Novel and recurrent LDLR gene mutations in Pakistani hypercholesterolemia patients
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The majority of patients with the autosomal dominant disorder familial hypercholesterolemia (FH) carry novel mutations in the low density lipoprotein receptor (LDLR) that is involved in cholesterol regulation. In different populations the spectrum of mutations identified is quite different and to date there have been only a few reports of the spectrum of mutations in FH patients from Pakistan. In order to identify the causative LDLR variants the gene was sequenced in a Pakistani FH family, while high resolution melting analysis followed by sequencing was performed in a panel of 27 unrelated sporadic hypercholesterolemia patients. In the family a novel missense variant (c.1916T > G, p.(V639G)) in exon 13 of LDLR was identified in the proband. The segregation of the identified nucleotide change in the family and carrier status screening in a group of 100 healthy subjects was done using restriction fragment length polymorphism analysis. All affected members of the FH family carried the variant and none of the non-affected members nor any of the healthy subjects. In one of the sporadic cases, two sequence changes were detected in exon 9, one of these was a recurrent missense variant (c.1211C > T; p.T404I), while the other was a novel substitution mutation (c.1214 A > C; N405T). In order to define the allelic status of this double heterozygous individual, PCR amplified fragments were cloned and sequenced, which identified that both changes occurred on the same allele. In silico tools (PolyPhen and SIFT) were used to predict the effect of the variants on the protein structure, which predicted both of these variants to have deleterious effect. These findings support the view that there will be a novel spectrum of mutations causing FH in patients with hypercholesterolaemia from Pakistan.
KeywordsHypercholesterolemia LDLR Mutation Pakistani
Familial hypercholesterolemia (FH; OMIM # 143890) is an inherited heterogeneous cardiovascular disorder that is manifested primarily due to elevated cholesterol levels in the blood. Clinically the disease is characterized by high blood cholesterol, tendon xanthomas, xanthelasmas, premature corneal arcus and atherosclerosis leading to coronary heart diseases [1, 2]. To date three FH causative genes have been identified, these are inherited as monogenic autosomal dominant hypercholesterolemia (ADH). These genes are the low density lipoprotein receptor (LDLR), apo-lipoprotein B-100 (ApoB-100) and proprotein convertase subtilisin/kexin type 9 (PCSK9) . Recently a novel locus HCHOLA4 on 16q22.1 has also been identified to be associated with the disease . Of all the associated genes, mutations in the LDLR gene account for more than 70% of all the FH cases and also result in elevated cholesterol levels in sporadic hypercholesterolemia patients, thus this gene could be used as a preliminary diagnostic marker for the screening of FH as well as sporadic hypercholesterolemia cases.
To date, besides our previous work [5, 6], no LDLR mutation spectrum data are available for the Pakistani population therefore in the present study a FH family and a panel of 27 unrelated hypercholesterolemia patients were studied to identify the causative mutations.
Materials and methods
The current study adhered to the tenants of the Declaration of Helsinki and was approved by the Ethics Committee and Institutional Review Board of Shifa College of Medicine and Shifa International Hospital, Islamabad. All patients and healthy individuals were informed about the study in their local language and informed written consent was also obtained from them prior to inclusion in the study.
Sample collection and DNA isolation
Candidate gene analysis
In the case of the proband of the affected family the promoter region and the eighteen coding exons of the LDLR gene including the exon–intron boundaries were sequenced using the primers and polymerase chain reaction (PCR) conditions as described previously .
Segregation analysis and mutation screening
Polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) analysis was carried out to confirm the segregation of the identified variant in all family members (Fig. 1b), in addition unaffected random controls were also screened using this technique. The samples were PCR amplified with the primers that were used for sequencing above. The c.1916T > G variant results in the elimination of a HincII restriction site, thus for the homozygous wild type sequence the amplified product of exon 13 (596 bp) was digested into two fragments (467 and 129 bp), while the homozygous mutant sequence remained undigested. In the case of heterozygotes all three fragments were obtained after digestion (596, 467 and 129 bp).
