Thr Change Research Articles

Novel Loci (EIF4A2, ADIPOQ, TPRG1) for Triglyceride / High-density Lipoprotein Cholesterol Ratio Longitudinal Change (ΔTHR) among Subjects without Type 2 Diabetes: Evidence from the Long Life Family Study (LLFS) and the Framingham Heart Study (FHS) Offspring Cohort (OS).

Triglyceride (TG) /High density lipoprotein cholesterol (HDL-C) ratio (THR) represents a single surrogate predictor of hyperinsulinemia or insulin resistance that is associated with premature aging processes, risk of diabetes and increased mortality. To identify novel genetic loci for THR change over time (ΔTHR), we conducted genome-wide association study (GWAS) and genome-wide linkage scan (GWLS) among subjects of European ancestry who had complete data from two exams collected about seven years apart from the Long Life Family Study (LLFS, n=1384), a study with familial clustering of exceptional longevity in the US and Denmark. Subjects with diabetes or using medications for dyslipidemia were excluded from this analysis. ΔTHR was derived using growth curve modeling, and adjusted for age, sex, field centers, and principal components (PCs). GWAS was conducted using a linear mixed model accounted for familial relatedness. Our linkage scan was built on haplotype-based IBD estimation with 0.5 cM average spacing. Heritability of ΔTHR was moderate (46%). Our GWAS identified a significant locus at the LPL (p=1.58e-9) for ΔTHR; this gene locus has been reported before influencing baseline THR levels. Our GWLS found evidence for a significant linkage with a logarithm of the odds (LODs) exceeding 3 on 3q28 (LODs=4.1). Using a subset of 25 linkage enriched families (pedigree-specific LODs>0.1), we assessed sequence elements under 3q28 and identified two novel variants (EIF4A2/ADIPOQ-rs114108468, p=5e-6, MAF=1.8%; TPRG1-rs16864075, p=3e-6, MAF=8%; accounted for ~28% and ~29% of the linkage, respectively, and 57% jointly). While the former variant was associated with EIF4A2 (p=7e-5) / ADIPOQ (p=3.49e-2) RNA transcriptional levels, the latter variant was not associated with TPRG1 (p=0.23) RNA transcriptional levels. Replication in FHS OS observed modest effect of these loci on ΔTHR. Of 188 metabolites from 13 compound classes assayed in LLFS, we observed multiple metabolites (e.g., DG.38.5, PE.36.4, TG.58.3) that were significantly associated with the variants (p<3e-4). our linkage-guided sequence analysis approach permitted our discovery of two novel gene variants EIF4A2/ADIPOQ-rs114108468 and TPRG1-rs16864075 on 3q28 for ΔTHR among subjects without diabetes selected for exceptional survival and healthy aging.

Whole Genome Linkage Scan Identifies a Novel Locus on 3q28 for TG/HDL-C Ratio Change Over Time

TG/HDL-C ratio (THR) represents a single inherited surrogate predictor of hyperinsulinemia or insulin resistance that is associated with premature aging processes, risk of diabetes and increased mortality. To identify genetic loci for THR change over time (ΔTHR), we conducted a whole genome linkage scan among subjects of European ancestry who had complete data from two exams collected about seven years apart from the Long Life Family Study (LLFS, n=3091), a study with familial clustering of exceptional longevity in the US and Denmark. Subjects with diabetes or using medications for dyslipidemia were excluded from this analysis. ΔTHR was derived using growth curve modeling, and adjusted for age, sex, PCs, familial membership, and then log-transformed to approximate normality. Our linkage scan was built on haplotype-based IBD estimation with 0.5 cM average spacing. Heritability of ΔTHR was moderate (46%), and evidence for significant linkage (LOD>3) was identified on 3q28 (LOD=4.1). This locus harbors ADIPOQ among several other promising candidate genes. Interestingly, several studies previously reported suggestive evidence of linkage at this locus for relevant traits including adiponectin, dementia, AD and SBP. This linkage signal was not explained by significant GWAS SNPs for LPL or those under the peak (LOD attenuated to 3.7). In conclusion, we found a novel genetic locus on 3q28 for ΔTHR in subjects without diabetes selected for exceptional survival and healthy aging. Further query of sequence elements including rare functional and regulatory variants at this locus is underway which may reveal novel insights on insulin resistance mechanisms for aging.

