Defective respiratory capacity and mitochondrial protein synthesis in transformant cybrids harboring the tRNA(Leu(UUR)) mutation associated with maternally inherited myopathy and cardiomyopathy.

We studied the physiometabolic effects of a mitochondrial DNA (mtDNA) heteroplasmic point mutation, the A-->G3260 transition associated with maternally inherited myopathy and cardiomyopathy. To eliminate the possible influence of the autochthonous nuclear gene set, we fused myoblast-derived cytoplasts of a patient with a human tumoral cell line deprived of mtDNA (Rho degrees). The presence and amount of the mutant G3260 vs the wild-type A3260 were measured by solid phase minisequencing. We observed a marked reduction of the percentage of mutant mtDNA in the culture system compared with that measured in the donor's muscle biopsy, suggesting the presence of negative selection against the mutation. Furthermore, stable mitotic segregation of the two mtDNA populations was observed in 18 of 19 transformant clones, suggesting the presence of intraorganelle and possibly intracellular homoplasmy in the precursor cells of the donor. Several indexes of mtDNA-related respiratory capacity, including oxygen consumption, complex I- and complex IV-specific activities, and lactate production, were markedly abnormal in the clones containing a high proportion of mutant mtDNA, as compared with those containing homoplasmic wild-type mtDNA, possibly because of impaired mitochondrial protein synthesis. We conclude that (a) the A-->G3260 transition is indeed responsible for the mitochondrial disorder identified in the donor patient, and (b) transformant cybrid system gives direct evidence of the mitochondrial origin of a genetic disorder and should be adopted for the evaluation of the pathogenic potential of the mtDNA mutations.     

Mitochondrial gene mutations in the tRNA(Leu(UUR)) region and diabetes: prevalence and clinical phenotypes in Japan

BACKGROUND: Mitochondrial gene mutations play a role in the development of diabetes mellitus. We have assessed the frequency of the A3243G and other mitochondrial mutations in Japan and in the relationship to clinical features of diabetes. METHODS: DNA was obtained from peripheral leukocytes of 240 patients with diabetes mellitus (39 with type 1; 188 with type 2; 13 with gestational diabetes) and 125 control subjects. We used PCR-restriction fragment length polymorphism analysis (ApaI) for A3243G and PCR-single-strand conformation polymorphism analysis to determine the mutations in the mitochondrial gene including nucleotide position 3243. RESULTS: The A3243G mutation was found in seven patients, and an inverse relationship was observed between the degree of heteroplasmy and the age at onset of diabetes. A3156G, G3357A, C3375A, and T3394C were detected in addition. Those who shared the same mutation showed similar clinical characteristics, thus representing a putative clinical subtype. The patients with A3156G had a sudden onset of hyperglycemia and showed a rapid progression to an insulin-dependent state with positive anti-glutamic acid decarboxylase antibody. Those with T3394C showed a mild defect in glucose-stimulated insulin secretion, and hyperglycemia appeared after adding such factors as aging or obesity. CONCLUSIONS: The identification of mitochondrial gene mutations allows preclinical diagnosis of diabetes and prediction of the age at onset by evaluating the degree of heteroplasmy in cases with A3243G. Mutation detection may also be important for patient management and identification of affected family members.     

Diminished insulin secretory response to glucose but normal insulin and glucagon secretory responses to arginine in a family with maternally inherited diabetes and deafness caused by mitochondrial tRNA(LEU(UUR)) gene mutation.

OBJECTIVE: The effects of glucose, arginine, and glucagon on beta-cell function as well as alpha-cell response to arginine were studied in a family with mitochondrial diabetes. RESEARCH DESIGN AND METHODS: The function of alpha- and beta-cells was assessed in all five siblings carrying the mitochondrial tRNA Leu(UUR) gene mutation at position 3243 and compared with six sex-, age-, and weight-matched control subjects. Insulin and C-peptide responses were evaluated by intravenous glucagon application, intravenous arginine stimulation test, and intravenous glucose tolerance test. Glucagon secretion was assessed during the arginine stimulation test. RESULTS: The glucose disappearance constant (K(g)) value (mean +/- SEM 0.61 +/- 0.04 vs. 1.1 +/- 0.04, P = 0.0002) as well as the acute insulin response to glucose (area under the curve [AUC] 0-10 min, 77.7 +/- 50.7 vs. 1,352.3 +/- 191.5 pmol/l, P = 0.0004) were decreased in all patients. Similarly, glucagon-stimulated C-peptide response was also impaired (728 +/- 111.4 vs. 1,526.7 +/- 157.7 pmol/l, P = 0.005), whereas the insulin response to arginine (AUC) was normal (1,346.9 +/- 710.8 vs. 1,083.2 +/- 132.5 pmol/l, P = 0.699). Acute glucagon response to arginine (AUC) was normal but tended to be higher in the patients than in the control subjects (181.7 +/- 47.5 vs. 90.0 +/- 21.1 pmol/l, P = 0.099). CONCLUSIONS: This study shows impaired insulin and C-peptide secretion in response to a glucose challenge and to glucagon stimulation in diabetic patients with mitochondrial tRNA Leu(UUR) gene mutation, although insulin and glucagon secretory responses to arginine were normal.     

