) gene; or (4) it is possible that polygenic factors interacting with environmental factors may lead to a clinical diagnosis mimicking the FH phenotype [30]. It is also likely that some patients with no identified mutation do not have true monogenic autosomal dominant hypercholesterolemia. The clinical diagnostic criteria for FH are not precise, and no family studies have been carried out in patients with any detected mutations to clarify this.
There were several limitations of the present study. FH patients are generally seen by cardiologists when cardiovascular complications appear, but the disease's etiology is not always investigated. This could explain the low number of FH patients recruited in the study sample. This cohort was therefore representative of FH patients who are referred to specialists because of a severe lipid phenotype, but not of those who remain undiagnosed, who may have milder phenotypes [31]. The DHPLC method used here has been reported to show a sensitivity of 96% [32], and an additional 4% of LDLR mutations may thus have remained undetected. Furthermore, several exons with a reportedly low mutation detection rate were not examined; the detection rate may have been increased by a further 3-4% (representing the percentage of mutations in exon 1, 16,17 and 18 [5] if all exons had been included. Additionally, only 30-50 base pairs of the LDLR introns were examined by the primers used here, and mutations may exist in other regions, although few have been reported [5].