Molecular analysis of three novel G6PD variants: G6PD Pedoplis-Ckaro, G6PD Piotrkow and G6PD Krakow.

We present three novel mutations in the G6PD gene and discuss the changes they cause in the 3-dimensional structure of the enzyme: 573C-->G substitution that predicts Phe to Leu at position 191 in the C-terminus of helix alphae, 851T-->C mutation which results in the substitution 284Val--> -->Ala in the beta+alpha domain close to the C-terminal part of helix alphaj, and 1175T-->C substitution that predicts Ile to Thr change at position 392.

Genetic polymorphisms in the human selenoprotein P gene determine the response of selenoprotein markers to selenium supplementation in a gender‐specific manner (the SELGEN study)

Selenium (Se), a micronutrient essential for human health, is incorporated into at least 25 selenoproteins including selenoprotein P (SePP), which transports Se within the body. This research identified two single nucleotide polymorphisms (SNPs) in the SePP gene, one in the coding region (position 24731, causing an Ala to Thr change) and one in the 3'untranslated region (position 25191). Their frequency was similar in Caucasian, Chinese, and South Asian populations. Prospectively genotyped volunteers were supplemented for 6 wk with 100 microg sodium selenite/day. Blood samples were analyzed for plasma Se and selenoprotein biomarkers at baseline, after supplementation, and during a washout period. Plasma Se, SePP, and glutathione peroxidase 3 (GPx3) levels increased with supplementation. Baseline plasma Se content depended on both SePP genotypes and body mass index. Presupplementation SePP concentration was associated with gender and genotype at SNP 24731 and postsupplementation concentration with SNP 25191. Both SNPs and gender were associated with differences in GPx3 activity, plasma, and erythrocyte thioredoxin reductase 1 concentrations and lymphocyte glutathione peroxidase 1 and 4 activities and concentrations. In conclusion, the data reveal two common functional SNPs within the human SePP gene that may predict behavior of biomarkers of Se status and response to supplementation and thus susceptibility to disease.

Mutations in the conserved woodchuck hepatitis virus polymerase FLLA and YMDD regions conferring resistance to lamivudine

During more than 104 weeks of treatment with lamivudine (3TC) in chronic woodchuck hepatitis virus (WHV) carrier woodchucks, viral recrudescence occurred. Analysis of WHV DNA polymerase from woodchuck serum samples by PCR followed by DNA sequencing demonstrated that all samples were wild type at the conserved YMDD motif in domain C. Four of the six 3TC-treated woodchucks showed a mixture of the wild-type Ala (GCT) and the mutant Thr (ACT) at the conserved amino acid residue 566 (FLLA) in domain B of the WHV polymerase region. The appearance of the A566T mutation was temporally associated with viral recrudescence. This change is analogous with the amino acid 181 (FLLA) in HBV where 3TC selects for a change from Ala to Thr in humans. In the woodchuck, the Ala to Thr change in the polymerase gene results in a mutation of the WHV surface protein (amino acid 377) from Trp (TGG) to an opal codon (TGA), which may prematurely terminates the polypeptide. Three WHV molecular infectious clones were constructed to study this mutation in greater detail in vitro: A566T, analogous to A181T in HBV; M589V, analogous to the M204V in HBV; and the double mutant A566T/M589V, analogous to A181T/M204V in HBV. These mutants exhibited drug-sensitivity and replication profiles that paralleled those reported for analogous HBV variants. In transfected Huh7 cells, WHV containing the M589V mutation conferred at least 100-fold increased resistance to 3TC, but replicated approximately 5-fold less efficiently than wild-type virus as judged by both extracellular virus production and intracellular DNA replicative forms. In contrast, A566T mutant was approximately 10-fold more resistant to 3TC, replicated intracellularly as well as wild type, but produced 10-fold lower levels of virions than wild type. These findings are consistent with the observation that the A566T mutation alters the overlapping WHV surface antigen reading frame. WHV carrying mutations in the conserved YMDD motif, while not directly selected during lamivudine therapy in WHV carrier woodchucks, are replication competent in cell culture indicating the potential for their emergence in treated animals. These results further illustrate the utility of the WHV/woodchuck model to studies of HBV-drug resistance.

