Analysis of a genetic mutation in an electrophoretic variant of the human lactate dehydrogenase-B(H) subunit

An electrophoretic variant of the lactate dehydrogenase (LDH)-B(H) subunit was discovered in a patient with diabetes mellitus. His LDH activity in serum was slightly lower than normal and the LDH isozyme pattern showed an abnormal migration indicating an LDH-B subunit variant of the fast type. The LDH containing the variant subunit revealed a decreased heat stability. DNA analysis of the variant allele detected a base substitution, an A to G transition, at codon 6 (AAA-->GAA). The mutation resulted in the replacement of a lysine by a glutamic acid (K6E). The change may cause the heat instability and affect the net charge of the variant subunit, resulting in an electrophoretic LDH-B subunit variant of the fast type.     

Functional consequences of a Na+ channel mutation causing hyperkalemic periodic paralysis

Hyperkalemic periodic paralysis (HYPP), one of several inheritable myotonic diseases, results from genetic defects in the human skeletal muscle Na+ channel. In some pedigrees, HYPP is correlated with a single base pair substitution resulting in a Met replacing Thr704 in the fifth transmembrane segment of the second domain. This region is totally conserved between the human and rat channels. We have introduced the human mutation into the corresponding region of the rat muscle Na+ channel cDNA and expressed it in human embryonic kidney 293 cells. Patch-clamp recordings show that this mutation shifts the voltage dependence of activation by 10-15 mV in the negative direction. The shift results in a persistent Na+ current that activates near -70 mV; this phenomenon could underlie the abnormal muscle activity observed in patients with HYPP.     

Metabolic consequences of phosphotransferase (PTS) mutation in a phenylalanine-producing recombinant Escherichia coli

E. coli strain PPA305, which has a wild-type PTS system, and PPA316, which utilizes a proton-galactose symport system for glucose uptake, were used as host strains to harbor a phenylalanine overproduction plasmid pSY130-14 and to study the effects of using different glucose uptake systems on phenylalanine production. The non-PTS strain (PPA316/pSY130-14) produced much less phenylalanine, ranging from 0 to 67% of that produced by the PTS strain (PPA305/pSY130-14) depending on cultivation conditions used. The non-PTS strain PPA316/pSY130-14 had an intracellular PEP concentration only one-sixth that of the PTS strain, PPA305/pSY130-14. Additionally, PPA316/pSY130-14 had a substantially lower energy state in terms of the size of the pool of high-energy phosphate compounds and the magnitude of the pH difference across the cytoplasmic membrane. The non-PTS strain consumed oxygen at a higher rate, attained lower biomass concentration, and produced no acetate and phenylalanine during fermentation, suggesting more carbon was oxidized to CO2, most likely through the TCA cycle. Analysis of intracellular fluxes through the central carbon pathways was performed for each strain utilizing exponential phase data on extracellular components and assuming quasi-steady state for intermediate metabolites. The non-PTS strain had a higher flux through pyruvate kinase (PYK) and TCA cycle which, in agreement with the observed higher oxygen uptake rate, suggests that more carbon was oxidized to CO2 through the TCA cycle. Further analysis using rate expression data for PYK and NMR data for the intracellular metabolites identified the regulatory properties of PYK as the probable cause for lower intracellular PEP levels in PPA316/pSY130-14.     

Restricted geographical extension of the association of a glucagon receptor gene mutation (Gly40Ser) with non-insulin-dependent diabetes mellitus

In the present work the effects of i.c.v. administration of La3+ and Gd3+ on the motor stimulant effect of cocaine in rats were studied. Both La3+ and Gd3+ failed to influence basal motor activity. However, the two metal ions differ in modulation of cocaine-induced activation of motor activity. While Gd3+ (10.0, 20.0 and 40.0 mM/1 ml) did not influence significantly the cocaine effect, La3+ (0.1, 1.0 and 10 mM/1 ml) inhibited cocaine-induced motor activation in a dose-dependent manner. The results suggest the possible involvement of La(3+)-but not Gd(3+)-sensitive calcium channels in the locomotor stimulant effect of cocaine.     

