Molecular genetic evidence for a founder effect in familial hypercholesterolemia among French Canadians

Summary Familial hypercholesterolemia (FH), at a prevalence of about 1 in 200 in the French-Canadian population, is caused by a 10-kb deletion in the low-density lipoprotein (LDL) receptor gene in 60% of French-Canadian FH heterozygotes. We genotyped 159 FH patients who carry this common mutation and 221 healthy French-Canadian controls for five DNA restriction fragment length polymorphisms (RFLPs) of the LDL receptor gene. The allele numbers for four of the five RFLPs differed significantly (P < 0.001) between FH patients and control subjects. Our results suggest that the 10-kb deletion carrier allele is associated with a single haplotype (called the B haplotype). In a family study including a patient homozygous for the 10-kb deletion, we showed that the B haplotype cosegregates with the deletion in affected members and that the B haplotype is also associated with the normal allele in some members of the family. We identified 15 different haplotypes for the normal allele in 10-kb deletion carrier FH heterozygotes. These results offer strong support, at a molecular level, for the hypothesis of a founder effect for the 10-kb deletion in the French-Canadian population.This work was presented in part at the meeting: Advances in Gene Technology: the molecular biology of human genetic disease, Miami, Florida, 1991     

Identification of a splice-site mutation in the low density lipoprotein receptor gene by denaturing gradient gel electrophoresis

We have applied the denaturing gradient gel electrophoresis (DGGE) technique to detect sequence variations in exon 9 of the low density lipoprotein receptor (LDLR) gene in individuals with heterozygous familial hypercholesterolemia (FH). A fragment containing exon 9 and 25 base pairs (bp) of the intron boundary sequence at either side was amplified. To this fragment a 40-bp GC-clamp was attached by the polymerase chain reaction (PCR). We have analyzed a total of 165 DNA samples of FH patients and have detected a mutation in three cases. Two patients were found to have the previously described South African G to A transition in codon 408. In a third patient, we observed a different banding pattern of the DNA fragments on DGGE indicating a different mutation. The mutant homoduplex band of this sample was purified from the gel, cloned in an AT-vector and sequenced. Sequence analysis demonstrated a G to A transition of the consensus G-nucleotide at the intron 9 splice donor site. Cosegregation between this mutation and elevated plasma cholesterol levels was observed in family members of this FH patient. This mutation probably prevents normal splicing of the mRNA and represents the first identified splice-site mutation in the LDLR gene. We conclude that the use of DGGE of GC-clamped PCR-amplified exon sequences offers a general strategy for the detection of disease-producing mutations in the LDLR gene.     

Cloning of the breakpoints of a deletion associated with choroideremia

Summary In order to characterize a previously described submicroscopic deletion encompassing (part of) the choroideremia (tapetochoroidal dystrophy: TCD) gene, we have cloned a 10.5-kb EcoRI fragment from the patient's DNA: this fragment carries the junction between both deletion endpoints (junction fragment). The distal portion of this fragment defines a new marker within, or just distal to the TCD gene. This marker has been employed to confirm the diagnosis in several affected family members, and to rule out carriership in a female at risk with conspicuous clinical signs.This work was presented in part at the 5th International Retinitis Pigmentosa Congress, Melbourne 1988     

Low density lipoprotein receptor founder mutations in Afrikaner familial hypercholesterolaemic patients: a comparison of two geographical areas

Summary Afrikaners with familial hypercholesterolaemia (FH) were screened for the presence of three point mutations in the low density lipoprotein receptor gene that were previously described as being relatively common in this population. The prevalence and distribution of the mutations were compared in 27 unrelated homozygous and 79 unrelated heterozygous FH Afrikaner patients from two regions in South Africa, the Transvaal and Cape Provinces. The relative distribution of the three mutations was similar in the two regions, with the FH1 mutation being the most prevalent (66%), followed by the FH2 mutation (27%) and the FH3 mutation (7%). Interestingly, defects other than the three common mutations are more common in the Cape than in the Transvaal; thus the three known mutations account for 98% of FH alleles in the Transvaal and only 74% in the Cape Province. None of the patients carried the recently described familial defective apolipoprotein B100 mutation. These results establish that three founder mutant genes occur amongst the Afrikaner and are responsible for the overall high prevalence of FH in this population.     

