Erythrocyte phosphofructokinase deficiency associated with an unstable variant of muscle phosphofructokinase.

A case of chronic non-spherocytic hemolytic anemia due to partial erythrocyte phosphofructokinase deficiency (61% of normal) is reported. Immunological studies in hemolystates, using anti-muscle and anti-leukocyte phosphofructokinase antisera, seemed to indicate that an isozyme of the muscle type was deficient in the patient. This hypothesis was confirmed by the studies of muscle phosphofructokinase; this enzyme was an unstable and fast variant. There was no deficiency in muscle because of the active synthesis of proteins by this tissue, but the deficiency could be detected in erythrocytes, old cells which are no longer able to synthesize proteins.     

Activities of hexokinase, phosphofructokinase, fructose bisphosphatase and 2-oxoglutarate dehydrogenase in muscle of normal subjects and very ill surgical patients

1. The activities of hexokinase, phosphofructokinase, fructose bisphosphatase and 2-oxoglutarate dehydrogenase have been measured in the vastus lateralis and rectus abdominus muscle of normal human subjects and in very ill surgical patients. 2. The activities of these enzymes in the muscle of control subjects were similar to the pattern seen in the skeletal muscle of other mammals and lower vertebrates. 3. Fructose bisphosphatase and phosphofructokinase activities were significantly lower in the muscle of ill patients although the depression of the activity of fructose bisphosphatase was much greater than that of phosphofructokinase in both muscle types of ill patients. 4. The maximum rate of cycling in the fructose 6-phosphate--fructose, 1,6-diphosphate cycle may be altered in the ill. 5. This decreased cycling may have a direct influence on the sensitivity of glycolysis to regulators such as the adenine nucleotides and may reduce the ability to maintain body temperature. 6. Increased glycogen synthesis in these muscles may indicate that the role of fructose bisphosphatase is unlikely to be solely in glycogen resynthesis.     

Muscle citrate content and the regulation of metabolism in fed and fasted human skeletal muscle

Summary. Blood and muscle metabolite levels were measured in seven healthy young adult male subjects in a fed state and again following a 24-hour fast. Skeletal muscle samples were obtained under local anaesthesia from m. vastus lateralis using a needle biopsy technique. The blood glucose concentration fell during fasting; the blood lactate concentration remained unchanged. Plasma free fatty acid concentrations rose, as did 3-hydroxybutyrate and acetoacetate levels. There was no change in the amount of carbohydrate stored as glycogen in the muscle in response to fasting. The muscle content of phos-phagens (adenosine triphosphate and phosphocreatine), glycolytic intermediates (glucose-6-phosphate, fructose-6-phosphate, fructose-1, 6-diphosphate, triose phosphates and lactate) and citrate was also not affected by fasting. There was a significant increase in the muscle content of 3-hydroxybutyrate. These results give no indication as to the mechanism by which a decreased rate of carbohydrate degradation might occur in muscle in the fasted state. It is clear, however, that an intracellular accumulation of citrate and a consequent inhibition of glycolysis at the level of phosphofructokinase does not take place in fasting human skeletal muscle.     

腓骨肌萎缩综合征患者MFN2基因突变检测的研究

目的 对1个腓骨肌萎缩综合征核心家系的致病突变进行鉴定和遗传学分析.方法 采用全外显子组测序技术筛选先证者中的致病突变,用Sanger测序技术检测核心家系中致病突变位点的基因型,并对其进行生物信息学分析.结果 先证者MFN2基因存在单碱基杂合突变c.1090C>T p.R364W,母亲与妹妹均为该位点单碱基杂合突变,且...     

Muscle phosphofructokinase deficiency. Biochemical and immunological studies of phosphofructokinase isozymes in muscle culture.

Muscle cultures from three unrelated patients with muscle phosphofructokinase (PFK; EC 2.7.1.11) deficiency (Glycogenosis type VII; Tarui disease) had normal PFK activity and normal morphology. Chromatographic and immunological studies showed that normal muscle cultures express all three PFK subunits, M (muscle-type), L (liver-type), and P (platelet-type) and contain multiple homotetrameric and heterotetrameric isozymes. Muscle cultures from patients lack catalytically active M subunit-containing isozymes, but this is compensated for by the presence of P- and L-containing isozymes. Despite the lack of muscle-type PFK activity, presence of immunoreactive M subunit was demonstrable by indirect immunofluorescence, suggesting a mutation of the structural gene coding for the M-subunit of PFK.     