Mutations in the 4-Hydroxyphenylpyruvic Acid Dioxygenase Gene Are Responsible for Tyrosinemia Type III and Hawkinsinuria

The enzyme 4-hydroxyphenylpyruvic acid dioxygenase (HPD) catalyzes the reaction of 4-hydroxyphenylpyruvic acid to homogentisic acid in the tyrosine catabolism pathway. A deficiency in the catalytic activity of HPD may lead to tyrosinemia type III, an autosomal recessive disorder characterized by elevated levels of blood tyrosine and massive excretion of tyrosine derivatives into urine. It has been postulated that hawkinsinuria, an autosomal dominant disorder characterized by the excretion of ‘hawkinsin,’ may also be a result of HPD deficiency. Hawkinsin is a sulfur amino acid identified as (2-l-cystein-S-yl, 4-dihydroxycyclohex-5-en-1-yl)acetic acid. Patients with hawkinsinuria excrete this metabolite in their urine throughout their life, although symptoms of metabolic acidosis and tyrosinemia improve in the first year of life. We performed analyses of the HPD gene in a patient with tyrosinemia type III and two unrelated patients with hawkinsinuria. A homozygous missense mutation predicting an Ala to Val change at codon 268 (A268V) in the HPD gene was found in the patient with tyrosinemia type III. A heterozygous missense mutation predicting an Ala to Thr change at codon 33 (A33T) was found in the same HPD gene in the two patients with hawkinsinuria. These findings support the hypothesis that alterations in the structure and activity of HPD are causally related to two different metabolic disorders, tyrosinemia type III and hawkinsinuria.

Association of HPC2/ELAC2 Genotypes and Prostate Cancer

HPC2/ELAC2 has been identified as a prostate cancer (CaP) susceptibility gene. Two common missense variants in HPC2/ELAC2 have been identified: a Ser-->Leu change at amino acid 217, and an Ala-->Thr change at amino acid 541. Tavtigian et al. reported that these variants were associated with CaP in a sample of men drawn from families with hereditary CaP. To confirm this report in a sample unselected for family history, we studied 359 incident CaP case subjects and 266 male control subjects that were frequency matched for age and race and were identified from a large health-system population. Among control subjects, the Thr541 frequency was 2.9%, and the Leu217 frequency was 31.6%, with no significant differences in frequency across racial groups. Thr541 was only observed in men who also carried Leu217. The probability of having CaP was increased in men who carried the Leu217/Thr541 variants (odds ratio = 2.37; 95% CI 1.06-5.29). This risk did not differ significantly by family history or race. Genotypes at HPC2/ELAC2 were estimated to cause 5% of CaP in the general population of inference. These results suggest that common variants at HPC2/ELAC2 are associated with CaP risk in a sample unselected for family history or other factors associated with CaP risk.

Low prevalence of germline CDKN2A and CDK4 mutations in patients with early-onset melanoma.

