Heterozygosity for lysosomal acid lipase E8SJM mutation and serum lipid concentrations.

BACKGROUND AND AIM: The complete absence of the lysosomal acid lipase (LAL) enzyme function causes Wolman's Disease that is fatal within the first six months of life. Subtotal defects cause Cholesteryl ester storage disease (CESD), an autosomal recessive disorder leading to hepatic steatosis, fibrosis, micronodular cirrhosis, combined hyperlipidemia with low HDL-cholesterol, increased risk for atherosclerosis, premature death. Since the frequency of the Exon 8 splice junction mutation (c.894 G > A, E8SJM), the CESD leading mutation, is not rare in the general population (allele frequency 0.0025), we investigated the impact of this mutation on serum lipid profile in E8SJM carriers. METHODS AND RESULTS: We collected E8SJM carriers both form genetic study-population analysis and from Outpatient Lipid Clinics and then we assessed their serum lipid profile. We found thirteen individuals heterozygote for E8SJM. Most of them were Germans, three Spanish and two Italian. We found a significant increase in total cholesterol levels in both sexes with E8SJM mutation, leading to a significant increase in LDL cholesterol in males. CONCLUSIONS: Our results show that LAL E8SJM carriers have an alteration in lipid profile with a Polygenic Hypercholesterolemia phenotype, leading to an increase in cardiovascular risk profile.

Assignment of Tangier disease to chromosome 9q31 by a graphical linkage exclusion strategy.

A low level of high density lipoprotein (HDL) cholesterol is a strong predictor of ischaemic heart disease (IHD) and myocardial infarction. One cause of low HDL-cholesterol is Tangier disease (TD), an autosomal codominant inherited condition first described in 1961 in two siblings on Tangier Island in the United States of America. Apart from low HDL-cholesterol levels and an increased incidence of atherosclerosis, TD is characterized by reduced total cholesterol, raised triglycerides, peripheral neuropathy and accumulation of cholesteryl esters in macrophages, which causes enlargement of the liver, spleen and tonsils. In contrast to two other monogenic HDL deficiencies in which defects in the plasma proteins apoA-I and LCAT interfere primarily with the formation of HDL (refs 7-10), TD shows a defect in cell signalling and the mobilization of cellular lipids. The genetic defect in TD is unknown, and identification of the Tangier gene will contribute to the understanding of this intracellular pathway and of HDL metabolism and its link with IHD. We report here the localization of the genetic defect in TD to chromosome 9q31, using a genome-wide graphical linkage exclusion strategy in one pedigree, complemented by classical lod score calculations at this region in a total of three pedigrees (combined lod 10.05 at D9S1784). We also provide evidence that TD may be due to a loss-of-function defect.

Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1.

Tangier disease (TD) was first discovered nearly 40 years ago in two siblings living on Tangier Island. This autosomal co-dominant condition is characterized in the homozygous state by the absence of HDL-cholesterol (HDL-C) from plasma, hepatosplenomegaly, peripheral neuropathy and frequently premature coronary artery disease (CAD). In heterozygotes, HDL-C levels are about one-half those of normal individuals. Impaired cholesterol efflux from macrophages leads to the presence of foam cells throughout the body, which may explain the increased risk of coronary heart disease in some TD families. We report here refining of our previous linkage of the TD gene to a 1-cM region between markers D9S271 and D9S1866 on chromosome 9q31, in which we found the gene encoding human ATP cassette-binding transporter 1 (ABC1). We also found a change in ABC1 expression level on cholesterol loading of phorbol ester-treated THP1 macrophages, substantiating the role of ABC1 in cholesterol efflux. We cloned the full-length cDNA and sequenced the gene in two unrelated families with four TD homozygotes. In the first pedigree, a 1-bp deletion in exon 13, resulting in truncation of the predicted protein to approximately one-fourth of its normal size, co-segregated with the disease phenotype. An in-frame insertion-deletion in exon 12 was found in the second family. Our findings indicate that defects in ABC1, encoding a member of the ABC transporter superfamily, are the cause of TD.

Apolipoprotein A-I variants. Naturally occurring substitutions of proline residues affect plasma concentration of apolipoprotein A-I.

