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Title: Detection of a novel mutation Y468X in exon 10 of the low-density lipoprotein receptor gene causing heterozygous familial hypercholesterolemia among French Canadians

Abstract

Familial hypercholesterolemia (FH) is caused by mutations in the low-density lipoprotein (LDL) receptor gene and characterized by raised plasma LDL-cholesterol (C) and premature coronary heart disease. FH has higher frequency among French Canadians (FC) in northeastern Quebec than in most other populations, 1:154 vs. 1:500. In FC, five mutations account for all the mutant alleles in homozygous FH and 81% in heterozygous FH; thus 19% are uncharacterized at the molecular level. We investigated the possibility of additional mutations(s), and direct sequencing of asymmetric PCR fragments showed a novel mutation (468 stop-codon) in the heterozygous form in exon 10 of the LDL receptor gene. This mutation results from cytosine to guanine transversion, converting codon 468 (TAC) encoding tyrosine into TAG stop-codon (Y468X). This nonsense mutation will result in a truncated protein shortened by 371 amino acids which will be rapidly degraded. However, we did not ascertain the functional aspects. We rather assessed its effects on the extent of elevation of LDL-C in heterozygous FH children. The Y468X mutation resulted in raised LDL-C levels which were comparable to subjects with a non-functional `null` allele due to deletion of the promoter region and exon 1 (237{plus_minus}49 vs. 248 {plus_minus}41 mg/dl; mean{plus_minus}SD, p<0.05). Themore » relative frequency of the Y468X mutation in a cohort of 343 children suspected for FH is 4.1% and it ranks number 4 in term of its prevalence. High frequency of FH among FC is attributed to a founder effect due to a high prevalence of one mutation; it is suggested that this novel mutation with low prevalence may be of later entry in this population.« less

Authors:
; ;  [1]
  1. Laval Univ., Quebec (Canada)
Publication Date:
OSTI Identifier:
134742
Report Number(s):
CONF-941009-
Journal ID: AJHGAG; ISSN 0002-9297; TRN: 95:005313-1480
Resource Type:
Journal Article
Resource Relation:
Journal Name: American Journal of Human Genetics; Journal Volume: 55; Journal Issue: Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; PATIENTS; METABOLIC DISEASES; HEREDITARY DISEASES; PHENOTYPE; CARDIOVASCULAR DISEASES; GENES; GENE MUTATIONS; DNA SEQUENCING; MUTATION FREQUENCY; DETECTION; RECEPTORS; LIPOPROTEINS; QUEBEC; EXONS; POLYMERASE CHAIN REACTION; CHOLESTEROL; CODONS; AMINO ACIDS; STATISTICS

