A Chinese homozygote of familial hypercholesterolemia: identification of a novel C263R mutation in the LDL receptor gene

Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene; it is characterized by a high concentration of LDL, which frequently gives rise to tendon xanthomas and premature coronary artery disease (CAD). Individuals with heterozygous FH in China often exhibit a milder phenotype than those in other countries. The diagnosis of heterozygous FH relies on the clinical phenotype and this does not always permit unequivocal diagnosis of the disease. In the course of investigation of FH in a Chinese population sample, we found a family whose proband showed a markedly raised concentration of LDL cholesterol in plasma, and the presence of skin and tendon xanthomata. We used single-strand conformation polymorphism (SSCP) analysis to screen all the 18 exons and the exon-intron boundaries of the LDLR gene. One novel homozygous mutation, replacing T by C at nucleotide 850 in exon 6 was identified. This change substituted cysteine for arginine at codon 263 (C263R) of the LDLR. By means of mutant allele-specific amplification, we unequivocally diagnosed six heterozygotes with this novel mutation in the proband's family. Received: October 30, 2000 / Accepted: December 18, 2000     

Identification of the valine 408 to methionine mutation in the LDL receptor in a Greek patient with homozygous familial hypercholesterolemia

We have identified the cytosine to thymine change in the codon for amino acid 408 which causes valine to be replaced by methionine in exon 9 of the LDL receptor gene in a 12-year-old Greek boy living in Germany, with homozygous familial hypercholesterolemia, by using polymerase chain reaction-amplified genomic DNA and subsequent restriction digestion. Homozygosity was confirmed by direct DNA sequencing. The mutation was present in both his parents, and his brother, grandmother, uncle and cousin. Six restriction fragment length polymorphisms of the LDL receptor gene were used to determine the haplotype of the defective allele. The haplotype was different from the one reported earlier in African Afrikaners and from Holland. We conclude that the mutation in the Greek boy probably occurred as an independent mutation. Because the parents are from different areas in Greece, this mutation may be common in Greeks.     

A novel LDLR mutation, H190Y, in a Utah kindred with familial Hypercholesterolemia

Heterozygous familial hypercholesterolemia (FH) is a serious disorder causing twice normal low-density lipoprotein (LDL) cholesterol levels early in childhood and very early coronary disease in both men and women. Treatment with multiple medications together with diet can normalize cholesterol levels in many persons with FH and prevent or delay the development of coronary atherosclerosis. Previously published blood cholesterol criteria greatly under-diagnosed new cases of FH among members of known families with FH and over-diagnosed FH among participants of general population screening. Thus, there is a need for accurate and genetically validated criteria for the early diagnosis of heterozygous FH. In the course of investigations of coronary artery disease in Utah, we identified a family whose proband showed elevated plasma levels of LDL cholesterol. To carry out molecular genetic diagnosis of the disease, we screened DNA samples for mutations in all 18 exons and the exon-intron boundaries of the LDL receptor gene (LDLR). Novel point mutations were identified in the proband: a C-to-T transversion at nucleotide position 631, causing substitution of tyrosine for histidine at codon 190 in exon 4 of the LDLR gene. The mutant allele-specific amplification method was used to examine 12 members of the family recruited for the diagnosis. This method helped to unequivocally diagnose 7 individuals as heterozygous for this particular LDLR mutation, while excluding the remaining 5 individuals from carrier status with FH. Received: March 29, 1999 / Accepted: June 18, 1999     

Identification of recurrent and novel mutations in the LDL receptor gene in Japanese familial hypercholesterolemia

We used the denaturing gradient gel electrophoresis (DGGE) method to investigate 120 Japanese patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequence of the low density lipoprotein (LDL) receptor gene. Fourteen aberrant DGGE patterns were found, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations (C317S, F382L A410T, L547V, and E693K), two nonsense mutations (W512X and K790X), four frameshift mutation (355del7, 1246ins5, 1687ins1, and 2035ins1), one splicing mutation (1845+2 T→C), and two inframe mutations (661ins21 and 1115del9/ins6) were identified. Six of these mutations (L547V, E693K, W512X, 355del7, 1687ins1, and 20354ins1) have not been described before in FH. These newly identified mutations cosegregated in their family members with defective LDL receptor activity and hypercholesterolemia, and are thought to be causal for the FH phenotype. These results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Japanese population. Hum Mutat 14:87, 1999. © 1999 Wiley‐Liss, Inc.     