A gel electrophoresis method for detection of mitochondrial DNA mutation (3243 tRNA(Leu (UUR))) applied to a Norwegian family with diabetes mellitus and hearing loss

Blood cells of selected patients from a large Norwegian family with maternally transmitted diabetes mellitus, hearing loss and muscular dysfunction were screened for possible A3243G mutation tRNA(Leu (UUR)) in mitochondrial DNA. We selected 7 patients from 3 of the 4 generations of the family and 10 unrelated healthy control subjects for mutation analysis using denaturing gradient gel electrophoresis (DGGE) and both manual and automated DNA sequencing. The A3243G mutation was found in peripheral blood cells of all 7 patients, but in none of the controls. The mutation was in the form of heteroplasmy and the amount of mutant DNA was found to be between 10% and 35% of total mtDNA in individual patients. This is the first report of a Norwegian family with maternally inherited diabetes and hearing loss carrying the A3243G mutation in mitochondrial DNA.     

Site-specific recombination with the chromosomal tRNA(Leu) gene by the large conjugative Haemophilus resistance plasmid

Characterization of the sequences involved in recombination of the Haemophilus plasmid p1056 with the Haemophilus influenzae chromosome produced evidence indicating site-specific recombination with chromosomal tRNA(Leu). attP sequences identical to those of p1056 were found in six plasmids of diverse origin, suggesting that a family of Haemophilus plasmids recombines with chromosomal tRNA(Leu).     

Isolated mitochondrial long-chain ketoacyl-CoA thiolase deficiency resulting from mutations in the HADHB gene

BACKGROUND: The human mitochondrial trifunctional protein (MTP) complex is composed of 4 hydroacyl-CoA dehydrogenase-alpha (HADHA) and 4 hydroacyl-CoA dehydrogenase-beta (HADHB) subunits, which catalyze the last 3 steps in the fatty acid beta-oxidation spiral of long-chain fatty acids. The HADHB gene encodes long-chain ketoacyl-CoA thiolase (LCTH) activity, whereas the HADHA gene contains the information for the long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) functions. At present, 2 different biochemical phenotypes of defects in the mitochondrial trifunctional protein complex are known: isolated LCHAD deficiency and generalized MTP deficiency, with decreased activities of all 3 enzymes. Isolated LCTH deficiency with mutations in the HADHB gene has not been reported. Patient and RESULTS: We report a male newborn who presented with lactic acidosis, pulmonary edema, and cardiomyopathy leading to acute heart failure and death at the age of 6 weeks. Routine newborn screening by tandem mass spectrometry showed increased concentrations of the acylcarnitines tetradecenoylcarnitine, hexadecenoylcarnitine, hydroxypalmitoylcarnitine, and hydroxyoctadecenoylcarnitine, suggesting LCHAD deficiency or complete MTP deficiency. Enzyme investigations revealed very low LCTH (4% of normal) and normal LCHAD activities, whereas molecular analysis showed compound heterozygosity for 185G > A (R62H) and 1292T > C (F431S) mutations in the HADHB gene. CONCLUSION: We describe the first case of isolated LCTH deficiency based on a mutation in the HADHB gene.     

Familial amyloidosis in a large Spanish kindred resulting from a D38V mutation in the transthyretin gene

BACKGROUND: Transthyretin amyloidosis, also known as familial amyloidotic polyneuropathy, is an autosomal dominant disorder that results from a mutation in the gene encoding plasma transthyretin (TTR). Distinct clinical presentations of the disease have been related so far to different point mutations, polyneuropathy being the predominant clinical feature in the majority of cases. Nevertheless, misdiagnosis of familial forms of amyloidosis is still common. MATERIALS AND METHODS: A 71-year-old man was admitted to our hospital for heart failure. He had been previously diagnosed of AL amyloidosis with predominant polyneuropathic, cardiac and laryngeal involvement on the basis of clinical data and amyloid deposition in tissue specimens. During admission, suspicion of transthyretin amyloidosis was raised due to the absence of renal involvement, monoclonal protein and plasma cell dyscrasia. Complete clinical evaluation and sequence analysis of the TTR gene of the patient and his family were performed. RESULTS: Gene sequence analysis revealed a rare A-to-T transition in exon 2 resulting in the substitution of aspartic acid by valine at position 38 (D38V) in the index case and in two other members of the family. Clinical study of the kindred showed a predominant late-onset heart involvement with variable polyneuropathy. CONCLUSIONS: Here we report a large pedigree from Spain with three members affected by a severe late-onset form of amyloidosis due to a rare D38V TTR mutation. The variations on the natural history of this form of amyloidosis may have important consequences on genetic counselling, follow-up, and therapeutic approaches for these patients.     