High resolution melting (HRM) analysis of sporadic cases
A panel of 27 sporadic hypercholesterolemia patients were screened using HRM analysis, which uses melting temperature curves to differentiate between ancestral and variant alleles . HRM was performed using Quanta Accumelt kit (Quanta BioSciences, Inc. Gaithersburg) as per the manufacturer’s instructions on a Corbett Rotor Gene 6000 system (Corbett Life Sciences, NSW, Australia).
Cloning to determine the allelic status of the double heterozygous mutations
Designing of oligonuclotides for cloning and PCR amplification
For cloning the fragments of the sporadic patient (HCS-39) containing the two mutations, oligonucletides containing SacI restriction site (underlined) were designed covering exon 9 as well as the exon–intron boundaries. The forward oligonucleotide sequence was: 5′-ATC GAG CTC TCC ATC GAC GGG TCC CCT CTG ACC C-3′ and the reverse was: 5′-ATC GAG CTC AGC CCT CAT CTC ACC TGC GGG CCA A-3′. PCR amplification of the 291 bp DNA fragment was carried out by G Strom Thermocycler (GS4) in 30 μl reaction volume using 60 ng template DNA, 0.2 mM dNTPs, 1× polymix, 4 pM of each primer, 2 mM MgCl2 and 1 U Taq DNA polymerase. The thermal profile was as follows, initial denaturation at 93°C for 4 min followed by 35 cycles of denaturation at 94°C for 20 s, annealing at 60°C for 30 s and elongation at 72°C for 45 s. The final elongation was carried out at 72°C for 7 min, the amplified product was then electrophoretically separated on 2% agarose gel at 120 V for 45 min.
Cloning into pGEM vector and sequencing of the cloned fragments
The PCR product and pGEM11 vector were digested with SacI at 37°C for 3 h followed by dephosphorylation of the sticky ends and ligation overnight at 16°C using DNA ligase. The ligation mixture was then used to transform competent E coli strain (DH5α) using a classical heat shock mechanism followed by growth of the cells for an hour at 37°C in Luria Broth (LB). The transformed cells were then plated on LB agar containing ampicillin as a selection marker. After an overnight incubation at 37°C the colonies from each plate were sub-cultured overnight with shaking at 37°C in LB. This was followed by plasmid DNA preparation using Miniprep Plasmid DNA extraction Kit (Qiagen, Hilden; Germany) and sequencing of the recombinant vectors to determine the allelic status of the cloned fragments.
In order to determine the predicted effect of the variants in silico web based tool Polymorphism phenotyping (polyPhen; http://genetics.bwh.harvard.edu/pph/) was used, which predicts the possible impact of substituted amino acid residues on the structure and function of the protein, based upon physical and comparative considerations . Moreover HOPE (Homotopy optimization using perturbations and ensembles) , tool was used to determine the effect of variation on the 3D structure of the protein.
Statistical analysis was carried out for the determination of the association of the identified variant with the disease in the FH family, for this Fisher Exact Probability test (http://faculty.vassar.edu/lowry/odds2x2.html) was used, where a p value of less than 0.05 was taken to be statistically significant.
Analysis of FH family
The mode of inheritance in the clinically diagnosed FH family was autosomal dominant with 7 affected members (Fig. 1a). The proband (IV:5) had undergone coronary artery bypass graft (CABG) before the age of 45 years, in addition the affected members were on statin treatment at the time of blood collection and had no xanthomas when clinically assessed.
The genotype and lipid profiles of the FH family members
Fischer exact probability test for the association of mutant G allele of c.1916T > G mutation in the Familial Hypercholesterolemia family
N = 6 (%)
N = 7 (%)
RM > 0.05
N = 6 (%)
N = 7 (%)
Analysis of sporadic hypercholesterolemia patients
Under normal physiological conditions LDLR plays a crucial role in maintaining the cholesterol level in the body [12, 13]. LDLR is a cell surface receptor that is responsible for the uptake of LDL-C from the circulating blood. This uptake process is mediated by apolipoprotein B100 (ApoB), which is part of the LDL particle carrying ~65–70% of the cholesterol in the blood [14, 15]. The internalization of ApoB-LDL-LDLR complex is carried out through clathrin coated pit mediated endosome formation. The low pH inside the endosome results in release of LDL from the receptor, which is then recycled back to the outer membrane . Most of the LDLR mutations identified so far in different world populations result in malfunctioning of the receptor. Defective LDLR usually leads to elevated TC and LDL-C levels, which is the major risk factor for the development of coronary artery disease (CAD) and its related complications .