A missense mutation in the low density lipoprotein receptor gene causes familial hypercholesterolemia in Sephardic Jews

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the low density lipoprotein (LDL) receptor gene. Here, we characterize an LDL receptor mutation that is associated with a distinct haplotype and that causes FH in the Jewish Sephardic population originating from Safed, a town in northern Israel. The mutation was found in eight FH families originating from this community comprising 10% of heterozygote FH index cases screened in Israel. The mutation was not found in four additional FH heterozygotes whose hypercholesterolemia co-segregated with an identical LDL receptor gene haplotype. A guanine to cytosine substitution results in a missense mutation (asp¹⁴⁷ to his) in the fourth repeat of the binding domain encoded by exon 4 of the LDL receptor gene. The mutant receptor protein was synthesized in cultured cells as a 120kDa precursor form that failed to undergo normal processing to a mature cell surface form. Most of the receptor precursors were degraded in the endoplasmic reticulum. The small number of mutant receptors on the cell surface were unable to bind LDL or very low density lipoprotein. The abnormal behavior of the mutant receptor was reproduced by site-directed mutagenesis and expression of the mutant protein in CHO cells. The mutation can be diagnosed by allele-specific oligonucleotide hybridization of polymerase chain reaction amplified DNA from FH patients.     

Identification of a deletion in the LDL receptor gene A Finnish type of mutation

A cDNA probe for the low density lipoprotein (LDL) receptor gene was used to screen DNA samples from 52 unrelated Finnish patients with the heterozygous form of familial hypercholesterolemia (FH) and 51 healthy controls. Southern blot analysis using the restriction enzyme PvuII revealed an abnormal 11 kb (kilo base-pair) restriction fragment in 16 (31%) of the patients but none of the controls. A more detailed restriction enzyme analysis of the DNA from patients revealed a mutation which apparently is due to an 8 kb deletion extending from intron 15 to exon 18 of the LDL receptor gene. Co-segregation of FH with the mutated gene was demonstrated in three families. These data are consistent with a ‘founder gene effect’ and support the assumption that recombinant DNA methods may have great impact on the diagnostics of FH in genetically homogeneous populations.     

Screening for a prevalent LDL receptor mutation in patients with severe hypercholesterolaemia

Summary A rapid new method for the diagnosis of familial hypercholesterolaemia (FH) detects the deletion extending from intron 15 to exon 18 in the low density lipoprotein (LDL) receptor gene, i.e. the FH-Helsinki mutation responsible for a major portion of FH in Finland. Amplification of the DNA sequences flanking the deletion in the mutant allele generated an abnormal 391-bp product that could be detected by photographing the ethidium-bromide-stained agarose gel after electrophoresis. Up to 50 samples can be analysed in about 8h. The method was validated by comparison with a routine Southern blot technique. The deletion was found in 23 out of 37 patients with a clinical diagnosis of FH (62%) and in 2 out of 73 with primary hypercholesterolaemia without a clinical diagnosis of FH within a series of 110 consecutvie patients with severe hypercholesterolaemia (serum cholesterol > 8mmol/l). The data indicate that DNA techniques may provide a supplementary aid for the routine diagnosis of FH and suggest that the polymerase chain reaction in particular may offer major advantages because of its simplicity and rapidity.     

A new missense mutation (Cys297→Phe) of the low density lipoprotein receptor in Italian patients with familial hypercholesterolemia (FHTrieste)

During a survey of the mutations of the low density lipoprotein receptor (LDL-R) gene in Italian patients with familial hypercholesterolemia (FH), we identified a novel point mutation, that creates a new EcoRI site at the 5 end of exon 7, in a heterozygous FH subject (FH-100). The sequence of a cDNA fragment encompassing exon 7 showed the presence of a GT transversion in codon 297; this created a new EcoRI site and produced a missense mutation, leading to a Cys₂₉₇Phe substitution in repeat A of the epidermal growth factor (EGF) precursor homology domain of LDL-R. Since the substitution of Cys₂₉₇ disrupts the intracellular transport of the LDL-R protein, as previously demonstrated by site-directed mutagenesis, we suggest that this mutation is the cause of FH in the FH-100 proband. We screened the DNA of 303 Italian FH patients by amplification of exon 7 from genomic DNA followed by digestion with EcoRI or by Southern blotting. Two individuals (FH-64 and FH-127) were found to be carriers of the Cys₂₉₇Phe mutation. Restriction fragment length polymorphism analysis demonstrated that, in two kindreds (FH-64 and FH-100), the haplotype in linkage with the Cys₂₉₇Phe mutation was the same, suggesting the presence of a common ancestor. The Cys₂₉₇Phe mutation has been designated FH_Trieste after the name of the city in Northern Italy from which probands FH-100 and FH-127 originate.     