In patients with cutaneous melanoma, early age at disease onset is characteristic in familial cases and in individuals with multiple primary melanomas. Both subsets of patients with melanoma are at risk for harboring germline CDKN2A or CDK4 mutations. We set out to prospectively determine the prevalence of CDKN2A and CDK4 mutations in a group of young patients with melanoma. We prospectively screened 913 patients over a 6-month period and identified 519 patients with invasive melanomas. We invited 172 patients with melanoma who were younger than 40 years to participate in the study, and 49 patients consented and donated peripheral blood samples. Forty-nine percent (n = 24) of our patients developed cutaneous melanoma before the age of 30 years. A melanoma clinic in the Boston, Mass, area. We used a combination of single-strand conformation analysis and direct sequencing of samples of peripheral blood leukocyte DNA to search for mutations in exons 1alpha, 1beta, 2, and 3 of CDKN2A and in exon 2 of CDK4. The mean and median ages at diagnosis in our group were 30 and 32 years, respectively. Among a group of 49 patients, we detected 1 (2%; 95% confidence interval, 0.07%-10.8%) Met 53 Ile CDKN2A mutation, which was found in a patient with a strong family history of melanoma. This alteration has been previously shown to impair p16 function. One patient had an Ala 148 Thr change in CDKN2A, which has also been shown to be a polymorphism. We also detected a sequence polymorphism (in the 3' untranslated region [3'UTR] of CDKN2A) in 27% of our patients. A similar incidence of this 3'UTR polymorphism was observed in a control population. We found no CDK4 mutations. Germline CDKN2A and CDK4 mutations are not common in patients who develop melanoma at an early age. This finding contrasts with other cancer-predisposition syndromes, in which there is an increased incidence of germline mutations among young patients. Selection of patients with melanoma for genetic testing based solely on age at onset may not be warranted at the current time.

The Pathogenic Role of Point Mutations Affecting the Translational Initiation Codon of Mitochondrial Genes

The mutation T3308C results in a Met → Thr change at the highly conserved amino acid position 1 of the mtDNA ND1 gene (M1T). To study its potential pathogenic effect we have carried out a combination of mitochondrial protein synthesis and Northern and Western analyses. Our data demonstrate that M1T mutation does not affect the efficiency of the synthesis of the ND1 polypeptide and suggest that any codon specifying methionine located close to the 5′ end of mitochondrial mRNAs may be used as translational initiator.

Mutational analysis of X-linked amelogenesis imperfecta in multiple families

Seven mutations in the amelogenin gene are associated with X-linked amelogenesis imperfecta. These mutations can produce reductions in the amount of enamel and the degree of mineralization. Two families have been identified from western North Carolina exhibiting features of amelogenesis imperfecta, characterized by brown enamel in affected males and interposed vertical bands of normal appearing and brown enamel in presumably heterozygous females. Mutational analysis reveals a C–A mutation in exon 6 at codon 41 of the X-chromosomal amelogenin gene, resulting in a pro–thr change in all individuals having the amelogenesis imperfecta phenotype. This mutation was previously reported in a family with X-linked hypomaturation amelogenesis imperfecta. There is no known relationship between any of the three families but the presence of similar phenotypes and common mutations suggests they may be distantly related. For individuals from all three families, the haplotype for six highly polymorphic loci flanking the amelogenin gene was determined. A common haplotype was demonstrated among two of the three families, suggesting that the mutation may have been inherited from a common ancestor. The finding that the third family had a distinct haplotype may indicate that the C–A mutation at codon 41 represents a mutational hotspot that occurs with greater frequency than other known amelogenin gene mutations. The phenotype resulting from this mutation was highly consistent in affected male members of the same family and between families.

Bilateral Striatal Necrosis and MELAS Associated with a New T3308C Mutation in the Mitochondrial ND1 Gene

We found a novel maternally inherited T3308C mutation in the mtDNA ND1 gene in a patient with bilateral striatal necrosis and stroke-like episodes. Muscle biopsy from the proband showed mitochondrial proliferation in blood vessels and normal respiratory chain activities. The mutation, which was not present in 100 normal controls or in 30 patients with mitochondrial disease, was heteroplasmic in both muscle and blood of the proband and in blood from her asymptomatic mother. This mutation results in a Met → Thr change at the highly conserved amino acid position 1. The T3308C mutation may alter the hydrophobicity and antigenicity of the N-terminal peptide of ND1.