Portal and mesenteric vein thrombosis in a patient heterozygous for a mutation (Arg506-->Gln) in the factor V gen (factor V Leiden)

Neurological complications such as spinal cord compression has rarely been reported both in the solitary type of osteochondroma or in multiple osteochondromas. We report a a 65-year-old patient with a thoracic osteochondroma involving the neural arch of T6, and the corresponding rib, who was followed-up for 5 years. Approximately 3 years after partial surgical removal, the lesion manifested as a dumbbell mass passing through the neural foramen leading to cord compression, and a hemilaminectomy was performed with subtotal tumor excision. A clinical follow-up 2 years later revealed normal findings.     

Apolipoprotein A-I(Zavalla) (Leu159-->Pro): HDL cholesterol deficiency in a kindred associated with premature coronary artery disease

We investigated the molecular defect causing high density lipoprotein cholesterol (HDL-C) deficiency in a male proband and his family members. Amplification and sequencing of genomic DNA disclosed a novel base-pair substitution at residue 159 in the apolipoprotein (apo) A-I gene. This substitution resulted in the loss of an AviII restriction site and a predicted substitution of leucine with proline at residue 159. Restriction enzyme analysis demonstrated absence of the AviII site in 19 of 40 biological family members. Compared with familial controls, subjects with the apoA-I(Zavalla) variant had reduced HDL-C (1.16 versus 0.27 mmol/L, P<0.0001), apoA-I (38.7 versus 124.4 mg/dL, P<0.0001), and apoA-II (14.3 versus 19.0 mg/dL, P<0.0001) levels. Two subjects who have developed coronary artery disease to date possess additional cardiovascular risk factors. Other heterozygotes for apoA-I(Zavalla) are presently without symptomatic coronary artery disease. This study identifies a monogenic cause of hypoalphalipoproteinemia, with the single base-pair substitution having a dominant effect on the low HDL-C phenotype. In addition, it extends recent observations that HDL-C deficiency states may be more prone to the development of premature coronary artery disease when accompanied by additional cardiovascular risk factors.     

A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S

Human prothrombin, factor IX, and factor X have been idolated in high yield and characterized as the their amino-terminal sequence, molecular weight, amino acid composition, and migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional human plasma protein, called protein S, has also been purified and its properties have been compared with those of prothrombin, factor IX, and factor X. Prothrombin (mol wt 72 000), factor IX (mol wt 57 000), and protein S (mol wt 69 000) are single-chain glycoproteins, while factor X (mol wt 59 000) is a glycoprotein composed of two polypeptide chains held together by a disulfide bond(s). The amino-terminal sequence of the light chain of human factor X is homologous with prothrombin, factor IX, and protein S. The heavy chain of human factor X is slightly larger than the heavy chain of bovine factor X and differs from bovine factor X in its amino-terminal sequence.     

Carboxy-terminal amino acid sequence of a human fibrinogen gamma-chain variant (gamma')

A normal human fibrinogen gamma-chain variant, termed gamma', is larger than the gamma chain (51 500 vs. 49 500) due to an extended COOH-terminal sequence. The extended COOH-terminal cyanogen bromide peptide (CNBr e') was isolated by high-pressure liquid chromatography, and its amino acid sequence was determined. Comparison with the corresponding COOH-terminal gamma-chain peptide (CNBr e) showed that the last four amino acids of the gamma chain were replaced in gamma' chains by a 20-residue fragment rich in aspartic and glutamic acids, having the sequence Val-Tyr-Pro-Glu-His-Pro-Ala-Glx-Thr-Glx-Tyr-Asx-Ser-Leu-Arg-Pro-Glx-Asx-Asx-Leu . Mutant gamma chains (gamma Paris I) from a congenitally dysfunctional fibrinogen molecule (fibrinogen Paris 1) express both gamma and gamma' features, suggesting that both gamma and gamma' chains are produced from a single gene. If this suggestion is correct, the observed differences in amino acid sequence could be explained by the existence of different mRNAs for gamma and gamma' chains, respectively, which are transcribed from one gene by differential RNA splicing.     