Six unrelated families with genetically determined structural variants of apo A-I were found in the course of an electrophoretic screening program for apo A-I variants in dried blood samples of newborns. The following structural variations were identified by the combined use of HPLC, time-of-flight secondary ion mass spectrometry (TOF-SIMS), and automated gas phase sequencing: Pro3----Arg (1x), Pro4----Arg (1x), and Pro165----Arg (4x). All variant carriers were heterozygous for their mutant of apo A-I. Subjects heterozygous for apo A-I(Pro165----Arg) (n = 12) were found to exhibit lower mean values for apo A-I (109 +/- 16 mg/dl) and HDL cholesterol (37 +/- 9 mg/dl) than unaffected family members (n = 9): 176 +/- 41 and 64 +/- 18 mg/dl, respectively (P less than 0.001). In 9 of 12 apo A-I(Pro165----Arg) variant carriers the concentrations of apo A-I were below the fifth percentile of sex-matched controls. By two-dimensional immunoelectrophoresis as well as by densitometry the relative concentration of the variant apo A-I in heterozygous carriers of apo A-I(Pro165----Arg) was determined to account for only 30% of the total plasma apo A-I mass instead of the expected 50%. Thus, the observed apo A-I deficiency may be largely a consequence of the decreased concentration of the variant apo A-I. In the case of the apo A-I(Pro3----Arg) mutant, densitometry of HDL apolipoproteins demonstrated a distinctly increased concentration of the variant proapo A-I relative to normal proapo A-I. This phenomenon was not observed in the apo A-I(Pro4----Arg) mutant or in other mutants. This suggests that the interspecies conserved proline residue in position 3 of mature apo A-I is functionally important for the regular enzymatic conversion of proapo A-I to mature apo A-I.

Apolipoprotein C-III(Lys58----Glu). Identification of an apolipoprotein C-III variant in a family with hyperalphalipoproteinemia.

Apolipoprotein C-III is a major protein constituent of triglyceride rich lipoproteins and HDL. It occurs in plasma in three isoforms differing by their sialic acid content. Apo C-III putatively inhibits lipolysis and the apo E mediated hepatic uptake of remnants from triglyceride rich particles. We identified a heterozygous carrier of an apolipoprotein C-III variant by the presence of additional bands after isoelectric focusing (IEF) of VLDL. Structural analysis of the variant protein by HPLC, time-of-flight secondary ion mass spectrometry, and automated gas phase sequencing revealed a lysine to glutamic acid replacement in position 58. The underlying A to G exchange was verified by direct sequencing subsequent to amplification by polymerase chain reaction of exon 4 of the apo C-III gene. Family studies revealed vertical transmission of this defect. The two variant carriers exhibited plasma concentrations of HDL cholesterol and apo A-I above the 95th percentiles of sex matched controls whereas the unaffected father and sister showed normal values. The plasma concentrations of apo C-III in the two variant carriers were decreased by 30-40% compared with those of the two unaffected family members and to random controls. Using two-dimensional immunoelectrophoresis as well as IEF and subsequent scanning densitometry, we found that the low serum concentration of apo C-III was a consequence of diminished concentrations of the variant apo C-III isoproteins in both VLDL (15% of normal) and HDL (25% of normal). Apo C-III(Lys58----Glu) heterozygotes possessed unusual HDL as demonstrated by nondenaturing gradient gel electrophoresis. They consisted mainly of HDL2b and contained a proportion of atypically large particles, enriched in apo E, with a Stokes diameter of 13-18 nm and resembling HDLc. In conclusion, heterozygosity for a structural apo C-III variant--apo C-III(Lys58----Glu)--was identified in two hyperalphalipoproteinemic subjects characterized by the presence of low plasma apo C-III concentrations and atypically large HDL.

A frameshift mutation in the human apolipoprotein A-I gene causes high density lipoprotein deficiency, partial lecithin: cholesterol-acyltransferase deficiency, and corneal opacities.

Epidemiologic data of recent years have identified an important role of HDL deficiency in the etiology of atherosclerosis. Biochemical data suggest that some of these deficiencies may be a consequence of defects in the structural genes of HDL apolipoproteins or of plasma enzymes that modify HDL. We analyzed the genetic defect in a 42-yr-old patient suffering from corneal opacities and complete absence of HDL cholesterol but not of coronary artery disease, thus clinically resembling fish eye disease. The observation of an abnormal immunoblot banding pattern of apolipoprotein A-I (apo A-I) and of reduced lecithin: cholesterol acyltransferase (LCAT) activity in plasma led to sequence analysis of the genes for apo A-I and LCAT in this patient and his family. Direct sequencing of polymerase chain reaction amplified DNA segments containing the exons of the candidate genes, resulted in the identification of a frameshift mutation in apo A-I while the LCAT sequence was identical to the wild type. The apo A-I mutation was predictive for an extensive alteration of the COOH-terminal sequence of the encoded protein. Evidence for the release of this mutant protein into the plasma compartment and for the absence of normal apo A-I was derived from ultraviolet laser desorption/ionization mass spectrometry analysis. Our results suggest that a defective apo A-I is the causative defect in this case of HDL deficiency with corneal opacities.

