Identification of heterozygous and homozygous insertion

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ABSTRACT

Background: Inherited forms of abnormally elevated blood lipids especially total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) with normal triglycerides (TG), also known as familial hypercholesterolemia (FH), is a major contributor of an early onset of coronary heart disease (CHD). The aim of the present study was to identify the genes responsible for causing FH in Pakistani population.

Methods: A large consanguineous family was identified and recruited for genetic analysis, which included sequencing of all the exons and introns of the low density lipoprotein receptor (LDLR) gene, in addition the exon containing the Apolipoprotein-B (APOB) mutations R3500Q and R3500W was also sequenced. Serum lipids including TC, TG, LDL-C and high density lipoprotein cholesterol (HDL-C) were determined in each individual.

Results: An insertion (2416-2417InsG) in exon 17 of the LDLR gene was found in all the affected individuals of the family. Common FH causing APOB mutations were not present in this family. Heterozygous individuals had TC levels ranging from ~300-500 mg/dL and the only homozygous individual with typical xanthomas had TC levels exceeding 900 mg/dL.

Conclusions: This is the first report of a known LDLR gene mutation causing FH in the Pakistani population. Despite a large heterogeneity of LDLR mutations there are still some common mutations which are responsible for FH throughout the world.

Keywords: low density lipoprotein receptor, familial hypercholesterolemia, mutation, heterozygous, homozygous, genetic testing

INTRODUCTION

Familial Hypercholesterolemia (FH) is a metabolic disorder which affects cholesterol metabolism and is inherited co-dominantly in a Mendelian pattern of inheritance, i.e., as a single gene disorder. It is characterized by elevated levels of total cholesterol and Low Density Lipoprotein-Cholesterol (LDL-C), and in some cases skin and tendon xanthomas, the disease manifests itself into premature Coronary Heart Disease (CHD) . Autosomal Dominant Hypercholesterolemia (ADH) is the most common form of FH with most of the individuals suffering from the disease due to mutations in LDLR, which has a worldwide prevalence of 1 in 500 heterozygotes and 1 in 106 for homozygotes . Mutations in three genes have been shown to cause ADH these are: Low Density Lipoprotein Receptor (LDLR), Apolipoprotein-B (APOB) and Pro-Protein Convertase Subtilisin like Kexin Type 9 (PCSK9) . In addition environmental factors like high fatty diet, family history etc. also plays an important role in modifying the disease condition.

Most of the cases diagnosed with FH have mutations in LDLR, in which more than 1064 variants have been listed in the LDLR database of University College London (UCL) (http://www.ucl.ac.uk/ldlr/LOVDv.1.1.0/). Most of these are point mutations, in addition other types of mutations including insertions, deletions and major rearrangements have also been reported .

In the present study we report a large multigenerational Pakistani family suffering from FH. The 38 years old male proband had significantly elevated TC levels, he had suffered from Myocardial Infarction (MI) at an early age and had undergone a coronary artery bypass, his 5 year old son also had elevated TC in addition to multiple xanthomas on his skin and tendons.

MATERIALS AND METHODS

Ethics Declaration

This study conforms 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 were informed about the study in their local language and informed written consent was also obtained from them prior to inclusion in the study.

Patient Selection and Screening

A large family having a clinically diagnosed FH patient (proband, IV-4) was selected for genetic testing and mutation screening of the genes involved in the disease. The proband's lipid profile was typical of a heterozygous FH and he had also undergone a coronary artery bypass at an early age. All the members of the family were screened for lipid profile to check their clinical status.

A five year old child (V-6) of the proband had xanthomas on his skin and his total cholesterol level was more typical of a homozygous status.

Diagnostic Criteria

US Make Early Diagnosis Prevent Early Death (MEDPED) criteria were used to diagnose the FH patients.

Blood Sampling

Approximately 10 ml of blood was drawn after 12-14 hours fasting and collected in separate tubes for DNA extraction by organic method and serum separation for the determination of lipid profile. For DNA extraction blood was collected in Acid Citrate Dextrose (ACD) vacutainer (Becton Dickinson product no 364606, Franklin Lakes, NJ, http://www.bd.com/) and for serum separation blood was collected in Z serum sep Clot Activator vacutainer tubes (Greiner bio-one, Munich, Germany). Serum was separated from clotted blood by centrifuging the vacutainers at 3000 rpm for 10 minutes.