Functional consequences of a posttransfection mutation in the H2-H3 cytoplasmic loop of the alpha subunit of Na,K-ATPase

During kinetic studies of mutant rat Na,K-ATPases, we identified a spontaneous mutation in the first cytoplasmic loop between transmembrane helices 2 and 3 (H2-H3 loop) which results in a functional enzyme with distinct Na,K-ATPase kinetics. The mutant cDNA contained a single G950 to A substitution, which resulted in the replacement of glutamate at 233 with a lysine (E233K). E233K and alpha1 cDNAs were transfected into HeLa cells and their kinetic behavior was compared. Transport studies carried out under physiological conditions with intact cells indicate that the E233K mutant and alpha1 have similar apparent affinities for cytoplasmic Na+ and extracellular K+. In contrast, distinct kinetic properties are observed when ATPase activity is assayed under conditions (low ATP concentration) in which the K+ deocclusion pathway of the reaction is rate-limiting. At 1 microM ATP K+ inhibits Na+-ATPase of alpha1, but activates Na+-ATPase of E233K. This distinctive behavior of E233K is due to its faster rate of formation of dephosphoenzyme (E1) from K+-occluded enzyme (E2(K)), as well as 6-fold higher affinity for ATP at the low affinity ATP binding site. A lower ratio of Vmax to maximal level of phosphoenzyme indicates that E233K has a lower catalytic turnover than alpha1. These distinct kinetics of E233K suggest a shift in its E1/E2 conformational equilibrium toward E1. Furthermore, the importance of the H2-H3 loop in coupling conformational changes to ATP hydrolysis is underscored by a marked (2 orders of magnitude) reduction in vanadate sensitivity effected by this Glu233 --> Lys mutation.     

Radioimmunoassay of human Hageman factor (factor XII)

A secific, sensitive, and reproducible radioimmunoassay for human Hageman factor (HF, factory XII) has been developed with purified human HF and monospecific rabbit antibody. Precise measurements of HF antigen were possible for concentrations as low as 0.1% of that in normal pooled plasma. A good correlation (correlation co-efficient = 0.82) existed between the titers of HF measured by clot-promoting assays and radioimmunoassays among 42 normal adults. Confirming earlier studies, HF antigen was absent in Hageman trait plasma, but other congenital deficient plasmas, including those of individuals with Fletcher trait and Fitzgerald trait, contained normal amounts of HF antigen. HF antigen was reduced in the plasmas of patients with disseminated intravascular coagulation or advanced liver cirrhosis, but it was normal in those of patients with chronic renal failure or patients under treatment with warfarin. HF antigen was detected by this assay in plasmas of primates, but not detectable in plasmas of 11 nonprimate mammalian and one avian species.     

A mutation in plasma platelet-activating factor acetylhydrolase (Val279-->Phe) is a genetic risk factor for stroke

BACKGROUND AND PURPOSE: Platelet-activating factor (PAF) is a phospholipid with multiple actions that include thrombosis and inflammation. It is inactivated by a plasma enzyme, PAF acetylhydrolase. Deficiency of this enzyme in plasma is caused by a missense mutation in the gene (Val279-->Phe). We have studied a possible association of this mutation with the risk of stroke. SUBJECTS AND METHODS: We studied 120 consecutive patients with cerebral thrombosis. The control group consisted of 134 patients matched for age and sex with minor complaints but without stroke. Genomic DNA was analyzed for the mutant allele by a specific polymerase-chain reaction. Plasma PAF acetylhydrolase activity was determined by the method of Stafforini et al. RESULTS: The prevalence of the mutant gene was 43.4% in stroke patients (39.2% heterozygotes and 4.2% homozygotes), which was significantly higher than the 25.4% in control subjects (22.4% heterozygotes and 3.0% homozygotes) (chi 2 = 9.22, P < .01). The prevalence was slightly higher in stroke patients without hypertension than those with hypertension, but the difference was not significant. The patients with family histories of stroke had a slightly higher but not a significant prevalence of the mutant gene as compared with those without family histories of stroke. Plasma PAF acetylhydrolase activity was higher in patients than in control subjects, in normal subjects, or patients with a heterozygous genotype. CONCLUSIONS: These results suggest that plasma PAF acetylhydrolase deficiency may be a risk factor for stroke. This may explain the relatively high prevalence of stroke in Japan, as the mutation is more common among Japanese than Caucasians.     