Citation Formats

Couture, P., Simard, J., and Moorjani, S.. Detection of a novel mutation Y468X in exon 10 of the low-density lipoprotein receptor gene causing heterozygous familial hypercholesterolemia among French Canadians. United States: N. p., 1994. Web.
Couture, P., Simard, J., & Moorjani, S.. Detection of a novel mutation Y468X in exon 10 of the low-density lipoprotein receptor gene causing heterozygous familial hypercholesterolemia among French Canadians. United States.
Couture, P., Simard, J., and Moorjani, S.. Thu . "Detection of a novel mutation Y468X in exon 10 of the low-density lipoprotein receptor gene causing heterozygous familial hypercholesterolemia among French Canadians". United States. doi:.
@article{osti_134742,
title = {Detection of a novel mutation Y468X in exon 10 of the low-density lipoprotein receptor gene causing heterozygous familial hypercholesterolemia among French Canadians},
author = {Couture, P. and Simard, J. and Moorjani, S.},
abstractNote = {Familial hypercholesterolemia (FH) is caused by mutations in the low-density lipoprotein (LDL) receptor gene and characterized by raised plasma LDL-cholesterol (C) and premature coronary heart disease. FH has higher frequency among French Canadians (FC) in northeastern Quebec than in most other populations, 1:154 vs. 1:500. In FC, five mutations account for all the mutant alleles in homozygous FH and 81% in heterozygous FH; thus 19% are uncharacterized at the molecular level. We investigated the possibility of additional mutations(s), and direct sequencing of asymmetric PCR fragments showed a novel mutation (468 stop-codon) in the heterozygous form in exon 10 of the LDL receptor gene. This mutation results from cytosine to guanine transversion, converting codon 468 (TAC) encoding tyrosine into TAG stop-codon (Y468X). This nonsense mutation will result in a truncated protein shortened by 371 amino acids which will be rapidly degraded. However, we did not ascertain the functional aspects. We rather assessed its effects on the extent of elevation of LDL-C in heterozygous FH children. The Y468X mutation resulted in raised LDL-C levels which were comparable to subjects with a non-functional `null` allele due to deletion of the promoter region and exon 1 (237{plus_minus}49 vs. 248 {plus_minus}41 mg/dl; mean{plus_minus}SD, p<0.05). The relative frequency of the Y468X mutation in a cohort of 343 children suspected for FH is 4.1% and it ranks number 4 in term of its prevalence. High frequency of FH among FC is attributed to a founder effect due to a high prevalence of one mutation; it is suggested that this novel mutation with low prevalence may be of later entry in this population.},
doi = {},
journal = {American Journal of Human Genetics},
number = Suppl.3,
volume = 55,
place = {United States},
year = {Thu Sep 01 00:00:00 EDT 1994},
month = {Thu Sep 01 00:00:00 EDT 1994}
}
  • The frequency of familial hypercholesterolemia (FH) is 1:500 in most populations and 1:154 among French Canadians (FC) in northeastern Quebec. FH is caused by numerous mutations in the LDL receptor gene; however, among FC five mutations are responsible for FH and these are two large deletions and three missense mutations. Due to the limited number of mutations, the diagnosis of FH at the molecular level is feasible in FC and, therefore, we have developed new approaches for rapid detection of the three point mutations using PCR amplification of genomic DNA with specific primers and size separation of fragments after digestionmore » with appropriate restriction enzymes. The point mutation in exon 3 (W66G) results from a T to G transversion. After PCR amplification of exon 3 with an exonic and a mismatch primer, DNA samples from normal subjects contain a mismatch three nucleotides upstream the mutant nucleotide 259, thus creating a BglII restriction site. The presence of the mutation at nucleotide 259 abolishes the BglII site. Another missense mutation in FC is due to a G to A transition in exon 4 (E207K). The same approach was used for the deletion of this missense mutation. After amplification, DNA samples from normal subjects contains a mismatch two nucleotides downstream from the mutant nucleotide at position 682, thus creating an MboII restriction site. In FH patients bearing the E207K mutation, the MboII site is abolished. The third missense point mutation in FH patients is located in exon 14 and is caused by a G to A transition (C646Y). The mismatch primer approach was used for rapid detection. The presence of a mismatch one nucleotide upstream from the 2000 mutant and the C646Y mutation create a NlaIII restriction site. In normal subjects, the absence of the mutation abolishes this restriction site. These new methodologies for rapid detection will be useful for both early screening and population genetic studies of FH.« less
  • The coding region of the low density lipoprotein (LDL)-receptor gene from a patient (MM) with homozygous familial hypercholesterolemia (FH) has been sequenced from six overlapping 500-base-pair amplified fragments of the cDNA from cultured skin fibroblasts. Two separate single nucleotide base changes from the normal sequence were detected. The first involved substitution of guanine for adenine in the third position of the codon for amino acid residue Cys-27 and did not affect the protein sequence. The second mutation was substitution of thymine for cytosine in the DNA for the codon for amino acid residue 664, changing the codon from CCG (proline)more » to CTG (leucine) and introducing a new site for the restriction enzyme PstI. MM is a true homozygote with two identical genes, and the mutation cosegregated with clinically diagnosed FH in his family in which first cousin marriages occurred frequently. LDL receptors in MM's skin fibroblasts bind less LDL than normal and with reduced affinity. Thus this naturally occurring single point mutation affects both intracellular transport of the protein and ligand binding and occurs in growth factor-like repeat C, a region that has not previously been found to influence LDL binding.« less
  • Familial hypercholesterolemia (FH) is a widespread human disease. FH is caused by a disturbance in the catabolism of low density lipoproteins (LDL), which results from mutations in the LDL receptor gene (LDLR). The majority of mutations in the LDLR locus is represented by large-scale reorganizations in the above gene. In this study, we describe a novel 5 kb deletion, which eliminates exons 4 to 6 in the LDLR gene. 16 refs., 2 figs., 1 tab.
  • The ligand-binding domain of the low density lipoprotein (LDL) receptor is composed of seven cysteine-rich repeats, each {approx} 40 amino acids long. Previous studies showed that if the first repeat of the ligand-binding domain (encoded by exon 2) is deleted, the receptor fails to bind an anti-LDL receptor monoclonal antibody (IgG-C7) but continues to bind LDL with high affinity. Cultured fibroblasts from a Black South African Xhosa patient (TT) with the clinical syndrome of homozygous familial hypercholesterolemia demonstrated high-affinity cell-surface binding of {sup 125}I-labeled LDL but not {sup 125}I-labeled IgG-C7. previous haplotype analysis, using 10 restriction fragment length polymorphic sites,more » suggested that the patient inherited two identical LDL receptor alleles. The polymerase chain reaction technique was used to selectively amplify exon 2 of the LDL receptor gene from this patient. Sequence analysis of the amplified fragment disclosed a deletion of six base pairs that removes two amino acids, aspartic acid and glycine, from the first cysteine-rich ligand binding repeat. The mutation creates a new Pst I restriction site that can be used to detect the deletion. The existence of this mutant allele confirms that the epitope of IgG-C7 is located in the first cysteine-rich repeat and that this repeat is not necessary for LDL binding. The mutant gene produced a normally sized 120-kilodalton LDL receptor precursor protein that matured to the 160-kilodalton form at less than one-fourth the normal rate.« less
  • We report the detection of low-density lipoprotein (LDL) receptors by the technique of receptor blotting in fibroblasts from a patient with homozygous familial hypercholesterolemia (FHC) previously classified as ''receptor negative.'' Solubilized receptors were electrophoresed, transferred to nitrocellulose paper, treated with LDL followed by radiolabeled antibody to LDL, and visualized by autoradiography. GM 2000 FHC fibroblasts revealed LDL receptors with an apparent molecular weight of approximately 140,000, the same as in normal cells. LDL receptor activity by blotting in GM 2000 cells was greatly diminished in comparison with normal cells, but was calcium dependent. Receptor activity was also detectable by conventionalmore » monolayer binding and degradation assays. Thus, GM 2000 cells have profoundly diminished LDL receptor activity, but retain the genetic capacity to make LDL receptor material of normal molecular weight that is capable of binding LDL. Previous studies have demonstrated the presence of trace amounts of immunoreactive LDL receptor protein in fibroblasts from some receptor-negative FHC homozygotes. These studies are extended by demonstrating the ability of this material to bind LDL.« less