Efficient in vivo gene delivery using modified Tat peptide with cationic lipids

A combination of modified HIV-1 Tat (mTat) peptide and cationic lipids, FuGENE HD (FH), dramatically enhanced transfection efficiency across a range of cell lines when compared to mTat or FH alone (Biomaterials 35:1705–1715 2014). The efficiency of this Tat peptide combination was significantly higher than many commercial non-viral vectors. In this present study, we tested the feasibility of this non-viral vector, mTat/FH, in vivo using plasmid DNA encoding a luciferase gene. The results of the in vivo studies showed that animals administered mTat/FH/DNA intramuscularly had significantly higher and longer luciferase expression (≈7 months) than those with mTat/DNA, FH/DNA, or DNA alone. Histological evaluation showed little immune response in the muscles, livers, and kidneys of mice administered with the mTat/FH. The combination of mTat with FH could significantly improve transfection efficiency, expanding the potential use of non-viral gene vectors in vivo.     

SNTG1, the gene encoding γ1-syntrophin: a candidate gene for idiopathic scoliosis

Idiopathic scoliosis (IS) affects approximately 2%–3% of the population and has a heritable component. The genetics of this disorder are complex. Here, we describe a family in which a pericentric inversion of chromosome 8 co-segregates with IS. We have used fluorescence in situ hybridization to identify cloned DNAs that span the breakpoints on the two arms of the chromosome. We have identified a bacterial artificial chromosome (BAC) of 150 kb that crosses the q-arm breakpoint and a BAC of 120 kb that crosses the p-arm breakpoint. The complete genomic DNA sequence of these BACs has been analyzed to identify candidate genes and to localize further the precise breakpoints. This has revealed that the p-arm break does not interrupt any known gene and occurs in a region of highly repetitive sequence elements. On the q-arm, the break occurs between exons 10 and 11 of the -1 syntrophin (SNTG1) gene. Syntrophins are a group of cytoplasmic peripheral membrane proteins that associate directly with dystrophin, the Duchenne muscular dystrophy gene; 1-syntrophin has been shown to be a neuronal cell-specific protein. Mutational analysis of SNTG1 exons in 152 sporadic IS patients has revealed a 6-bp deletion in exon 10 of SNTG1 in one patient and a 2-bp insertion/deletion mutation occurring in a polypyrimidine tract of intronic sequence 20 bases upstream of the SNTG1 exon 5 splice site in two patients. These changes were not seen in a screen of 480 control chromosomes. Genomic DNAs from seven affected individuals within the family of a patient carrying the 6-bp deletion were typed to determine whether the alteration co-segregated with IS. The deletion was only observed in five out of these seven individuals. Thus, although genetic heterogeneity or multiple alleles cannot be ruled out, the 6-bp deletion does not consistently co-segregate with the disease in this family.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Recurrent and novel LDL receptor gene mutations causing heterozygous familial hypercholesterolemia in La Habana

The molecular basis of familial hypercholesterolemia (FH) in three families of Spanish descent from La Habana was investigated by the candidate gene approach. The Arg3500Gln mutation of apolipoprotein B-100 was not found. Identification of low density lipoprotein receptor (LDLR) gene haplotypes segregating with FH guided the characterisation of three point mutations by automated sequencing. One, a Val408Met missense mutation, a founder mutation in Afrikaner FH patients, was recurrent, being associated with a distinct DNA haplotype. The other two, Glu256Lys and Val776Met missense mutations, were novel and modified highly conserved residues. These mutations were absent in normolipidemic subjects and were associated in heterozygous carriers with twice the cholesterol levels observed in noncarriers. Noticeably, cardiovascular complications were rarely observed in older heterozygotes, even in those with the Afrikaner FH-2 mutation. These findings confirm the molecular heterogeneity of LDLR gene mutations causing FH and the variability of their expression across different populations.     

Familial adenomatous polyposis: A submicroscopic deletion at the APC locus in a family with mentally normal patients

Cytogenetically visible deletions that include the adenomatosis polyposis coli (APC) locus have repeatedly been reported in mentally handicapped polyposis patients. We report on a family with a submicroscopic deletion of about 200 kb including more than the 3 half of the APC gene and the adjacent DPI gene. The deletion was detected by linkage analysis with flanking and intragenic markers and proven by in situ hybridisation with intragenic cosmid clones. All the familial adenomatous polyposis (FAP) patients and persons at risk in the family show normal behaviour and intelligence. Thus, it is conceivable that at least some of the FAP patients in whom mutations could not be identified by routine methods may have large but submicroscopic deletions.     