Dual therapy with IL-12 and IFNα provides a novel approach to the management of chronic viral disease

During more than 104 weeks of treatment with lamivudine (3TC) in chronic woodchuck hepatitis virus (WHV) carrier woodchucks, viral recrudescence occurred. Analysis of WHV DNA polymerase from woodchuck serum samples by PCR followed by DNA sequencing demonstrated that all samples were wild type at the conserved YMDD motif in domain C. Four of the six 3TC-treated woodchucks showed a mixture of the wild-type Ala (GCT) and the mutant Thr (ACT) at the conserved amino acid residue 566 (FLLA) in domain B of the WHV polymerase region. The appearance of the A566T mutation was temporally associated with viral recrudescence. This change is analogous with the amino acid 181 (FLLA) in HBV where 3TC selects for a change from Ala to Thr in humans. In the woodchuck, the Ala to Thr change in the polymerase gene results in a mutation of the WHV surface protein (amino acid 377) from Trp (TGG) to an opal codon (TGA), which may prematurely terminates the polypeptide. Three WHV molecular infectious clones were constructed to study this mutation in greater detail in vitro: A566T, analogous to A181T in HBV; M589V, analogous to the M204V in HBV; and the double mutant A566T/M589V, analogous to A181T/M204V in HBV. These mutants exhibited drug-sensitivity and replication profiles that paralleled those reported for analogous HBV variants. In transfected Huh7 cells, WHV containing the M589V mutation conferred at least 100-fold increased resistance to 3TC, but replicated approximately 5-fold less efficiently than wild-type virus as judged by both extracellular virus production and intracellular DNA replicative forms. In contrast, A566T mutant was approximately 10-fold more resistant to 3TC, replicated intracellularly as well as wild type, but produced 10-fold lower levels of virions than wild type. These findings are consistent with the observation that the A566T mutation alters the overlapping WHV surface antigen reading frame. WHV carrying mutations in the conserved YMDD motif, while not directly selected during lamivudine therapy in WHV carrier woodchucks, are replication competent in cell culture indicating the potential for their emergence in treated animals. These results further illustrate the utility of the WHV/woodchuck model to studies of HBV-drug resistance.

A Novel Thrombomodulin Gene Mutation in a Patient Suffering from Sagittal Sinus Thrombosis

Thrombomodulin is an endothelial cell membrane glycoprotein that promotes protein C activation. It has been clearly demonstrated that the anticoagulant functions of the protein C system are important in the prevention of thromboembolic disease. Patients with protein C or protein S deficiency and/or resistance to activated protein C (APC resistance) are at higher risk for developing thromboembolic disease. The first mutation in the thrombomodulin gene was discovered in an American patient suffering from pulmonary embolism at the age of 45 (Ohlin and Marlar 1995). Here we report a case of sagittal sinus thrombosis in a 42-year-old Swedish woman. She was found to carry a heterozygous point mutation changing G127 to A, predicting an Ala25 to a Thr change in the mature thrombomodulin protein. This mutation was also found in her 16-year-old daughter, who so far has not suffered from any thrombotic events. The patient had no other detectable prothrombotic genetic defects associated with the coagulation system. This case supports the hypothesis of an association between mutations in the thrombomodulin gene and venous thrombosis.

Genetic polymorphisms of the renin-angiotensin system and angiographic extent and severity of coronary artery disease: the CORGENE study.

Genetic polymorphisms of the renin-angiotensin system (RAS) have been associated with coronary artery disease (CAD) but no relation between these polymorphisms and coronary atherosclerosis has yet been systematically evaluated. The CORGENE study is a cross-sectional study involving 463 Caucasians who underwent standardized coronary angiography for established or suspected CAD [156 patients with a previous myocardial infarction (MI), 307 without MI]. Four angiographic scores assessing the extent and severity of the coronary lesions were obtained from a double visual analysis of each angiogram, arbitration being achieved by a quantitative measurement. Three different genotypes were analyzed: the angiotensin I-converting enzyme insertion/deletion (ACE I/D) polymorphism, the Met to Thr change at position 235 of the angiotensinogen gene (AGT M235T) and the A to C transition at position 1166 of the angiotensin II type-1 receptor gene (AT1R A1166C). No significant association was observed between these polymorphisms and the clinical characteristics of MI and non-MI subjects. While most classical risk factors were positively correlated with the angiographic scores, no significant relationship could be established with the three genotypes (r ranging from -0.08 to 0.05). Only one significant correlation was observed: between the presence of the AGT 235T allele and the extent of the coronary lesions (r = -0.19, P = 0.04) in patients with low-risk status. These overall results are not in favor of a role of these RAS genetic polymorphisms in the development of coronary atherosclerosis.