An intronic mutation in a lariat branchpoint sequence is a direct cause of an inherited human disorder (fish-eye disease).

The first step in the splicing of an intron from nuclear precursors of mRNA results in the formation of a lariat structure. A distinct intronic nucleotide sequence, known as the branchpoint region, plays a central role in this process. We here describe a point mutation in such a sequence. Three sisters were shown to suffer from fish-eye disease (FED), a disorder which is caused by mutations in the gene coding for lecithin:cholesterol acyltransferase (LCAT). Sequencing of the LCAT gene of all three probands revealed compound heterozygosity for a missense mutation in exon 4 which is reported to underlie the FED phenotype, and a point mutation located in intron 4 (IVS4:T-22C). By performing in vitro expression of LCAT minigenes and reverse transcriptase PCR on mRNA isolated from leukocytes of the patient, this gene defect was shown to cause a null allele as the result of complete intron retention. In conclusion, we demonstrated that a point mutation in a lariat branchpoint consensus sequence causes a null allele in a patient with FED. In addition, our finding illustrates the importance of this sequence for normal human mRNA processing. Finally, this report provides a widely applicable strategy which ensures fast and effective screening for intronic defects that underlie differential gene expression.

A unique genetic and biochemical presentation of fish-eye disease.

This paper describes a novel genetic defect which causes fish-eye disease in four homozygous probands and its biochemical presentation in 34 heterozygous siblings. The male index patient presented with premature coronary artery disease, corneal opacification, HDL deficiency, and a near total loss of plasma lecithin:cholesterol acyltransferase (LCAT) activity. Sequencing of the LCAT gene revealed homozygosity for a novel missense mutation resulting in an Asp131 - Asn (N131D) substitution. Heterozygotes showed a highly significant reduction of HDL-cholesterol and apolipoprotein A-I levels as compared with controls which was associated with a specific decrease of LpA-I:A-II particles. Functional assessment of this mutation revealed loss of specific activity of recombinant LCAT(N131D) against proteoliposomes. Unlike other mutations causing fish-eye disease, recombinant LCAT(N131D) also showed a 75% reduction in specific activity against LDL. These unique biochemical characteristics reveal the heterogeneity of phenotypic expression of LCAT gene defects within a range specified by complete loss of LCAT activity and the specific loss of activity against HDL. The impact of this mutation on HDL levels and HDL subclass distribution may be related to the premature coronary artery disease observed in the male probands.

Genetic and phenotypic heterogeneity in familial lecithin: cholesterol acyltransferase (LCAT) deficiency. Six newly identified defective alleles further contribute to the structural heterogeneity in this disease.

The presence of lecithin:cholesterol acyltransferase (LCAT) deficiency in six probands from five families originating from four different countries was confirmed by the absence or near absence of LCAT activity. Also, other invariate symptoms of LCAT deficiency, a significant increase of unesterified cholesterol in plasma lipoproteins and the reduction of plasma HDL-cholesterol to levels below one-tenth of normal, were present in all probands. In the probands from two families, no mass was detectable, while in others reduced amounts of LCAT mass indicated the presence of a functionally inactive protein. Sequence analysis identified homozygous missense or nonsense mutations in four probands. Two probands from one family both were found to be compound heterozygotes for a missense mutation and for a single base insertion causing a reading frame-shift. Subsequent family analyses were carried out using mutagenic primers for carrier identification. LCAT activity and LCAT mass in 23 genotypic heterozygotes were approximately half normal and clearly distinct from those of 20 unaffected family members. In the homozygous patients no obvious relationship between residual LCAT activity and the clinical phenotype was seen. The observation that the molecular defects in LCAT deficiency are dispersed in different regions of the enzyme suggests the existence of several functionally important structural domains in this enzyme.

Polymorphic site study at codon 347 of apolipoprotein A-IV in a Japanese population.

In a population of Japanese subjects, we surveyed codon 347 of the apolipoprotein (apo) A-IV gene and found that the frequency of a rare allele at this point was extremely low compared to that in western populations. Only one of 850 unrelated samples showed mutation at the enzyme recognition site by agarose gel electrophoresis. However, direct sequencing of the coding region revealed that it did not result from the ACT (Thr) to TCT (Ser) mutation which has been reported in western countries, but from an ACT to ACG (Thr) mutation, which does not affect the primary structure of apo A-IV. Two additional family members showed the same point mutation at codon 347.

De novo mutation in the SCN5A gene associated with early onset of sudden infant death.