Lipid Profile

Lipid profile including TC, TG, LDL-C and HDL-C of all the subjects were obtained with Roche/Hitachi automated system by using commercial kits CHOL (Cholesterol CHOD-PAP) Cat. No. 11489232216, LDL-C plus 2nd generation (LDL-Cholesterol) Cat. No. 03038696122, HDL-C plus 3rd generation (HDL-Cholesterol) Cat. No. 04713109190 and TG (Triglyceride GPO-PAP) Cat. No. 11488872216 (Roche Diagnostics GmbH, Germany).

Primer Designing

Polymerase Chain Reaction (PCR) primers for amplifying the promoter sequence and the eighteen coding exons of the LDLR gene including the exon-intron boundaries were designed using the software Primer 3 primer designing tool (http://www.primer3.com). For the exons 4, 5, 6, 7, 10, 11 and 13 primer sequences were obtained from Tommy Hyatt (personal communication). All the primers were checked for specificity against LDLR gene by using the NCBI Blast tool (http://www.ncbi.nlm.nih.gov).

Polymerase Chain Reaction (PCR)

Promoter sequence, all the coding exons and exon-intron boundaries of the LDLR gene were amplified by PCR in 25 µl final volume, which consisted of 0.3mM deoxyribo nucleotide triphosphates (dNTPs), 1x Taq buffer (10mM Tris-HCl pH 9.0, 50mM KCl, 0.1% Triton X-100 and 0.01% w/v gelatin), 2.0mM MgCl2, 0.5µM of each primer (forward and reverse), 1.5U of Taq polymerase and 50 ng of genomic DNA. The thermal cycling consisted of an initially denaturation at 95oC for 10 minutes, followed by 40 cycles of amplification consisting of denaturing at 95oC for 1 minute, primer annealing at 57oC for 1 minute and primer extension at 72oC for 1 minute. A final extension step was performed at 72oC for 7 minutes.

Electrophoresis

Amplified PCR products were eletrophoretically separated on 2% agarose gel containing ethidium bromide. Bands of DNA were visualized by UV transillumination and bands of desired length were excised from the gel.

DNA purification from gel

PCR Amplified DNA fragments were extracted and purified from the agarose gel using the DNA extraction kit from Fermentas Life Sciences (Cat. No. k0513, Burlington, Ontario).

DNA Sequencing and Mutation Screening

The purified fragments were then sequenced in both the forward and reverse directions. Sequences of all the exons and promoter region were analyzed for detection of mutation by using Vector NTI Advance 11 software (Invitrogen, Carlsbad, CA).

Part of exon 26 of the APOB gene from codon 3473 to 3562 containing the two common FH causing mutations R3500Q and R3500W was also sequenced, using the primers of Yang et al. .

One hundred control individuals having normal lipid profile levels and no known history of any cardiac disease were also sequenced to find the frequency of the mutation in the general population. In addition probands from a cohort of three FH families were also sequenced to see if they carried this LDLR mutation.

Statistical Analysis

        Fisher Exact Probability Test was used to calculate the significance of association between the identified mutation and the disease (http://faculty.vassar.edu/lowry/odds2x2.html). A p value of less than 0.05 was taken to be statistically significant.

RESULTS

Lipid profile test results and clinical symptoms of all the family members are given in Table 2. Sequencing results revealed a point mutation (2416_2417insG) in the LDLR gene of this family. This mutation (2416_2417insG) is an insertion of a G in a stretch of 5Gs (c2412-2416), thus the 5G being the normal allele and the 6G being the mutant allele. The proband's father (III-1) is homozygous wild type (5G/5G) , while his mother (III-2) is a carrier heterozygote (5G/6G) for the mutation. His brother (IV-3) and sister (IV-1) were also found to carry both the wild type alleles. His wife (IV-5) and two children (V-4, V-5, V-6) are heterozygous (5G/6G) affected, while the youngest child (V-6) was homozygous (6G/6G) for the mutant allele and only one child (V-3) was found to be normalhomozygous wild type. The individuals with the normal genotype (5G/5G) have normal TC levels, affected heterozygotes (5G/6G) have their TC level in the range of 300-500 mg/dL, while the only homozygous mutant (6G/6G) child (V-6) of the family has highly raised TC level of more than 900 mg/dL .