Isoenzymes of human phosphofructokinase

Human liver phosphofructokinase has been isolated in order to obtain antibodies against human liver phosphofructokinase. With the antiserum it could be shown in human liver that two types of phosphofructokinase exist with no subunits in common. Leucocytes, erythrocytes and kidney also possess the liver (L-) type subunit and a small amount of L-type is present in brain. There is no L-type of phosphofructokinase present in muscle and heart tissue.     

Glycogen content and activities of key glycolytic enzymes in muscle biopsies from control subjects and patients with chronic alcoholic skeletal myopathy

The capacity for glycolysis in muscle biopsies obtained from long-term heavy alcohol drinking patients has been compared with tissue from control subjects by assay in vitro of the total activities of glycogen phosphorylase, phosphofructokinase and fructose 1,6-bisphosphatase, key regulatory enzymes in the anaerobic glycolytic pathway. Biopsies from 13 of 22 patients had type II fibre atrophy, and the activities of all three enzymes were reduced in these biopsies, when expressed in terms of DNA content, the most striking reduction being in phosphofructokinase activity. The amount of glycogen in the tissue correlated closely with these enzyme activities and was slightly lower in the most atrophic tissue, when expressed in terms of DNA content. The activities of acid and neutral alpha-glucosidases were similar in biopsies from control subjects and patients with various severities of alcohol myopathy. The reduced activities are consistent with a reduced proportion of type II fibre muscle mass in these patients, and suggest that there may be a reduced capacity for glycolysis with resultant reduced lactate production. Whether the changes in enzyme activities are primary to the selective atrophy remains to be established.     

1 例RHD*weak D type 72 患者的血型鉴定及家系RHD 基因序列分析

目的 研究1 例RhD 血型鉴定部分凝集结果个体及其家系血清学表现和RHD 基因。方法 通过血型微柱凝胶卡检测先证者ABO 及RhD 血型;盐水试管法检测先证者及其父母RhCcEe 抗原;间接抗人球蛋白试验(indirect antihumanglobulin test,IAT) 及流式细胞术检测先证者RhD 抗原。PCR 序列特异性引物(PCR sequence specific primer, PCRSSP)检测RHD 基因以及RhD 杂合型分析,基因测序方法分析RHD 基因编码区序列。结果 血清学检测发现先证者血型为A 型RhCcee,血型微柱凝胶卡、盐水试管法以及IAT 法检测RhD 抗原,结果呈部分凝集现象。流式细胞术结果显示先证者RhD 抗原性减弱。经RHD 基因编码序列分析发现,RHD 基因第9 外显子上的第1212 位碱基发生C ＞A 纯合突变,为RHD*weak D type 72 的特征性突变点。家系调查显示,先证者父亲为O 型RhCCDee,母亲为A 型RhCcDee。父亲携带RHD*weak D type 72 等位基因,基因型为RHD*weak D type 72 / RHD+;母亲一条染色体缺失了全部的RHD 基因,基因型为RHD+/ RHD-。证明先证者分别从父亲和母亲遗传RHD*weak D type 72 和RHD-等位基因,基因型为RHD*weak D type 72 / RHD-。结论 发现了1 例RHD*weak D type 72/RHD-基因型个体,丰富了RHD*weakD type 72 变异型的研究数据。根据家系调查证明,RHD*weak D type 72 等位基因由遗传获得,而非由个体基因变异形成。     