BACKGROUND: Congenital long QT syndrome (LQTS), a cardiac ion channel disease, is an important cause of sudden cardiac death. Prolongation of the QT interval has recently been associated with sudden infant death syndrome, which is the leading cause of death among infants between 1 week and 1 year of age. Available data suggest that early onset of congenital LQTS may contribute to premature sudden cardiac death in otherwise healthy infants. METHODS AND RESULTS: In an infant who died suddenly at the age of 9 weeks, we performed mutation screening in all known LQTS genes. In the surface ECG soon after birth, a prolonged QTc interval (600 ms(1/2)) and polymorphic ventricular tachyarrhythmias were documented. Mutational analysis identified a missense mutation (Ala1330Pro) in the cardiac sodium channel gene SCN5A, which was absent in both parents. Subsequent genetic testing confirmed paternity, thus suggesting a de novo origin. Voltage-clamp recordings of recombinant A1330P mutant channel expressed in HEK-293 cells showed a positive shift in voltage dependence of inactivation, a slowing of the time course of inactivation, and a faster recovery from inactivation. CONCLUSIONS: In this study, we report a de novo mutation in the sodium channel gene SCN5A, which is associated with sudden infant death. The altered functional characteristics of the mutant channel was different from previously reported LQTS3 mutants and caused a delay in final repolarization. Even in families without a history of LQTS, de novo mutations in cardiac ion channel genes may lead to sudden cardiac death in very young infants.

A novel long-QT 5 gene mutation in the C-terminus (V109I) is associated with a mild phenotype.

Mutations in the human minK gene KCNE1 have been linked to autosomal dominant and autosomal recessive long-QT (LQT) syndrome, a cardiac condition predisposing to ventricular arrhythmias. minK and KvLQT1, the LQT1 gene product, form a native cardiac K+ channel that regulates the slowly delayed rectifier potassium current I(Ks). We used single-strand conformation polymorphism and sequencing techniques to identify novel KCNE1 mutations in patients with a congenital LQT syndrome of unknown genetic origin. In 150 unrelated index patients a missense mutation (V109I) was identified that significantly reduced the wild-type I(Ks) current amplitude (by 36%) when coexpressed with KvLQT1 in Xenopus oocytes. Other biophysical properties of the I(Ks) channel were not altered. Since we observed incomplete penetrance (only one of two mutation carriers could be diagnosed by clinical criteria), and the family's history was unremarkable for sudden cardiac death, the 109I allele most likely causes a mild phenotype. This finding may have implications for the occurrence of "acquired" conditions for ventricular arrhythmias and thereby the potential cardiac risk for asymptomatic mutation carriers still remains to be determined.

Molecular analysis at the Harvey Ras-1 gene in patients with long QT syndrome.

Patients with long QT syndrome (LQTS; MIM 1921500) frequently suffer from syncope and are threatened by sudden cardiac death due to ventricular arrhythmias, typically of the torsade de pointes type. Initial progress in revealing the molecular basis of the disease was made by the observation of genetic linkage of the disease locus to the Harvey Ras-1 gene (HRAS 1) on chromosome 11p15.5. More recently loci on chromosomes 3, 4, and 7 have also been found to be linked to LQTS, thus demonstrating heterogeneity in the causes for this disease. The present study performed sequence analysis on the HRAS 1 gene in patients with congenital and acquired LQTS to determine the frequency of HRAS 1 mutations in patients with this disease. In neither group were no mutations identified in the coding regions or in the splice donor and acceptor sites. Alleles characterized by a T to C transition in exon 1 and an insertion/deletion polymorphism upstream of exon 1 showed no significant difference in their frequencies between LQTS patients and normal controls. No quantitative influence of the such characterized genotypes on the QT duration was observed. These results demonstrate that structural mutations in the HRAS 1 gene are not a frequent cause of LQTS. Also, since there was no association of different alleles at the HRAS 1 locus with changes in QT duration, it appears unlikely that this gene is a major contributor to this disease.

Electrophoretic screening for human apolipoprotein C-II variants: repeated identification of apolipoprotein C-II(K19T).

Screening for apolipoprotein (apo) C-II variants in the plasma of 400 students, 600 patients of a cardiological rehabilitation center, and 1200 patients of an outpatient lipid clinic by isoelectric focusing and subsequent anti-apo C-II immunoblotting led to the identification of four individuals whose plasma samples contained an apo C-II isoform with an abnormal isoelectric point. In all cases direct sequencing of PCR-amplified DNA assessed a heterozygous A to C transversion in codon 19 of the apo C-II gene which leads to the replacement of lysine with threonine. Two of the four index patients presented with moderate hypertriglyceridemia; one suffered from severe hyperlipidemia, with triglyceride levels ranging between 180 and 1900 mg/dl, depending on dietary changes. Sequencing of this proband's lipoprotein lipase gene showed no alteration compared to the wild-type sequence. A study in his family revealed that heterozygosity for apo C-II(K19T) is not associated with differences in mean lipid and lipoprotein concentrations. In conclusion, apo C-II(K19T) occurs in Germany at a frequency of approximately 1 in 550. Although this variant is not sufficient to cause hypertriglyceridemia, it may be possible that apo C-II(K19T) cause hypertriglyceridemia in the presence of additional as yet unidentified environmental and/or genetic factors.