Soluble epoxide hydrolase variant (Glu287Arg) modifies plasma total cholesterol and triglyceride phenotype in familial hypercholesterolemia: intrafamilial association study in an eight-generation hyperlipidemic kindred

Plasma lipid and lipoprotein in general reflect the complex influences of multiple genetic loci, for instance, even familial hypercholesterolemia (FH), a representative example of monogenic hyperlipidemia, often presents with phenotypic heterogeneity. In the course of investigating familial coronary artery disease in Utah, we studied 160 members of an eight-generation extended family of FH in which 69 members were affected with type IIa hyperlipoproteinemia (HLPIIa; high plasma cholesterol) and ten with type IIb hyperlipoproteinemia (HLPIIb; high plasma cholesterol as well as plasma triglyceride). Soluble epoxide hydrolase (EPHX2, sEH) plays a role in disposition of epoxides in plasma lipoprotein particles. Intrafamilial correlation analysis of the modifier effect of Glu287Arg substitution in the EPHX2 gene was carried out among 79 LDLR mutation carriers and 81 noncarriers. In the carriers, plasma cholesterol levels were elevated among carriers of the 287Arg allele (mean±SD=358 ± 72 mg/dl) in comparison with 287Glu homozygotes (mean±SD=302 ± 72 mg/dl) (p=0.0087). Similarly, in the LDLR mutation carriers, the plasma triglyceride levels were elevated among carriers of the 287Arg allele (mean ± SD=260 ± 100 mg/dl) in comparison with 287Glu homozygotes (mean ± SD=169 ± 83 mg/dl) (p=0.020). No such gene-interactive effect was observed among noncarriers of the LDLR mutation. Half of the patients who presented with HLPIIb had inherited a defective LDLR allele as well as an EPHX2-287Arg allele, whereas the majority who presented with HLPIIa had a defective LDLR allele but not an EPHX2-287Arg allele. These results indicate a significant modification of the phenotype of FH with defective LDLR allele by EPHX2-287Arg variation in our studied kindred.     

Eight novel mutations and functional impairments of the LDL receptor in familial hypercholesterolemia in the north of Japan

In the course of investigations of familial coronary artery disease in Hokkaido, the northland of Japan, we identified 13 families affected by familial hypercholesterolemia. Among them, we identified eight novel mutations of the low-density lipoprotein (LDL) receptor gene, four of which caused frameshifts: (1) a 7-bp deletion at nucleotide (nt) 578–584 (codon 172–174, exon 4); (2) a 14-bp insertion at 682 nt (codon 207–208, exon 4); (3) a 49-bp deletion at nt 943–991 (codon 294–310, exon 7); and (4) a one-base insertion of C to a stretch of C₃ at nucleotides 1687–1689 or codon 542. The others included (5) a T-to-C transition at nt 1072 causing substitution of Cys for Arg at codon 337 (C337R, exon 8); (6) a splice-site G-to-T substitution in intron 11; (7) a splice-site G-to-C substitution in intron 11; and (8) a G-to-T transition at nt 1731 causing substitution of Trp for Cys at codon 556 (W556C, exon 12). To disclose the functional consequences of novel mutations, we characterized each of these mutations by two assays in peripheral lymphocytes, i.e., uptake of fluorescently labeled LDL by LDL receptors, and measurement of cell surface-bound LDL receptor protein using specific monoclonal antibody against LDL receptor. Received: September 27, 2001 / Accepted: November 12, 2001     

A novel deletion in the low-density lipoprotein receptor gene in a patient with familial hypercholesterolemia from Petersburg

Fifty St. Petersburg patients with type IIa hyperlipoproteinemia were screened for the presence of structural rearrangements in the low-density lipoprotein receptor (LDLR) gene. One novel deletion of the length about 5 kilobases (kb) was found. This deletion seems to remove completely exons 4, 5, and 6 from the LDLR gene, coding for the largest part of the receptor ligand-binding domain. © 1993 Wiley-Liss, Inc.     

Characterization of a disease-causing Glu119-Lys mutation in the low-density lipoprotein receptor gene in two Danish families with heterozygous familial hypercholesterolemia