三个遗传性血小板无力症家系临床特性和基因分析

目的 对三个遗传性血小板无力症(GT)家系进行整合素αⅡbβ3临床特性分析和基因突变检测.方法 经BPC、血涂片、出血时间(BT)、凝血常规检查、血小板聚集试验和流式细胞术进行实验检测;用PCR法对先证者αⅡbβ3基因所有外显子及其侧翼序列进行扩增;PCR产物纯化后直接测序,检测其基因突变.突变位点经直接测序排除基因多态性,103名健康人作对照.结果 三个家系的先证者BPC正常,血小板不聚集,BT延长,凝血常规指标检查正常,对ADP、凝血酶、肾上腺素、胶原、花生四烯酸等多种诱聚剂反应低下,而对瑞斯托霉素反应基本正常;家系1和家系3先证者的血小板膜表面αⅡbβ3的含量极度降低,家系2先证者的血小板膜表面αⅡb阳性血小板为63%,β3阳性血小板为76%.家系1先证者αⅡb基因存在G10A纯合突变,β3基因存在G1412T纯合突变.家系2先证者β3基因存在G1199A和1525delC复合杂合突变.家系3先证者在αⅡbβ3基因未检测到突变.结论 G10A和G1412T复合纯合突变是导致家系1先证者发生GT的原因,G1199A和1525delC复合杂合突变是导致家系2先证者发生GT的原因.G10A、G1412T和1525delC为国际首次报道的突变.     

Activities of key enzymes in the energy metabolism of human myocardial and skeletal muscle

Summary. Activities of total creatine kinase (CK), its isoenzyme MB (CK-MB), total lactate dehydrogenase (LD) and its isoenzyme LD₁, phosphofructokinase (PFK), asparate aminotransferase (ASAT) and citrate synthase (CS) were determined in skeletal muscle biopsies obtained from physically trained and untrained men and in myocardial biopsies from patients subjected to open heart surgery because of valve disease. The LD₁, ASAT and CS activities were higher in trained than in untrained skeletal muscle and still higher in heart muscle than in either trained or untrained skeletal muscle. The CK-MB activity was higher in trained than untrained skeletal muscle and the myocardial CK-MB activity was similar to that in trained skeletal muscle. Total CK activity was slightly lower in trained than in untrained skeletal muscle and the myocardial CK activity was approximately one third of the skeletal muscle CK. Both the PFK and the total LD activity was of similar magnitude in the different muscle types. In conclusion, as estimated by enzyme activities, the oxidative capacity is 2–3 times larger in myocardial than in skeletal muscle, while the glycolytic capacity as estimated by PFK appears to be the same.     

伴肌肉痉挛、疼痛的脊髓小脑性共济失调3型家系研究

目的 分析伴有肌肉痉挛的脊髓小脑性共济失调3型（SCA3） 一家系的临床特点.方法 采取家系18名成员的血清,提取DNA进行基因PCR扩增及检测,确定其类型,并对家系成员的临床表现、头颅磁共振、药物疗效等进行分析.结果 该家系6代人中,17人有步态不稳等表现,其中6人已死亡,在世的家系成员中有6人除步态不稳外,还伴有不同程度肌肉痉挛、疼痛等表现,对家系中的18人进行基因检测,其中12人PCR扩增发现相应位点CAG异常重复,证实为SCA3型,先证者头颅MRI发现小脑萎缩,给予卡马西平片治疗,可明显缓解肌肉痉挛、疼痛等症状.结论 报道一个以肌肉痉挛、疼痛为主要临床表现的SCA3型一家系,并发现卡马西平片对缓解SCA3伴发的肌肉痉挛有一定疗效.     

Correlative studies on the effect of carbachol on myo-inositol trisphosphate accumulation, myosin light chain phosphorylation and contraction in sphincter smooth muscle of rabbit iris