The ligand binding domain of LDL receptor is located near the N-terminal and is encoded by exons 2–6 of the gene. This domain consists of seven LDL class A motifs that are responsible for electrostatic attachment of LDL-C-ApoB to LDLR via the receptor binding motif of ApoB. Adjacent to the ligand binding domain, is the epidermal growth factor precursor homology (EGFP) domain that is encoded by exons 7–14 and consists of three EFG-like motifs in which the last two have 5 YWTD repeats in between. These repeats along with two flanking EGF-like motifs form a six bladed β-propeller that is responsible for the release of LDL particle in the acidic environment of the endosome and also in recycling of the receptor .
Mutations in the β-propeller and C terminal region of the last EGF-like motif has been previously reported to cause hypercholesterolemia due to defects in recycling of the receptors because of disruption of the β-propeller structure [15, 17]. As a result the LDL particles are not released from the receptor in the endosomes, which result in the degradation of the LDLR-LDL complex inside the endosomes. This defective recycling may ultimately lower the quantity of cell surface LDLR, which subsequently results in elevation of TC and LDL-C in the blood of the patients.
The identified variants (p.V639G, p.T404I and p.N405T) in the present study are in close proximity of the YWTD repeats and are present at the C terminal region of the third EGF-like motif and are therefore highly likely to be responsible for higher LDL levels in the patients. The combined effect of the latter two variants is difficult to assess, as neither Polyphen nor SIFT allow the analysis of two variants next to each other on the same allele, but it seems unlikely that these two damaging variants would compensate for each other. The V639 in LDLR is at a common site for substitution mutations, Fouchier et al.  and Nauck et al.  have previously reported a missense substitution (c.1916T > A, p.(V639D)) at the same site in the Dutch and German populations. When we performed in silico analysis of the p.V639D variant, it revealed that both the changes i.e. V639G and V639D were comparable because both the changes affect hydrogen bonding in the 3D structure of the protein. The amino acid conservation of the V639 shows the Val to be conserved only in higher vertebrates, while in most of the vertebrates the V639 is often replaced with Ile (Fig. 2). We also performed protein modeling for this change, which revealed that although Ile is larger in size as compared to the Val but this change does not have any effect on the hydrogen bonding potential of the protein.
To date 1,122 mutations have been identified in the promotor, exons and intron–exon boundaries of the LDLR gene , where most of them result in elevated cholesterol levels, the novel c.1916T > G variant in exon 13 in the current Pakistani family is also believed to be the causative variant involved in the higher than normal levels of serum cholesterol in the patients, where the homozygous individual IV:5 had 372 mg/dL serum TC, whereas heterozygotes had levels that ranged between 222–280 mg/dL TC (Table 1). In the case of the sporadic patient (HCS-39) both the changes (p.T404I and p.N405T) were found to be on the same allele and as the cholesterol level (238 mg/dL) of this patient was similar to that of heterozygous cases, therefore this points to the possibility that both of the substitutions are acting as a single heterozygous mutant allele.
Despite the huge mutational data currently available for the LDLR gene there is a need to fully define all the pathogenic variants in the different world populations, especially Pakistan where there is no comprehensive data available for the disease. Among the identified variants most of the LDLR mutations are rare and occur in single families , as Pakistan is a multi-racial nation therefore this heterogeneous population is expected to carry a number of novel LDLR mutations. Recently we reported a triple nucleotide variant in the LDLR gene that results in the production of a truncated protein lacking the β-propeller region , which was predicted to cause the observed cardiac complications including premature coronary artery disease leading to myocardial infarction. Our current work further defines the LDLR mutation spectrum in sporadic patients and families, which hopefully will result in timely management of the disease and better understanding of the molecular mechanisms involved in pathogenesis.
This work was financially supported by grant No 934 from the Higher Education Commission of Pakistan, awarded to RQ. Part of this work was supported by Shifa College of Medicine through a core grant to RQ. WA was supported by an IRSIP grant from HEC for his studies at UCL. We would like to thank the FH families and healthy individuals for donating their blood. SEH, RW and WP are supported by the British Heart Foundation (RG008/08).
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