Mutations in the gene for the low-density lipoprotein receptor (LDL receptor) cause the autosomal dominant inherited disease familial hypercholesterolemia (FH). In 15 Danish patients with heterozygous FH we have screened exon 4 of the LDL receptor gene for point mutations and small rearrangements employing genomic DNA amplification and bidirectional solid-phase sequencing. Two subjects were found to be heterozygous for a guanine to adenine base substitution at nucleotide position 418 of the LDL receptor cDNA. This point mutation results in an amino acid change from glutamic acid to lysine at amino acid residue 119 in the third repeat of the cysteine-rich ligand binding domain of the mature LDL receptor. Disruption of LDL receptor function by the Glu¹¹⁹-Lys mutation was confirmed by site-directed rnutagenesis and expression in COS-7 cells. By Western blotting the mutation was found to affect the processing of the LDL receptor protein. Using flow cytometric analysis of the transfected cells a decreased binding and internalization of LDL by the mutant receptor was documented. By means of a mutation-specific PCR-based assay the Glu¹¹⁹-Lys mutation was not detected in another 85 apparently unrelated Danish heterozygous FH patients. We identified six persons in the index families with the Glu¹¹⁹-Lys mutation cosegregating with the clinical syndrome of FH in these families. Furthermore, haplotype analysis revealed that the haplotype [SfaNI+, StuI+, AvaII–, (dTA)₇] of the mutation carrying allele was the same in the two apparently unrelated patients. This indicates that the mutation has been inherited from a common ancestor. © 1994 Wiley-Liss, Inc.     

A novel point mutation (Pro84 ← Ser) of the low density lipoprotein receptor gene in a family with moderate hypercholesterolemia

To obtain insight into the possibility that genetic variation of the structure of the low density lipoprotein (LDL) receptor protein could result in subtle changes of serum cholesterol levels, we used single-strand conformation polymorphism (SSCP) to screen all 18 exons of the LDL receptor gene in a panel of subjects with moderate hypercholesterolemia. One novel mutation, replacing C to T at nucleotide 313 and predicted to cause a substitution of serine for proline at codon 84, was identified in a single proband. A convenient PCR assay based on the use of primer-introduced restriction fragment length polymorphism was set up for the detection of this mutation. However, the pathophysiologic significance of the Pro84 → Ser replacement remains to be clarified, as serum LDL cholesterol levels were not significantly higher in mutation carriers vs. non-carriers in the affected family, and no other proband was identified, on screening of DNA samples from 350 Finns. The Pro84 → Ser mutation represents the second single-amino acid change of the LDL receptor protein so far reported which is not associated with the clinical phenotype of familial hypercholesterolemia.     

Identification of the serine-156 to leucine mutation in the low-density lipoprotein receptor in a German family with familial hypercholesterolemia

Summary Familial hypercholesterolemia is caused by various mutations in the gene encoding the low-density lipoprotein receptor. To date more than 100 mutations have been identified, including insertions and deletions as well as single base changes. In the German population haplotype analysis using four restriction fragment length polymorphisms has recently suggested that there exist at least six different genetic defects. Screening 100 FH patients of German origin for the serine 156 to leucine mutation, originally described in a Puerto Rican family living in the United States, resulted in the identification of the mutation in one family. However, by haplotype analysis the mutation was found on a different haplotype from that reported originally. Based on comparison of the haplotypes and their frequencies we suggest that this mutation has occurred independently at least twice.Abbreviations FH familial hypercholesterolemia - LDLR low-density lipoprotein receptor - PCR polymerase chain reaction - RFLP restriction fragment length polymorphism Dedicated to Prof. Dr. N. Zöllner on the occasion of his 70th birthday     

High prevalence of a novel mutation in the exon 4 of the low-density lipoprotein receptor gene causing familial hypercholesterolemia in Belgium

In a cohort of 70 unrelated patients living in Southern Belgium with autosomal dominantly inherited hypercholesterolemia, 11 had a hitherto undescribed mutation in exon 4. It consisted inaC→A mutation at nucleotide 366, resulting in a stop codon at residue Cys₁₂₂. This C122X mutation is expected to cause a class I receptor defect. The biochemical and clinical data collected from the patients carrying the mutation were consistent with a severe form of familial hypercholesterolemia (FH). Some differences between generations were noted. Amongst the C122X carriers, those born after 1926 had cardiovascular complications earlier than those born before 1926. This raises the possibility that changes in environmental factors during the course of the century have had an unfavorable impact on the prognosis of the disease. The mutation was found in 16% of the suspected FH patients and less frequently (less than 3% of suspected FH) in Northern Belgium. The haplotype of the chromosomes carrying the mutation was the same in all C122X families, but extensive genealogical studies failed to reveal a common ancestor. We conclude that C122X is an old and common cause of FH in Belgium. Screening for this mutation may be useful in the diagnosis of FH in Belgium.     