Previously, we have reported that activation of muscarinic cholinergic receptors in the iris smooth muscle results in a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol and myo-inositol trisphosphate (IP3) and that the stimulated hydrolysis of this phospholipid correlates well with contraction. To determine whether or not there is a causal relationship between PIP2 breakdown and contraction, we have conducted correlative studies on the effects of carbachol (CCh) on PIP2 breakdown, measured as IP3 accumulation, myosin light chain (MLC) phosphorylation and contraction in the rabbit iris sphincter. We have also investigated the effects of time, temperature, atropine antagonism, Ca++ and C-kinase activators on the three measured responses. The data obtained can be summarized as follows: dose-response studies for IP3 accumulation, MLC phosphorylation and contraction revealed a close correlation between these responses; kinetic data on atropine antagonism showed that the three measured responses are competitively inhibited by the muscarinic antagonist; time course studies conducted at low temperature showed that the CCh-induced IP3 accumulation and MLC phosphorylation may precede contraction; time course studies on the effect of Ca++ on the three measured responses showed that IP3 release may account for the rapid phase of CCh-induced contraction and that extracellular Ca++ is essential for sustained MLC phosphorylation and the slow phase of contraction; the activity of phospholipase C, the enzyme involved in PIP2 hydrolysis, in membrane fragments from 32P-labeled sphincter muscle was found to be highly sensitive to Ca++, with half-maximal stimulation at about 1.1 microM Ca++; and phorbol 12,13-dibutyrate, but not phorbol 12-myristate 13-acetate, induced MLC phosphorylation and muscle contraction in a dose- and time-dependent manner. Phorbol 12,13-dibutyrate and ionomycin acted in a synergistic manner to elicit contraction. In conclusion, contractions by CCh in the iris sphincter may be explained on the basis of enhanced PIP2 turnover and its derived second messenger molecule(s); that there are consistent correlations, using different concentrations of CCh, atropine antagonism, time, temperature and Ca++, between the stimulated hydrolysis of PIP2, MLC phosphorylation and contraction. Finally, whereas the data presented favor the involvement of IP3 in the phasic component of the contractile response, the studies with phorbol 12,13-dibutyrate suggest that contractile regulation by 1,2-diacylglycerol, through activation of C-kinase, may be important during the tonic component of smooth muscle contraction.     

A novel COQ8A missense variant associated with a mild form of primary coenzyme Q10 deficiency type 4

BackgroundPrimary coenzyme Q10 deficiency refers to a group of diseases characterised by reduced levels of coenzyme Q10 in related tissues or cultured cells associated with the 9 genes involved in the biosynthesis of coenzyme Q10. A biallelic pathogenic variant of COQ8A gene causes the occurrence of the primary coenzyme Q10 deficiency type 4. The objective of this study was to investigate the genetic cause of muscle weakness in a proband who had a negative DMD gene test for Becker muscular dystrophy.MethodsThe DNA of the proband was sequenced using whole exome sequencing. With the help of the Human Phenotype Ontology (HPO), the range of related candidate pathogenic genes has been reduced to a certain extent based on “muscle weakness” (HP:0001324). In addition, family linkage analysis, phenotypic-genotype check and protein structure modeling were used to explore the genetic cause of the proband.ResultsThe compound heterozygous variant c.836A > C (p.Gln279Pro) and c.1228C > T (p.Arg410Ter) in the COQ8A gene was identified in the proband. According to the 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines for the interpretation of sequence variants, the novel variant c.836A > C could be classified as “likely pathogenic” for the proband.ConclusionThe p.Gln279Pro was detected in the KxGQ motif and the QKE triplet of the COQ8A protein, whose structures were crucial for the structure and function of the COQ8A protein associated with the biosynthesis of coenzyme Q10 and the proband's clinical symptoms were relatively milder than those previously reported.     

Direct enzymatic assay for %HbA1c in human whole blood samples

ObjectivesDevelopment and validation of a direct enzymatic HbA1c assay that utilizes a single channel on chemistry auto-analyzers without the need to run separate glycated hemoglobin and total hemoglobin assays.Design and methodsAn enzyme based single channel assay was developed to measure %HbA1c in human whole blood samples. The performance characteristics of the Diazyme Direct Enzymatic HbA1c Assay were evaluated on the Hitachi 917 auto-analyzer using whole blood samples, appropriate controls and a reference lot of manufactured reagents. Accuracy studies were completed by comparing the Direct Enzymatic Assay to existing HPLC and immunoassay methods. Interference testing was performed to determine the effect of total hemoglobin, glycated serum proteins, chemical substances and hemoglobin variants in patient samples.ResultsThe Direct Enzymatic HbA1c Assay showed within run precision and total precision results of ≤ 2% CV for both normal and abnormal level samples. Method comparison studies showed that there was a good correlation between the Direct Enzymatic HbA1c and the HPLC (R² = 0.98) or the immunoassay (R² = 0.97) methods. The assay measured within the range of 4–16% HbA1c and showed excellent performance with variant hemoglobin in samples.ConclusionsDiazyme Direct Enzymatic HbA1c Assay is accurate and precise when compared to currently marketed medical devices. The assay is designed to report %HbA1c values directly without need for a separate measurement of total hemoglobin and is not adversely affected by interferences from common hemoglobin variants in samples. It is a cost effective, user-friendly method and is adaptable to most general chemistry analyzers.     