No evidence for linkage of long QT syndrome and chromosome 11p15.5 markers in a Chinese family: evidence for genetic heterogeneity

Recently the defective gene locus in seven Caucasian families with the Romano-Ward form of long QT syndrome (LQT) has been mapped to chromosome 11p. To understand the molecular basis of LQT in Chinese, a three-generation family was investigated. Fourteen family members were studied and five individuals were diagnosed to be affected, according to electrocardiographic criteria. Two genomic DNA probes (c-Ha-ras-3-HVR and insulin-5-HVR) and one tetranucleotide repeat polymorphism (THZ) derived from chromosome 11p15.5 loci and previously demonstrated to be closely linked to LQT were used as probes to analyze this family. A lod score of less than -2 was noted for all three polymorphisms. Our data show that there was no evidence of linkage between these three loci and the gene for LQT in this studied family. We believe that this result provides additional evidence for genetic heterogeneity of LQT.     

Two partial deletion mutations involving the same Alu sequence within intron 8 of the LDL receptor gene in Korean patients with familial hypercholesterolemia

Twenty-eight unrelated persons heterozygous for familial hypercholesterolemia (FH) were screened to assess the frequency and nature of major structural rearrangements at the low-density lipoprotein (LDL) receptor gene in Korean FH patients. Genomic DNA was analyzed by Southern blot hybridization with probes encompassing exons 1–18 of the LDL receptor gene. Two different deletion mutations (FH29 and FH110) were detected in three FH patients (10.7%). Each of the mutations was characterized by the use of exon-specific probes and detailed restriction mapping mediated by long-PCR (polymerase chain reaction). Mutation FH29 was a 3.83-kb deletion extending from intron 6 to intron 8 and FH110 was a 5.71-kb deletion extending from intron 8 to intron 12. In FH29, the translational reading frame was preserved and the deducible result was a cysteine-rich A and B repeat truncated protein that might be unable to bind LDL but would continue to bind β-VLDL. FH110 is presumed to be a null allele, since the deletion shifts the reading frame and results in a truncated protein that terminates in exon 13. Sequence analysis revealed that both deletions have occurred between two Alu-repetitive sequences that are in the same orientation. This suggested that in these patients the deletions were caused by an unequal crossing over event following mispairing of two Alu sequences on different chromatids during meiosis. Moreover, in both deletions, the recombinations were related to an Alu sequence in intron 8 and the deletion breakpoints are found within a specific sequence, 27 bp in length. This supports the hypothesis that this region might have some intrinsic instability, and act as one of the important factors in large recombinational rearrangements. Received: 3 April 1996 / Revised: 19 August 1996     

Distribution and characterization of a Sandhoff disease-associated 50-kb deletion in the gene encoding the human β-hexosaminidase β-chain

Summary A 50-kb deletion was demonstrated in the gene encoding for the -subunit of human hexosaminidase (HEXB), using field inversion gel electrophoresis (FIGE) of SfiI-digested chromosomal DNA from patients with Sandhoff disease. We investigated 14 patients from different parts of Europe and found no deletion in 5 patients, 2 patients homozygous for the deletion, and 7 patients with the deletion in one allele. The distribution of the 50-kb deletion was approximately in agreement with the Hardy-Weinberg equilibrium. The deletion was characterized using chromosomal DNA from one of the two homozygous patients. Restriction fragments were hybridized with a 1.6-kb (almost complete) and a 0.4-kb (5) HEXB cDNA clone. It appeared that the deletion started in intron 5, extending in the 5 direction and causing the loss of exon 1–5 and the promoter area of the HEXB gene.     

Identification of a nonsense mutation in the amelogenin gene (AMELX) in a family with X-linked amelogenesis imperfecta (AIH1)

A family with X-linked amelogenesis imperfecta (XAI) is described in which the disease is associated with a nonsense mutation in exon 5 of the amelogenin gene. This mutation involves a single base deletion (CCCCCCC) in the exon in an affected male, his sister and his mother. The effect of this deletion is to alter the reading frame and to introduce an inappropriate TGA stop codon (an opal mutation) into the exonic sequence of the amelogenin gene immediately 3 of the mutation. The clinical features in the examined members of this family indicate that, in some individuals, the most noticeable defect is of enamel hypoplasia. In others, the hypoplastic changes are subtle and might have been overlooked on cursory examination; the most noticeable change is of enamel colour, indicating a degree of hypomineralisation. We propose that the amelogenin gene is implicated in both the formation of enamel of normal thickness and in the normal mineralisation process.     