家族性腺瘤性息肉病一家系调查及APC基因胚系突变分析

目的 探讨家族性腺瘤性息肉病(familial adenomatous polyposis,FAP)家系调查及高危亲属基因筛查的意义,报道云南省一FAP家系发病相关基因APC基因的胚系突变结果.方法 查阅对2001年昆明医学院第一附属医院1例FAP患者病例,电话联系及登门随访进行其家系调查,绘制家系图谱.抽取该家系成员外周静脉血提取DNA,利用PCR方法扩增APC基因,应用DNA自动测序仪进行测序.结果 该家系三代共计9人,成员Ⅰ1、Ⅱ1、Ⅱ2、Ⅱ3、Ⅱ4、Ⅲ2、Ⅲ3、Ⅲ48人检出APC基因胚系突变c.3587C>A(S1196X),其中Ⅱ2、Ⅱ2、Ⅱ4、Ⅲ2、Ⅲ3经肠镜检查证实有结直肠多发息肉,Ⅲ4未检出息肉,为基因突变携带者.结论 通过家系调查对高危亲属进行基因筛查可以发现早期患者,尤其是无临床表现的FAP基因突变携带者,以早期进行医学干预及预防性手术治疗,降低FAP的癌变率、病死率;APC基因c.3587C>A(S1196X)胚系突变是引起该家系FAP患者发病的原因.     

Effects of apoE gene polymorphism on Lp(a) concentrations depend on the size of apo(a): a study of 466 white men

 Polymorphisms in the genes for the low-density lipoprotein (LDL) receptor ligands, apolipoprotein E (apoE), and apolipoprotein B (apoB) are associated with variation in plasma levels of LDL cholesterol. Lp(a) lipoprotein(a) [Lp(a)] is LDL in which apoB is attached to a glycoprotein called apolipoprotein(a) [apo(a)]. Apo(a) has several genetically determined isoforms differing in molecular weight, which are inversely correlated with Lp(a) concentrations in blood. The interaction of apo(a) with triglyceride-rich lipoproteins differs with the size of apo(a), and therefore the effects of apoE gene polymorphism on Lp(a) levels could also depend on apo(a) size. We have investigated the possible effect of genetic variation in the apoE and apoB genes on plasma Lp(a) concentrations in 466 white men with different apo(a) phenotypes. Overall there was no significant association between the common apoE polymorphism and Lp(a), but in the subgroup with apo(a)-S4, concentrations of Lp(a) differed significantly among the apoE genotypes (P=0.05). Lp(a) was highest in the apoE genotypes ɛ₂ɛ₃ and ɛ₃ɛ₃ and lowest in genotype ɛ₃ɛ₄, and the apoE polymorphism was estimated to account for about 2.4% of the variation in Lp(a). In contrast, in the subgroup with apo(a)-S2 Lp(a) was significantly lower (P=0.04) in apoE genotype ɛ₂ɛ₃ than in genotype ɛ₃ɛ₃. Lp(a) concentrations did not differ among the XbaI (P=0.65) or SP 24/27 (P=0.26) polymorphisms of the apoB gene. The expected effects of both apoE and apoB polymorphism on LDL levels were significant in the whole population sample and in subjects with large-sized apo(a) isoforms (P<0.01), whereas no effect was seen in those with low molecular weight apo(a) isoforms. We conclude that the influence of apoE genotypes on Lp(a) concentrations depends on the size of the apo(a) molecule in Lp(a), possibly because both apo(a)-S4 and apoE4 have high affinity for triglyceride-rich lipoproteins and may be taken up and degraded rapidly by remnant receptors. Received: 12 January 1995 / Accepted: 12 July 1996     

Clinical variant of Tangier disease in Japan: mutation of the <Emphasis Type="Italic">ABCA1</Emphasis> gene in hypoalphalipoproteinemia with corneal lipidosis

 Despite progress in molecular characterization, specific diagnoses of disorders belonging to a group of in-herited hypoalphalipoproteinemias, i.e., apolipoprotein AI deficiency, lecithin-cholesterol acyltransferase deficiency, Tangier disease (TD), and familial high-density lipoprotein (HDL) deficiency, remain difficult on a purely clinical basis. Several TD patients were recently found to be homozygous for mutations in the ABCA1 gene. We have documented here a clinical variant of TD in a Japanese patient who manifested corneal lipidosis and premature coronary artery disease as well as an almost complete absence of HDL-cholesterol, by identifying a novel homozygous ABCA1 mutation (R1680W). We propose that patients with apparently isolated HDL deficiency who are found to carry ABCA1 mutations may in fact belong to a category of TD patients whose phenotypic features are only partially expressed, and that a number of hidden clinical variants of TD might exist among other HDL deficiency patients who have escaped correct clinical diagnosis. Received: March 1, 2002 / Accepted: March 15, 2002     

Screening the human bradykinin B2 receptor gene in patients with cardiovascular diseases: Identification of a functional mutation in the promoter and a new coding variant (T21M)

