Screening of Mutations in the LDLR Gene in South indian Patients With Familial Hypercholesterolemia

INTRODUCTION
Familial hypercholesterolemia is a metabolic inherited autosomal dominant disorder
In 1938 Carl Muller described FH as an inborn error of metabolism Characterized by increased plasma low density lipoprotein (LDL) level Mainly caused due to loss of function mutations in LDL receptor (LDLR) gene Frequency of affected individuals is 1/500 worldwide, homozygous FH occurs in 1/106 individuals (Goldstein et al., 2001) In India still the exact distribution of FH mutations is unknown
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SYMPTOMS
FH affected patients present with High cholesterol level High LDL levels
above 260mg/dl in children under 16 290mg/dl in an adult Greater than 170-200mg/dl in children Greater than 220mg/dl in adults

Tendon xanthoma
Cholesterol deposits in the eyelids (xanthelasmas)

Familial history of myocardial infarction below age of 50

Premature atherosclerosis
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LDL RECEPTOR
LDL receptor is a cell surface glycoprotein synthesized in rough endoplasmic reticulum LDLR gene consists of 18 exons spanning 45kb on distal short arm of chromosome 19 (Sdhof et al., 1985) A mosaic protein of 840 amino acids

Mediates endocytosis of LDL

REVIEW OF LITERATURE
In 1979 Goldstein et al., stated that an elevated plasma LDL level due to FH leads to atherosclerosis and eventually death Over 1000 mutations in LDLR gene alters the functionality of the receptor (Radar et al., 2003)

Seven unrelated South African Indians with familial hypercholesterolemia revealed two novel (g.1215C>G and g. 2356A>T) and two recurrent missense mutations in CpG dinucleotides at bases 661 and 682 in the low density lipoprotein receptor (LDLR) gene (Kotze et.al., 1997)

Ashavaid et.al., (2000) identified two mutations (i.e. 242insG in exon 3 and 397insG in exon 4) in LDL receptor causing familial hypercholesterolemia in Indian subjects by a simplified rapid PCR- heteroduplex method.

Kulkarni et.al., (2011) reported 3 novel mutations : one in exon 3 (i.e. g.18298A>C) and two in exon 10 (i.e. g.29372_29373insC and g.29209A>G) by sequence analysis of the LDL receptor (LDLR) gene.

OBJECTIVES
To screen mutations in LDLR gene in the South Indian patients clinically diagnosed with Familial Hypercholesterolemia by using
Extraction of Genomic DNA from whole blood sample using salting out method Amplification of genomic DNA encoding LDLR gene using exon specific primers for all 18 exons Perform PCR- heteroduplex analysis for PCR products Identification of mutations by Sequencing

METHODOLOGY
Sample collection: I. Patients having characteristic features of FH, properly diagnosed and reported by clinicians will be taken for the study II. Ethnically matched unrelated Normal individuals sample will be taken as control Isolation of DNA from whole blood: I. II. I. Collection of 5-10 ml whole blood from FH patients. Isolation of genomic DNA by Salting out method (Miller et al.,1987) Quantification of DNA using photometer
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 Screening of mutation by PCR- Heteroduplex method

I. Primers available in the literature are taken for exon amplification II. Amplification of exons of LDLR gene using designed primers III. Heteroduplex analysis with corresponding mutant and wild type DNA fragments DNA sequencing for the candidate gene: I. Automated DNA Sequencing by ABI Prism Sequencer II. Analysis of sequencing data by Seqman, DNA Star Software
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PCR- Heteroduplex Analysis

PCR amplification Mixing and denaturation of PCR amplified product + Known normal control Annealing

Mismatch bps induce bends or kinks

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EXPECTED RESULTS
1. Screening of the recurrent mutations in LDLR gene

2. Identification of any novel mutations in the Indian patients affected with Familial Hypercholesterolemia
3. Distribution of frequency of mutation in the Indian patients of various ethnic groups

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REFERENCES
Ashavaid, Altaf A. Kondkar and Kappiareth G. Nair (2000); Identification of Two LDL-Receptor Mutations Causing Familial Hypercholesterolemia in Indian Subjects by a Simplified Rapid PCR-Heteroduplex Method. Clinical Chemistry 46 (8) : 1183-1185
Ashavaid T. F., Altaf A. K. and Nair K. G. (2000); Molecular basis of familial hypercholesterolemia: an indian experience. Indian Journal of Clinical Biochemistry 15: 11-19 Hattori H., Nagano M. et.al. (1999) Identification of Recurrent and Novel Mutations in the LDL Receptor Gene in Japanese Familial Hypercholesterolemia. HUMAN MUTATION Mutation in Brief 248 Kotze MJ, Loubser O, Thiart R, Nico J, de Villiers P, Langenhoven E, et al., (1997); CpG hotspot mutations at the LDL receptor locus is a frequent cause of familial hypercholesterolemia among South African Indians. Clin Genet 51:394398

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 Kulkarni S., Basavraj S.et.al. (2011); Mutation Analysis of the LDL Receptor Gene in Indian Families with Familial Hypercholesterolemia. Asian Journal of Medical Sciences 2: 82-86
Rubinsztein D. C., Coetzee G. A. et al. (1992) Identification and properties of the proline664-leucine mutant LDL receptor in South Africans of Indian origin. Journal of Lipid Research 33: 1647-1655 Setia N., Verma I. C., Khan B., and Arora A. (2012) Premature Coronary Artery Disease and Familial Hypercholesterolemia: Need for Early Diagnosis and Cascade Screening in the Indian Population. Cardiology Research and Practice doi:10.1155/2012/658526

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