Dimorphic markers for the human apolipoprotein CII gene locus.

Two restriction-site polymorphisms (RSP) have been detected when using a human apolipoprotein CII (apoCII) cDNA clone as a hybridization probe. These include a BanI and a TaqI RSP. Frequencies of the more common allele have been determined in a German population of 100 individuals and are 0.66 (BanI RSP) and 0.56 (TaqI RSP). Corresponding polymorphic information content (PIC) values are 0.36 and 0.37 for individual sites, and 0.58 for the BanI-TaqI pair of sites, making this locus a very informative (PIC-rich) marker for this region of chromosome 19. Haplotype studies also indicate the presence of allelic association (linkage disequilibrium) at the human apoCII gene locus.

A novel frameshift mutation in PMP22 accounts for hereditary neuropathy with liability to pressure palsies.

Peripheral myelin protein PMP22 deficiency is associated with hereditary neuropathy with liability to pressure palsies (HNPP). Most HNPP cases are caused by a 1.5-megabase deletion in chromosome 17p11.2-12, a region that contains the PMP22 gene, whereas point mutations leading to HNPP are extremely rare. We have identified a family with clinical and electrophysiologic features of HNPP,in which all affected members are heterozygous carriers of a single base insertion in codon 94. This mutation is predicted to alter the reading frame and to result in a delayed termination signal. We conclude that the functional consequences of the frameshift are equivalent to those of the PMP22 deletion allele.

PCR-based strategy for the diagnosis of hereditary neuropathy with liability to pressure palsies and Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are inherited peripheral neuropathies. In most cases these disorders are caused by either the duplication (in CMT1A) or the deletion (in HNPP) of a 1.5-megabase DNA fragment on chromosome 17p11.2, which contains the peripheral myelin protein 22 gene (PMP22). We developed a rapid and simple quantitative PCR assay for the detection of the CMT1A duplication or the HNPP deletion. The assay is based on the quantitative determination of the copy number of a 240-base pair DNA fragment from exon 4 of the PMP22 gene. Quantification was done on an automated fluorescence sequencer. Using this method we analyzed four families with the HNPP phenotype. In these families we identified the deletion in all affected individuals. To test the validity of the method, we compared the quantitative PCR results from 50 DNA samples, including 15 samples from individuals with HNPP, 15 samples from CMT1A patients, and 20 from normal controls, with the results obtained by Southern blot analysis. Concordant results were obtained in 49 of the 50 cases.

The contribution of candidate genes to the response of plasma lipids and lipoproteins to dietary challenge.

The possible role of four candidate genes in lipid and lipoprotein response to diet was examined in 214 members of two large kibbutz settlements in Israel. Four site polymorphisms (signal peptide insertion/deletion, XbaI, EcoRI and MspI) of the apo B gene, the common apo E genotypes, three common mutations (T-93G, S447stop and N291S) of the LPL gene and the CETP I405V RFLP were determined. The average reduction induced by diet in participants with the absence of the EcoRI restriction site (L4154) of the apo B gene compared with those found to be homozygotes for the restriction site (G/G4154) were: 16.2 and 8.0 mg/dl for total cholesterol (TC) (P=0. 01); and 15.6 and 6.2 mg/dl for LDL-C (P=0.007), respectively. TC and LDL-C baseline levels were significantly different among the apo-E genotypes, yet there were no significant effects on lipid and lipoprotein dietary response. Triglyceride baseline values were significantly lower (P=0.007) among subjects with the LPL S447stop mutation and HDL-C was significantly lower (P=0.008) among subjects found to be heterozygous for the LPL N291S mutation. A heterogeneous response for triglyceride was observed for individuals with the S291 allele as compared to those individuals who were found to be homozygous for the N291 allele. No differences in dietary responsiveness were observed among the apo E and CETP genotypes. In conclusion, our results suggest that sequence variation(s) in the coding region of the apo B gene linked to the EcoRI polymorphism are associated with total cholesterol and LDL-C responsiveness to dietary manipulation. In our study population, LPL mutations had a significant effect on TG and HDL-C baseline levels and on their response to diet.