To elucidate if genetic variants in the bradykinin B₂ receptor (B₂) gene occur that could affect receptor expression and function, we screened for mutations in the promoter and in the coding region of the human B₂ gene. In our initial study we analyzed 92 consecutive, unrelated subjects (including 25 patients with hypertrophic cardiomyopathy, 18 patients with dilated cardiomyopathy (DCM), 25 patients with hypertension, 18 patients with coronary heart disease, and 6 patients with valvular heart disease) using nonradioactive polymerase chain reaction–single-strand conformation polymorphism analysis as mutation screening method. We detected eight as yet unknown polymorphic sites in the promoter region of the B₂ gene (−845 C/T, −704C/T, −649 insG, −640 T/C, −536 C/T, −412 C/G, −143 C/T and −78 C/T) with allele frequencies between 0.5 and 13%. One of them (−412 C/G) destroys a Sp1 binding site and abolishes protein binding to this Sp1 site in human umbilical vein endothelial cells and human vascular smooth muscle cells. In the protein-coding region one new coding variant (T21M) with the potential to create a truncated receptor isoform was detected. We determined the frequency of the promoter variant at position −412 (C → G) and the newly identified coding variant (T21M) in extended samples of 69 patients with HCM, 163 patients with DCM, 109 patients with hypertension, and 173 healthy anonymous blood donors. The promoter variant (−412C/G) was found in one blood donor and the T21M mutation was not found in the control population. Therefore, it appears that these mutations are rare events and the determination of clinical significance will be a demanding task in the future. Am. J. Med. Genet. 80:521–525, 1998. © 1998 Wiley-Liss, Inc.     

p21 Expression in colorectal carcinomas: a study on 103 cases with analysis of p53 gene mutation/expression and clinic-pathological correlations

The WAF1/CIP1 gene product, p21, an inhibitor of cyclin-dependent kinases, is a critical downstream effector in the p53 pathway. The expression of p21 in human neoplasms is heterogeneous, and may be related to p53 functional status. We evaluated p21 immunoreactivity in 103 colorectal carcinomas (CC) in relation to the p53 gene and protein alterations and clinico-pathologic parameters. High p21 expression (more than 10% reactive cells) was seen in 39% of cases. p21 staining was heterogeneous and often detected in clusters of tumour cells; in some tumours p21 staining was more pronounced in superficial areas. No relation was seen between p21 immunoreactivity and site of the tumours (right vs left), TNM stage and grade. p21 expression was related to p53 status as evaluated with IHC or with SSCP analyses, low p21 expression usually being associated with p53 protein overexpression (P=0.048) and p53 gene alteration (P=0.005). The strongest associations were seen when the combined p53/p21 immunophenotype was compared with p53 gene alterations (P=0.0002). These data support the hypothesis that p21 expression in CC is mainly related to p53 functional status, suggesting that p21 expression could be an interesting adjunct in the evaluation of the functional status of the p53 pathway in CC. Received: 22 February 1999 / Accepted: 21 June 1999     

Protein targeting in the analysis of learning and memory: a potential alternative to gene targeting

Gene targeting using homologous recombination in embryonic stem (ES) cells offers unprecedented precision with which one may manipulate single genes and investigate the in vivo effects of defined mutations in the mouse. Geneticists argue that this technique abrogates the lack of highly specific pharmacological tools in the study of brain function and behavior. However, by now it has become clear that gene targeting has some limitations too. One problem is spatial and temporal specificity of the generated mutation, which may appear in multiple brain regions or even in other organs and may also be present throughout development, giving rise to complex, secondary phenotypical alterations. This may be a disadvantage in the functional analysis of a number of genes associated with learning and memory processes. For example, several proteins, including neurotrophins – cell-adhesion molecules – and protein kinases, that play a significant developmental role have recently been suggested to be also involved in neural and behavioral plasticity. Knocking out genes of such proteins may lead to developmental alterations or even embryonic lethality in the mouse, making it difficult to study their function in neural plasticity, learning, and memory. Therefore, alternative strategies to gene targeting may be needed. Here, we suggest a potentially useful in vivo strategy based on systemic application of immunoadhesins, genetically engineered fusion proteins possessing the Fc portion of the human IgG molecule and, for example, a binding domain of a receptor of interest. These proteins are stable in vivo and exhibit high binding specificity and affinity for the endogenous ligand of the receptor, but lack the ability to signal. Thus, if delivered to the brain, immunoadhesins may specifically block signalling of the receptor of interest. Using osmotic minipumps, the protein can be infused in a localized region of the brain for a specified period of time (days or weeks). Thus, the location and timing of delivery are controlled. Here, we present methodological details of this novel approach and argue that infusion of immunoadhesins will be useful for studying the role particular receptors play in behavioral and neural plasticity.