纤维蛋白原Bβ-链复合杂合突变导致的遗传性无纤维蛋白原血症

遗传性无纤维蛋白原血症是一种由于纤维蛋白原基因缺陷所致常染色体隐性遗传病.为了对1例遗传性无纤维蛋白原血症家系进行表型和基因型分析,采集了该家系三代10人外周血,吸取上层血浆用血凝仪检测活化部分凝血活酶时间（APTT）、凝血酶原时间（PT）和凝血酶时间（TI）;纤维蛋白原（Fg）含量分别用Clauss法和免疫比浊法进行检测;以常规酚-氯仿法抽提家系所有成员外周血基因组DNA,PCR扩增Fg基因FGA、FGB和FGG所有外显子及其侧翼序列和启动子区,PCR产物纯化后直接测序以检测基因突变.102例健康献血者作为正常对照.结果表明：先证者表型诊断为无纤维蛋白原血症;基因型呈Fg B β-链Arg17stop和Gly347Arg复合杂合突变,前者来源于母系,后者来源于父系.结论：Fg B β-链Arg17 stop和Gly347Arg复合杂合突变是引起该家系先证者产生无纤维蛋白原血症的原因.     

B(A)血型分子机制研究及其家系分析

目的 研究罕见B(A)血型的血清学特性和分子机制,为B(A)血型的临床输血提供理论基础.方法 利用单克隆抗体检测1例先证者、家系成员及献血者红细胞ABO血型抗原,用标准A、B、O红细胞检测其血清中的ABO抗体.采用盐水法、凝聚胺法和抗球蛋白法进行先证者与献血者交叉配合试验.采用PCR技术扩增ABO基因的第6、7外显子序列,对先证者、家系成员、献血者标本的ABO基因外显子6、7和侧翼内含子序列进行测序分析,并对先证者标本进行单倍体序列分析.结果 先证者及其2位家系成员红细胞上有A、B抗原,同时血清中存在抗A_1抗体,血清学表型为A_2B.直接测序分析发现先证者标本第6、7外显子存在261G/del、297A/G、526C/G、657C/T、700C/G、703A/G、796A/C、803G/C、930A/G杂合,可推断为B(A)_(02)/O_(01)基因型杂合子;家系中其母亲基因型为B(A)_(02)/B_(101),外祖母为B(A)_(02)/O_(01).先证者单倍体序列分析得到2个等位基因B(A)_(02)和O_(01);与B_(101)序列相比,B(A)_(02)位第700位C>G,导致1个氨基酸改变:第234位脯氨酸变成丙氨酸.既往血清学特性为A_2B的2个献血者,1个基因型为B(A)_(02)/O_(01),另1个基因型是A_(208)/B_(101).B(A)血型先证者与这2名献血者进行交叉配血试验均相合,临床输注后无不良反应.结论 α-1,3-半乳糖基转移酶等位基因(B等位基因)700C>G突变可导致形成B(A)血型,其血清学特性显示为A_2B表型.B(A)血型临床输血相配合的供者可选择A_2B表型的献血者.     

一种新的FⅩⅢ基因突变导致的遗传性FⅩⅢ缺陷症

目的 对1例遗传性凝血因子ⅩⅢ（FⅩⅢ）缺陷症患者及其家系成员FⅩⅢ基因[F（13）A]进行分析，探讨其分子致病机制。方法 尿素溶解法定性检测FⅩⅢ活性，抽提外周血基因组DNA．PCR扩增FⅩⅢA基因的15个外显子及其侧翼序列，PCR产物纯化后直接基因测序，并对家系成员F（13）基因相应的突变序列进行检测。结果 先证者FⅩⅢ定性试验阳性。基因测序发现先证者F（13）A存在纯合缺失，外显子10自127067位起缺失33个核苷酸（127067del33，GI：AF418272），导致阅读框内缺失11个氨基酸（406Met-416Ala），产生了由720个氨基酸残基组成的截短型FⅩⅢA蛋白。其父母FⅩⅢ定性试验为阴性，均显示为该序列的杂合缺失突变。结论 先证者为遗传性FⅩⅢ缺陷症患者，由F（13）A外显子10缺失突变所致。该突变为国际首次报道。     

Mutation of a highly conserved residue of betaI spectrin associated with fatal and near-fatal neonatal hemolytic anemia.