A Gly1127Ser mutation in an EGF-like domain of the fibrillin-1 gene is a risk factor for ascending aortic aneurysm and dissection

Ascending aortic disease, ranging from mild aortic root enlargement to aneurysm and/or dissection, has been identified in 10 individuals of a kindred, none of whom had classical Marfan syndrome (MFS). Single-strand conformation analysis of the entire fibrillin-1 (FBN1) cDNA of an affected family member revealed a G-to-A transition at nucleotide 3379, predicting a Gly1127Ser substitution. The glycine in this position is highly conserved in EGF-like domains of FBN1 and other proteins. This mutation was present in 9 of 10 affected family members and in 1 young unaffected member but was not found in other unaffected members, in 168 chromosomes from normal controls, and in 188 chromosomes from other individuals with MFS or related phenotypes. FBN1 intragenic marker haplotypes ruled out the possibility that the other allele played a significant role in modulating the phenotype in this family. Pulse-chase studies revealed normal fibrillin synthesis but reduced fibrillin deposition into the extracellular matrix in cultured fibroblasts from a Gly1127Ser carrier. We postulate that the Gly1127Ser FBN1 mutation is responsible for reduced matrix deposition. We suggest that mutations such as this one may disrupt EGF-like domain folding less drastically than do substitutions of cysteine or of other amino acids important for calcium-binding that cause classical MFS. The Gly1127Ser mutation, therefore, produces a mild form of autosomal dominantly inherited weakness of elastic tissue, which predisposes to ascending aortic aneurysm and dissection later in life.     

The inhibition of human factor VIIa-tissue factor by antithrombin III-heparin is enhanced by factor X on a human bladder carcinoma cell line

The effects of influenza A and B and RSV on mortality in England and Wales were assessed by regression analysis for the period 1975-90. Morbidity data from sentinel practices were used to calculate 4-weekly rates of aggregated upper respiratory tract infections (URTI); PHLS laboratory reports were used as indices of infection, and 4-weekly death rates from all causes, excluding childbirths, were used to study relationships with mortality. Deaths correlated strongly with influenza A and B reports, temperature, and interactions between aggregated URTI and temperature, and RSV outbreaks and temperature. Estimates of 'seasonal' 4-weekly mortality associated with URTI were made by substituting into primary regression models the mean of annual trough consultation rates for aggregated URTI and baseline values for RSV and influenza. Peak 4-weekly mortality associated with URTIs was estimated at c. 24000 and c. 28000 during combined influenza and RSV epidemics of 1975-6 and 1989-90 respectively. Secondary regression analysis was carried out with the estimated 'seasonal' 4-weekly deaths associated with URTI as dependent variable and laboratory data as regressors. Estimated excess mortality associated with influenza was considerable even during years without major epidemics. Overall during the 15 winters the estimated mortality associated with RSV was 60-80% more than that associated with influenza. The modelling permits only a crude estimate of RSV associated mortality. None the less it suggests that RSV is an important cause of winter mortality.     

The prevalence of factor V Leiden (1691 G-->A) mutation in Turkey

Resistance to activated protein C (APC), which is caused by a single point mutation in the gene for factor V, is a common risk factor for thrombosis. In this study, we screened factor V (FV) Leiden mutation in 81 subjects. The mutation in the heterozygous form was found in 7.1 percent of our normal population. This high frequency suggests that screening for the FV mutation should be considered in patients with a family history of thrombosis.     

Functional characterization of missense mutations in ATP7B: Wilson disease mutation or normal variant?

Wilson disease is an autosomal recessive disorder of copper transport that causes hepatic and/or neurological disease resulting from copper accumulation in the liver and brain. The protein defective in this disorder is a putative copper-transporting P-type ATPase, ATP7B. More than 100 mutations have been identified in the ATP7B gene of patients with Wilson disease. To determine the effect of Wilson disease missense mutations on ATP7B function, we have developed a yeast complementation assay based on the ability of ATP7B to complement the high-affinity iron-uptake deficiency of the yeast mutant ccc2. We characterized missense mutations found in the predicted membrane-spanning segments of ATP7B. Ten mutations have been made in the ATP7B cDNA by site-directed mutagenesis: five Wilson disease missense mutations, two mutations originally classified as possible disease-causing mutations, two putative ATP7B normal variants, and mutation of the cysteine-proline-cysteine (CPC) motif conserved in heavy-metal-transporting P-type ATPases. All seven putative Wilson disease mutants tested were able to at least partially complement ccc2 mutant yeast, indicating that they retain some ability to transport copper. One mutation was a temperature-sensitive mutation that was able to complement ccc2 mutant yeast at 30 degreesC but was unable to complement at 37 degreesC. Mutation of the CPC motif resulted in a nonfunctional protein, which demonstrates that this motif is essential for copper transport by ATP7B. Of the two putative ATP7B normal variants tested, one resulted in a nonfunctional protein, which suggests that it is a disease-causing mutation.