Human dehydroepiandrosterone sulfotransferase pharmacogenetics: quantitative Western analysis and gene sequence polymorphisms.

Dehydroepiandrosterone sulfotransferase (DHEA ST) catalyzes the sulfation of DHEA and other hydroxysteroids. DHEA ST enzymatic activity in individual human liver biopsy samples has been shown to vary over a five-fold range, and frequency distribution histograms are bimodal, with approximately 25% of subjects included in a high activity subgroup. We set out to characterize the molecular basis for variation in human liver DHEA ST activity. The first step involved performing quantitative Western analysis of cytosol preparations from 92 human liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. There was a highly significant correlation (r(s) = 0.635, P < 0.0001) between levels of DHEA ST activity and immunoreactive protein. We next attempted to determine whether the expression of DHEA ST might be controlled, in part, by a genetic polymorphism. DNA was isolated from three "low" and three "high" DHEA ST activity liver samples. Exons and the 5'-flanking region of the DHEA ST gene (STD) were amplified for each of these samples with the polymerase chain reaction (PCR). When compared with "wild type" STD sequence, some of the samples contained a T --> C transition at DHEA ST cDNA nucleotide 170, located within exon 2, resulting in a Met 57 --> Thr change in amino acid. Other samples contained an A --> T transversion at nucleotide 557 within STD exon 4 that resulted in a Glu 186 --> Val change. STD exons 2 and 4 were then sequenced for DNA isolated from an additional 87 liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. The allele frequency for the exon 2 polymorphism in these samples was 0.027, whereas that for the exon 4 polymorphism was 0.038, but neither polymorphism was systematically related to the level of enzyme activity in these samples. Transient expression in COS-1 cells of cDNA that contained the nucleotide 170 and 557 polymorphisms, either separately or together, resulted in decreased expression of both DHEA ST enzymatic activity and level of immunoreactive protein, but only when the nucleotide 557 variant was present. Identification of common genetic polymorphisms within STD will now make it possible to test the hypothesis that those polymorphisms might alter in vivo expression and/or function of this important human steroid-metabolizing enzyme.

Proteolytically cleaved mutant antithrombin-Hamilton has high stability to denaturation characteristic of wild type inhibitor serpins

The serpin family of proteins consists primarily of proteinase inhibitors which form tight complexes with target proteinases. Inhibitor serpins are cleaved by proteinase and undergo a large conformational change in which the polypeptide segment terminating at the target reactive site, at which cleavage takes place, inserts itself as an additional strand, s4A, in the center of a preexisting β-sheet. This change in conformation increases the stability towards denaturation of the cleaved serpin relative to the native uncleaved form. Mutant serpins with single amino acid changes in the s4A strand have been identified, and in most cases these are proteinase substrates but not inhibitors. We have measured the stability to denaturation of one of these non-inhibitor substrate mutants, antithrombin-Hamilton, which has an Ala → Thr change at position P12 in strand s4A. We find that it undergoes the transformation to the more stable form which is observed for inhibitor serpins, from which we conclude that the Ala → Thr change in antithrombin-Hamilton does not prevent insertion of s4A into β-sheet A in the cleaved form.