Vascular endothelial dysfunction resulting from L-arginine deficiency in a patient with lysinuric protein intolerance.

Although L-arginine is the only substrate for nitric oxide (NO) production, no studies have yet been reported on the effect of an L-arginine deficiency on vascular function in humans. Lysinuric protein intolerance (LPI) is a rare autosomal recessive defect of dibasic amino acid transport caused by mutations in the SLC7A7 gene, resulting in an L-arginine deficiency. Vascular endothelial function was examined in an LPI patient who was shown to be a compound heterozygote for two mutations in the gene (5.3-kbp Alu-mediated deletion, IVS3+1G-->A). The lumen diameter of the brachial artery was measured in this patient and in healthy controls at rest, during reactive hyperemia (endothelium-dependent vasodilation [EDV]), and after sublingual nitroglycerin administration (endothelium-independent vasodilation [EIV]) using ultrasonography. Both EDV and NO(x) concentrations were markedly reduced in the patient compared with those for the controls. They became normal after an L-arginine infusion. EIV was not significantly different between the patient and controls. Positron emission tomography of the heart and a treadmill test revealed ischemic changes in the patient, which were improved by the L-arginine infusion. Thus, in the LPI patient, L-arginine deficiency caused vascular endothelial dysfunction via a decrease in NO production.     

Mutations in mitochondrial DNA as a cause of exercise intolerance.

Exercise intolerance is a common presentation of metabolic myopathies, especially of congenital errors of glycogen and lipid metabolism. Recently, however, exercise intolerance has been associated with specific defects in protein-coding genes of mitochondrial DNA (mtDNA), including mutations in genes for complex I, complex III, and complex IV. Contrary to the general rules of mitochondrial genetics, all patients were sporadic cases and all mutations were restricted to skeletal muscle, suggesting that they were somatic mutations not affecting the germ line.     

中国人遗传性高铁血红蛋白血症NADH—细胞色素b5还原酶基因L …

目的：鉴定导致中国人遗传性高铁血红蛋白血症（ＲＣＭ）的ＮＡＤＨ－细胞色素ｂ５还原酶（ｂ５Ｒ）基因突变类型，探讨ＲＣＭ发病的分子基础。方法：逆转录－聚合酶链反应产物直接测序和ｃＤＮＡ克隆测定分析先证者的ｂ５Ｒ编码基因，限制性酶切分析其基因组ＤＮＡ。结果；发现一例ＲＣＭ患者ｂ５Ｒ基因第７２密码子存在新的错义突变（ＣＴＣ→ＣＣＣ）。结论：该突变导致ｂ５Ｒ蛋白第７２位亮氨酸被脯氨酸替换（Ｌ７２Ｐ）是该先证     

四引物扩增受阻突变体系快速检测Wilson病基因Arg778 Leu突变

目的 建立一种不需要限制性片段长度多态性或直接测序的新方法检测肝豆状核变性（WD）病人突变热点ATP7B基因的Arg778Leu突变基因型。方法 用四引物扩增受阻突变体系聚合酶链反应（tetra-primer amplification refractory mutation system-PCR,tetra-primer ARMS—PCR）检测47例WD患者和30例正常对照的ATP7B基因Arg778Leu突变,并用测序进行验证。结果 47例WD患者中检出Arg778Leu纯合突变4例,杂合突变14例,总检出率38．3％（18／47）;30例正常对照未发现突变;DNA测序结果与四引物ARMS—PCR结果完全一致。结论 ATP7B基因Arg778Leu突变是中国人WD突变热点;四引物ARMS—PCR法检测Arg778Leu突变有快速、简便、准确的优点,可以区分等位基因是否纯合,适于大样本的人群筛查。此法也可用于检测其他点突变。     

抗凝血酶基因C2759T(Leu99Phe)突变导致抗凝血酶缺陷症的分子机制研究

目的研究抗凝血酶(AT)基因C2759T(Leu99Phe)突变引起AT缺陷症的分子机制。方法用大引物法构建C2759T突变体AT重组表达质粒(ATM2759)，并将其和正常AT重组质粒(ATN)分别用Superfect试剂转染COS7细胞或CHO细胞，进行体外表达试验和细胞免疫荧光染色。、结果转染ATM2759的COS7细胞，培养上清液和细胞裂解液中的AT抗原(AT：Ag)分别为ATN的35．63％和174．97％，而培养上清液中的AT活性(AT：A)为ATN的47．73％。细胞免疫荧光试验证实，转染ATM2759的CHO细胞，其荧光强度明显高于转染ATN的CHO细胞。结论Leu99Phe 突变可能不是由于影响AT与肝素的结合而导致AT缺陷，而是由于突变导致AT分泌障碍和细胞内滞留所致。     