The molecular defect in a family with mild atypical osteogenesis imperfecta and extreme joint hypermobility: Exon skipping caused by an 11-bp deletion from an intron in one COL1A2 allele

Summary We have investigated a family with an autosomal dominantly inherited connective-tissue defect causing extreme joint hypermobility, premature osteoporosis and late-onset fractures. Analysis of collagenous proteins from affected individuals showed a deletion in some 2(I) chains. Peptide mapping localized this to the CB peptide 2CB4, which covers the N-terminal one-third of the protein chain. Polymerase chain reaction amplification and sequencing of cDNA derived from this region of the mRNA identified á heterozygous deletion of the 54 by comprising exon 9. Similar analysis of the genomic DNA revealed an 11-bp deletion from bp3 to bp13 of IVS-9. This disrupts the consensus 5 splice signal (GTAAGT) and leads to exon skipping. In a family study of 13 affected and unaffected family members using both heteroduplex formation and direct analysis for the deletion, all of the affected, but no unaffected individuals, were found to carry the deletion. This generated a positive Lod score of 2.6 with the Liped programme.     

Identification of a recurrent insertion mutation in the LDLR gene in a Pakistani family with autosomal dominant hypercholesterolemia

Familial Hypercholesterolemia (FH) results in elevated levels of blood lipids including total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) with normal triglycerides (TG). This disease is one of the major contributors towards 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, for this purpose a large consanguineous FH family was selected for genetic analysis. Serum lipid levels, including TC, TG, LDL-C and high density lipoprotein cholesterol (HDL-C), were determined in patients and healthy controls. In order to find the causative mutation in this family, direct sequencing of the low density lipoprotein receptor (LDLR) gene was performed. In addition the part of the Apolipoprotein-B (APOB) gene containing the mutations R3500Q and R3500W was also sequenced. Affected individuals of the family were found to have raised TC and LDL-C levels. Sequencing revealed an insertion mutation (c.2416_2417InsG) in exon 17 of the LDLR gene 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. 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.     

家族性高胆固醇血症患者低密度脂蛋白受体基因新突变位点的鉴定

家族性高胆固醇血症(hypercholesterolemia familial,FH)是由于低密度脂蛋白受体(low density lipoprotein receptor,LDLR)基因突变导致的常染色体显性遗传性疾病,临床上表现为多发黄色瘤、高水平血浆LDL、早发性冠心病及有阳性家族史。本研究通过临床症状结合血脂测定诊断出一个FH家系,其纯合子FH患者的血浆总胆固醇水平高达19．05mmol／L,LDL达17．06mmol／L,并有黄色瘤;而杂合子FH患者的血浆总胆固醇水平为7．96mmol／L,LDL为5．55mmol/L,并有心绞痛症状和黄色瘤。我们对该FH家系患者LDLR基因的PCR扩增DNA片段进行测序,发现纯合子FH患者LDLR基因Exon4区域内发生了GAG683GCG突变,即编码LDLR第683位的谷氨酸被丙氨酸替换,而杂合子FH患者该位点呈现杂合突变。此基因型与临床诊断遗传谱完全一致。同时,利用获得Epstein-Barr(EB)病毒转化型人永生淋巴细胞株(EBV-Ls)与荧光探针DiI标记的LDL结合反应,再通过流式细胞仪检测结果显示,具有功能性LDLR的EBV-Ls细胞比例,在纯合子FH患者(7．02％)和杂合子FH患者(62．64％)均比健康对照者(84．69％)低,纯合子FH患者LDLR活性仅为健康对照者的8．29％、而杂合子FH患者LDLR活性约为健康对照者的73．96％,前者呈现非常显著的降低。这些EBV-Ls细胞LDLR的功能变化分析,有力地支持了该FH家系的临床诊断和DNA测序结果。经查阅最新的UMD-LDLR完全版证实,本研究发现鉴定的GAG683GCG突变是人LDLR基因的新突变位点。     

Rapid detection of deletions in the Duchenne muscular dystrophy gene by PCR amplification of deletion-prone exon sequences

Summary Using the polymerase chain reaction (PCR) technique, we have screened the DNA of 42 patients with Duchenne or Becker muscular dystrophy for deletions within the DMD gene. Two regions within putative deletion hot spots of this gene were tested, and deletions were found in 16.6% of patients. The oligonucleotide primers employed in this study initiate the amplification of exon sequences and were used to test the suitability and reliability of PCR in deletion screening and prenatal diagnosis using various numbers of cycles and artificial contamination ratios. We compared our approach with both multiplex DNA amplification and Southern blot analysis. A comparative evaluation of currently available techniques is presented.