Fisher Exact Probability Test yielded a p-value of 7.62E-9 for the dominant model of inheritance, which indicates a strong association of the 6G allele (2416-2417InsG) with FH in this family , while the recessive model gave a nonsignificant association (p=0.294). The 6G allele frequency distribution was also found to be significantly associated with the disease (p=1.09E-7). No mutant allele was found in the control individuals, which also resulted in a strong association (p=6.24E-13) of the genotype with the disease when the controls and non-affected individuals were compared with the affected individuals. No APOB mutation was identified in exon 26 in this family.

DISCUSSION

Inherited defects involving cholesterol metabolism are becoming a leading cause of cardiac diseases and improper management of these disorders can lead to fatality. The LDLR is involved in lipid metabolism and cholesterol uptake by cells is dependent on the proper functioning of LDLR. Mutations in LDLR disrupt the normal functioning of the receptor thus disturbing the cholesterol balance in the blood. A large number of LDLR mutations have been identified for this gene and this number is increasing every day. In many populations of the world, including Pakistan, no data are available about the causative agent of the disease, in these countries the number of CAD related deaths is increasing rapidly.

We have identified an LDLR mutation (2416_2417insG) in a Pakistani family, which results in the production of a defective LDL-receptor containing the pVal806GlyfsX11 mutation. This frame shift mutation results in a premature stop codon at position 816 in exon 17 of the LDLR gene, which results in truncation of the protein. thus producing a defective receptor. With the help of DNA sequencing we have identified nine members as heterozygous for the FH causing LDLR mutation in this family.

LDLR is a glycoprotein and was initially identified by Brown and Goldstein in 1973 while they were trying to search the underlying cause of FH . The mutation (2416_2417insG) identified in our current study was first reported in the Swedish population and subsequently in other populations .

LDLR gene mutations are grouped into five classes which include ligand binding defects, transport defects, internalization defects, recycling defects and production of no receptors . In the case of the present study the causative mutation results in a frameshift of the gene causing the appearance of a premature stop codon at position 816 in exon 17 and results in the deletion of 45 amino acids from the C-terminus of the mature receptor. This deletion of the amino acids from the receptor results in a defective receptor belonging to the third group of mutants, which

include receptors that do not internalize .

Exon 17 of the LDLR gene encodes part of the trans-membranal and part of the cytoplasmic domain . The cytoplasmic domain of the LDLR has been shown to be involved in many processes including recycling of the LDLR to the cell surface, internalization of the LDLR-LDL-C complex, clustering of the receptors in clathrin-coated pits and sorting process . The 50 amino acids (residue 811-860) long cytoplasmic tail of the LDL receptor contains an NPXY sequence that is required for receptor internalization . Autosomal recessive hypercholesterolemia (ARH) which encodes LDLR adaptor protein 1 (LDLRAP1) is also required for clustering of the receptors in clathrin-coated pits and internalization but an intact six amino acid motif of FDNPVY on the LDLR cytoplasmic domain is required for LDLRAP1 binding . Mutations in this FDNPVY motif are known to cause LDLR clustering defects in clathrin-coated pits . Wu et al. have demonstrated using in vivo and in vitro techniques that the adaptor protein beta-arrestin 2 is an enhancer of LDLR endocytosis via its binding to the NPVY motif on the LDLR cytoplasmic tail and mutations in this region were found to impair the endocytosis process of LDLR by approximately 80% in transfected Chinese hamster ovary cells . Clustering motif of the LDLR has been shown to interact with the terminal domain of clathrin and receptor internalization rates are dependent on the hexapeptide motif affinity for clathrin while the reverse turn conformation of the receptor internalization motif regulates this affinity .