A novel mutation of the ceruloplasmin gene in a patient with heteroallelic ceruloplasmin gene mutation (HypoCPGM)

We found a novel missense mutation in the ceruloplasmin (Cp) gene in a patient with the heteroallelic Cp gene mutation (HypoCPGM). The patient was a 72-year-old woman who came to our hospital with a 1-year history of postural tremor of the hands. The diagnosis was made based on serum Cp and copper readings which were about half the normal levels, as well as MRI tests of her brain which showed characteristics for hereditary ceruloplasmin deficiency (HCD), known to be caused by the homoallelic Cp gene mutation. Polymerase chain reaction (PCR)-direct sequencing analysis of the Cp gene of the patient revealed a novel point mutation, A to T, at nucleotide position 82 in Exon 1. This mutation changes the Ile28 codon (ATT) to a Phe codon (TTT) (missense mutation). PCR-restriction analysis with restriction enzyme Tsp EI for the mutation revealed that both the patient and her son were heterozygotes for the mutation.     

The mutation in the mitochondrial aldehyde dehydrogenase (ALDH2) gene responsible for alcohol-induced flushing increases turnover of the enzyme tetramers in a dominant fashion.

Deficiency in mitochondrial aldehyde dehydrogenase (ALDH2), a tetrameric enzyme, results from inheriting one or two ALDH2*2 alleles. This allele encodes a protein subunit with a lysine for glutamate substitution at position 487 and is dominant over the wild-type allele, ALDH2*1. The ALDH2*2-encoded subunit (ALDH2K) reduces the activity of ALDH2 enzyme in cell lines expressing the wild-type subunit (ALDH2E). In addition to this effect on the enzyme activity, we now report that ALDH2*2 heterozygotes had lower levels of ALDH2 immunoreactive protein in autopsy liver samples. The half-lives of ALDH2 protein in HeLa cell lines expressing ALDH2*1, ALDH2*2, or both were determined by the rate of loss of immunoreactive protein after inhibition of protein synthesis with puromycin and by pulse-chase experiments. By either measure, ALDH2E enzyme was very stable, with a half-life of at least 22 h. ALDH2K enzyme had an enzyme half-life of only 14 h. In cells expressing both subunits, most of the subunits assemble as heterotetramers, and these enzymes had a half-life of 13 h. Thus, the effect of ALDH2K on enzyme turnover is dominant. These studies indicate that the ALDH2*2 allele exerts its dominant effect both by interfering with the catalytic activity of the enzyme and by increasing its turnover. This represents the first example of a dominantly acting allele with this effect on a mitochondrial enzyme's turnover.     

Platelet Gs hypofunction and abnormal morphology resulting from a heterozygous RGS2 mutation

Summary. Background: Regulator of G‐protein signaling (RGS) 2 negatively regulates Gs signaling by inhibiting the activation of adenylyl cyclase (AC). RGS2 mRNA contains four translation initiation sites, leading to four isoforms with different abilities to inhibit AC activity; the largest isoform is the most pronounced inhibitor. A role for RGS2 in platelets is not known. Objective: To describe a heterozygous RGS2 mutation (G23D) in three related patients, leading to Gs hypofunction in their platelets, and to study the mechanism behind the effect of the RGS2 mutation on platelet function and morphology. Methods: Gs signaling was studied ex vivo in platelets and in vitro in transfected cells. Translation initiation was evaluated in vitro, and the interaction of wild‐type and G23D RGS2 with AC was unraveled via immunoprecipitation. Platelet granule content was analyzed with proteomics. Results: The mutation leads to reduced cAMP production after stimulation of Gs‐coupled receptors. The largest RGS2 isoforms, with strong AC inhibitor activity, are enriched when the mutation is present, as compared with wild‐type RGS2. Moreover, the mutation results in a stronger interaction of RGS2 with AC. G23D RGS2 carriers have enlarged, round platelets with abnormal α‐granules. Proteomics of the platelet releasate revealed altered expression of some proteins involved in actin assembly, and carriers seemed to have a reduced platelet shape change. Conclusions: We present the first platelet Gs signaling defect caused by a heterozygous RGS2 variant that results in a unique mutational mechanism, such as the differential use of translation initiation sites resulting in different functional RGS2 isoforms.