Sequence analysis of thymidine kinase-defective mutants of equine herpesvirus-1 (EHV-1)

We have amplified, cloned and sequenced the gene encoding the thymidine kinase (TK) of a wild-type strain (Ab4) of equine herpesvirus-1 (EHV-1) and two mutants with defective TK activity isolated for resistance to penciclovir (PCV). One of the mutants, PR1, has suffered a 879-bp deletion which reduces the size of TK to 180 bp. The other mutant, PR3, has an adenine to cytosine mutation resulting in a Lys 38 → Thr change. This mutation modifies the amino acid sequence of a domain involved in binding ATP, leading to non-detectable enzymatic activity. Lys 38 thus appears to be essential for the activity of the TK of EHV-1.

The Structure and Characteristics of a Fourth Allele of Polymorphic N-Acetyltransferase Gene Found in the Japanese Population

Polymorphic N-acetyltransferase (NAT2), which exists in the human liver and metabolizes a wide spectrum of arylamine, is encoded by one gene (NAT2). We have identified the mutations of a fourth NAT2 allele (allele 4) which we previously found in the Japanese population. Compared with allele 1, which expresses a high enzyme activity, allele 4 has 3 nucleotide differences. 2 are missense mutations at nucleotide position (nt) 341 and 803. The other is a silent mutation at the KpnI site. From the result of expression studies of chimeric gene constructs between allele 1 and allele 4 in Chinese hamster ovary cells, nt 341 missense mutation of the allele 4 which causes the Ile-114 to Thr change is responsible for the low enzyme activity of NAT2 protein.

Mitochondrial 2-methylacetoacetyl-CoA thiolase deficiency: an inborn error of isoleucine and ketone body metabolism.

A review is presented of 22 published cases of verified or probable mitochondrial 2-methylacetoacetyl-CoA thiolase deficiency, a disorder of isoleucine and ketone body metabolism. The clinical expression, characterized by ketoacidosis, vomiting and lethargy, is highly variable. Typical age of onset is between 6 and 24 months. The disorder, which has been observed in several ethnic groups, is apparently inherited as an autosomal, recessive trait. The prognosis is relatively good if acute episodes of ketoacidosis and dehydration are adequately treated. There is abnormal urinary excretion of 2-methyl-3-hydroxybutyric acid, tiglylglycine, and in some instances also 2-methyl-acetoacetic acid. However, such a pattern of organic aciduria has also been found in cases with normal thiolase activity. Genetic complementation analyses have demonstrated considerable heterogeneity. The cDNA for human methyl-acetoacetyl-CoA thiolase has been cloned and sequenced. Studies in one patient showed a G-to-A substitution at position 1138 of the mRNA, causing 347Ala to Thr change in the mature enzyme. Studies in other patients have shown variable enzyme amount and/or stability.

A mutation present in the amino terminus of Sabin 3 poliovirus VP1 protein is attenuating

The attenuated phenotype of Sabin 3 poliovirus compared with its neurovirulent progenitor strain has been largely accounted for by mutations in the genome at positions 472 and 2034 (G. D. Westrop, K. A. Wareham, D. M. A. Evans, G. Dunn, P. D. Minor, D. I. Magrath, F. Taffs, S. Marsden, M. A. Skinner, G. C. Schild, and J. W. Almond, J. Virol. 63:1338-1344, 1989). By sequencing vaccine virus RNA, we recently identified another Sabin 3-specific mutation at position 2493 (U----C), which predicts an Ile----Thr change at the sixth residue of VP1 (C. Weeks-Levy, J. M. Tatem, S. J. DiMichele, W. Waterfield, A. F. Georgiu, and S. J. Mento, Virology 185:934-937, 1991). Viruses generated by using cDNAs which represent the vaccine sequence (LED3) and a derivative (VR318) possessing a single base change to the wild-type nucleotide (U) at 2493 were used to determine the impact of the 2493 mutation on virus phenotype. The VP1 proteins of LED3 and VR318 viruses were distinguishable by denaturing electrophoretic analysis. LED3 produced smaller plaques in Vero cells than VR318 virus did. Neurovirulence testing of these cDNA-derived viruses in monkeys demonstrated that the 2493 mutation in LED3 virus is attenuating.