Maurer and Cooper have proposed two different models of Disabled 2 (Dab2) and ARH binding mediated LDLR endocytosis. They have shown that Dab2 mediates endocytosis independently of AP-2 and ARH while ARH requires AP-2. Dab2 binds PIP2 and clathrin by itself replacing the function of AP-2 and is involved in sorting of LDL receptors into the clathrin coated pits thus facilitating internalization in subsequent step. They also concluded that there may be some yet unidentified adaptor protein which Dab2 may recruit during internalization process in the absence of AP-2 and ARH.

Removal of 45 amino acid residues from the cytoplasmic tail of the LDLR because of the truncation of the receptor by premature stop codon as a result of the pVal806GlyfsX11 mutation will make the receptor defective and interactions of all the accessory proteins involved in the internalization process with the cytoplasmic tail motif will be disturbed. This disturbance in the internalization process will ultimately affect other intracellular processes like feed back inhibition of cholesterol biosynthesis and continuous production of endogenous cholesterol will ultimately result in the accumulation of cholesterol in the blood and elevation of TC and LDL-C levels.

Heterozygous mutations result in less serious consequences with expected cholesterol levels of 300-500 mg/dL while homozygous mutations have deleterious effects with highly elevated cholesterol levels of more than 1000 mg/dL and individuals having homozygous mutations of LDLR have been documented to hardly survive into the second decade of life, with expected life span of approximately 14-20 years .

In this study 9 individuals of a family who were heterozygous for this mutation were found to have TC and LDL-C levels between 300-500 mg/dL, which exactly matched their mutational status (5G/6G), these individuals had till date not developed any symptoms of hypercholesterolemia including xanthomas on the skin and early onset of CHD except the proband who developed CHD and underwent coronary artery bypass. Genetic screening is the most powerful technique having the potential of diagnosing the inherited disorders explicitly. The affected heterozygous individuals in our study had ages between 8-60 years, however, the one LDLR homozygous (6G/6G) child in this family had markedly elevated TC and LDL-C levels (TC >900 mg/dL and LDL-C>700 mg/dL) and also had cutaneous xanthomas. Goharkhay et al. have shown that highly elevated levels of circulating blood cholesterol and vulnerability to atherosclerotic disease in FH offsprings of high cholesterol diet fed mice might be due to the hepatic cholesterol homeostasis reprogramming. This phenomenon might be working here to lower the life expectancy of the FH homozygotes due to early onset of the atherosclerotic disease. Early diagnosis of FH heterozygotes by LDLR mutation screening is very much helpful and essential for timely management of future drastic events by dietary intervention and in some cases statin or combined therapy with other drugs like ezitimibe. In case of homozygotes, who are resistant to statin or combined therapy, liver transplant can be a better option .

Our proband's TC and LDL-C is relatively lower than other affected individuals of the family, which is because of the use of statins and consumption of low fat diet for the management of the disease. His pretreated TC and LDL-C levels were >300 mg/dL. Slightly raised TC and LDL-C levels rather than the normal values are because of the use of proteinaceous diet .

In conclusion we have identified the first LDLR mutation in the Pakistani population, although this mutation has been reported previously in other world populations, it is of significant importance in our population as till date LDLR mutation spectrum in our population has not been defined. This is the sixth report of this mutation which shows that despite the heterogeneous nature of LDLR alleles there are still some common variants which are responsible for causing similar FH phenotypes throughout the world and thus common treatment efforts for such conditions can be devised. On the other hand a large number of known allelic variants are a challenge for devising a common diagnostic and treatment strategy. . Early diagnosis by genetic testing will be helpful in managing the disease condition very well and affected individuals may lead a normal life by dietary intervention and chemotherapy where required.

Keeping in view all these problems and hurdles there is a need for more comprehensive genetic screening strategies and individualized treatment protocols including the use of statin or combined therapies, liver transplants or gene therapy.

ACKNOWLEDGEMENT

        We are thankful to the family members who participated in this study. 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. We are also grateful to Tommy Hyatt, Lab Manager for Dr. Helen H. Hobbs's Laboratory, McDermott Center for Human Growth and Development, for providing the LDLR primer sequences.

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