We studied an infant with severe nonimmune hemolytic anemia and hydrops fetalis at birth. His neonatal course was marked by ongoing hemolysis of undetermined etiology requiring repeated erythrocyte transfusions. He has remained transfusion-dependent for more than 2 yr. A previous sibling born with hemolytic anemia and hydrops fetalis died on the second day of life. Peripheral blood smears from the parents revealed rare elliptocytes. Examination of their erythrocyte membranes revealed abnormal mechanical stability as well as structural and functional abnormalities in spectrin. Genetic studies revealed that the proband and his deceased sister were homozygous for a mutation of betaIsigma1 spectrin, L2025R, in a region of spectrin that is critical for normal function. The importance of leucine in this position of the proposed triple helical model of spectrin repeats is highlighted by its evolutionary conservation in all beta spectrins from Drosophila to humans. Molecular modeling demonstrated the disruption of hydrophobic interactions in the interior of the triple helix critical for spectrin function caused by the replacement of the hydrophobic, uncharged leucine by a hydrophilic, positively charged arginine. This mutation must also be expressed in the betaIsigma2 spectrin found in muscle, yet pathologic and immunohistochemical examination of skeletal muscle from the deceased sibling was unremarkable.     

一个多发性骨软骨瘤家系的EXT1基因突变分析

目的对1个多发性骨软骨瘤家系的致病基因EXT1进行突变分析。方法提取先证者外周血基因组DNA,PCR扩增EXT1基因全部外显子序列并进行序列测定;找到突变后,对相应PCR产物进行T克隆,以证实突变。对其他家庭成员的EXT1基因相应外显子也进行序列分析。结果先证者的临床表现符合多发性骨软骨瘤,其父也有类似临床表现,但明显轻于先证者,且11名同胞及其子女均无发病。先证者序列分析表明,其EXT1基因第6外显子存在杂合的缺失突变：1476-1477delTC。先证者父亲的血液组织、精子和口腔粘膜细胞也存在相同突变,但在PCR产物直接测序中突变体的测序信号明显弱于先证者;在PCR产物的T克隆-测序中,来自先证者父亲的血液、精子、口腔黏膜细胞突变体的重组菌分别占总数的10%、2.6%、13.3%。其他11名同胞未见EXT1基因突变。结论先证者父亲在胚胎早期发生了EXT1基因突变,使其体内部分细胞（包括部分软骨细胞）带有EXT1基因突变,导致症状较轻的多发性骨软骨瘤。     

Defective binding of spectrin to ankyrin in a kindred with recessively inherited hereditary elliptocytosis

The interaction of spectrin with spectrin-depleted inside-out membrane vesicles was studied in a kindred with an atypical variant of hereditary elliptocytosis inherited in a recessive manner. The probands are characterized by prominent elliptocytosis, decreased erythrocyte thermal stability, an altered limited tryptic peptide pattern of spectrin digested at low ionic strength, and defective spectrin dimer-dimer association. The parents are normal. The spectrin/band 3 ratio determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated membranes of the probands was decreased to approximately 70% of control values, and total erythrocyte spectrin content in one proband was also decreased on SDS-PAGE. When a monospecific antispectrin antibody was used, a faintly labeled fragment of molecular weight approximately 28,000 was detected on immunoblots of whole cell lysates of one proband and a control, but could not account for the decreased erythrocyte spectrin content of the proband on SDS-PAGE. Binding and competitive inhibition studies revealed an alteration in the spectrin-ankyrin interaction due to an abnormality of spectrin in the probands. No defect was found in the mother; the father's spectrin showed decreased binding affinity, although it was not so severe as in the probands. Separation of bound and unbound spectrin dimers from one proband and subsequent conversion to tetramers showed that the self-association defect was detectable only on the bound subpopulation of her spectrin. These findings demonstrate a hitherto undescribed functional abnormality of spectrin in this kindred which could result in decreased stability of the membrane skeleton and contribute to the elliptocytic shape of these erythrocytes.