Guanidinoacetate methyltransferase (GAMT) deficiency: non-invasive enzymatic diagnosis of a newly recognized inborn error of metabolism

Guanidinoacetate methyltransferase deficiency is a newly recognized inborn error of creatine biosynthesis. Manifestation of neurologic symptoms occurs in infancy and is partly reversible upon oral substitution of creatine. In the first two index patients, enzymatic diagnosis was established in a liver biopsy, and the underlying molecular defect in the GAMT gene has been identified. In order to provide non-invasive biochemical diagnosis, we have developed an enzyme assay based on the formation of radiolabeled creatine from ¹⁴C guanidinoacetate and S-adenosylmethionine in concentrated and dialyzed extracts from cultivated skin fibroblasts, Epstein–Barr virus transformed lymphoblasts, and cultivated amniotic cells. Cells were investigated from controls, from 1 index patient with proven GAMT deficiency and from 3 additional patients with clinical and biochemical signs of GAMT deficiency. Separation of ¹⁴C guanidinoacetate from ¹⁴C creatine in the reaction mixture was accomplished by HPLC on Hypersil ODS column and radioactivity was determined in fractions according to respective UV signals. GAMT activities in control fibroblasts (n=7), lymphoblasts (n=8) and in amniotic cells (n=2) were 0.38–0.56, 0.61–0.84 and 0.38–0.56 nmol/h/mg protein. Apparent Km values were 9.5–14.8 μM for guanidinoacetate and 68–78 μM for S-adenosylmethionine. In the index patient and in the three additional patients at risk, GAMT activity was <0.1 nmol/h/mg protein. The assay described here allows non-invasive diagnosis of GAMT deficiency in patients at risk.     

Guanidinoacetate and creatine plus creatinine assessment in physiologic fluids: an effective diagnostic tool for the biochemical diagnosis of arginine:glycine amidinotransferase and guanidinoacetate methyltransferase deficiencies

BACKGROUND: Disorders of creatine metabolism arise from genetic alterations of arginine:glycine amidinotransferase (AGAT), guanidinoacetate methyltransferase (GAMT), and the creatine transporter. We developed a strategy for the detection of AGAT and GAMT defects by measurement of guanidinoacetate (GAA) and creatine plus creatinine (Cr+Crn) in biological fluids. METHODS: Three patients with AGAT deficiency from the same pedigree and their eight relatives, as well as a patient affected by a GAMT defect and his parents were analyzed by a new HPLC procedure in comparison with 90 controls. The method, which uses precolumn derivatization with benzoin, separation with a reversed-phase column, and fluorescence detection, has shown good precision and sensitivity and requires minimal sample handling. RESULTS: In the three AGAT patients, plasma GAA was 0.01-0.04 micro mol/L [mean (SD) for neurologically normal controls was 1.16 (0.59) micromol/L], Cr+Crn was 15-29 micro mol/L [reference limit in our laboratory, 79 (38) micromol/L]. Urinary GAA was 2.4-5.8 micro mol/L [reference, 311 (191) micromol/L], and Cr+Crn was 2.1-3.3 mmol/L [reference, 9.9 (4.1) mmol/L]. We found a smaller decrease in GAA and Cr+Crn in some carriers of an AGAT defect. In the patient with GAMT deficiency, plasma and urine GAA was increased (18.6 and 1783 micromol/L, respectively), and Cr+Crn was decreased in plasma (10.7 micromol/L) and urine (2.1 mmol/L). GAA was increased in the parents' plasmas and in the mother's urine. CONCLUSION: The assessment of GAA is a new tool for the detection of both GAMT and AGAT deficiencies.     

Cholangiocarcinoma in two siblings with emphysema and alpha-1-antitrypsin deficiency

Two siblings with homozygous ZZ alpha-1-antitrypsin deficiency were discovered to have primary liver tumours (both cholangiocarcinomas). This lends support to the view that alpha-1-antitrypsin deficiency plays a role in the development of some primary liver tumours.     

17alpha-hydroxylase deficiency : biochemical and molecular findings in two sisters and their family.

OBJECTIVE: To search for molecular changes in two Argentinian sisters with a clinical and biochemical diagnosis of 17alpha-hydroxylase deficiency. SUBJECTS: Both patients had 46 XX karyotype, with sexual infantilism, primary amenorrhea, and hypertension. Other member of the first degree family did not have this deficiency. HORMONAL RESULTS: The patients showed high levels of gonadotrophins and progesterone along with very low cortisol and androgen levels. Basal levels of corticosterone were very high, but aldosterone was normal. Both steroids had a high response after adrenocorticotropic hormone (ACTH) stimulation, with no changes in 17-hydroxyl progesterone and cortisol levels. Progesterone, corticosterone, and aldosterone decreased with the dexamethasone test, without modifications in 17-hydroxyl progesterone and cortisol levels. A corticosterone/aldosterone ratio was calculated from the results of the stimulation test; the ratios were similar in both patients. On administration of the ACTH test, both parents and one sister (S2) showed a marked response in corticosterone levels, their corticosterone/aldosterone ratios were also similar to each other and similar to the patients. MOLECULAR RESULTS: Molecular studies in the cytochrome P450 17 (CYP17) gene showed that exon 8 had a 4 bp duplication at codon 480 (CATC) in the two patients and their mother and in exon 1, a C to T transition at codon 96 was identified, changing CGG into TGG in the two patients, S2, and their father. CONCLUSIONS: Both patients were shown to be compound heterozygous, carrying different alleles in exon 1 and exon 8, inherited from their father and mother, respectively. The molecular results obtained on S2 confirmed the heterozygosity suggested by the stimulated hormonal test and corticosterone/aldosterone ratio.     

Apolipoprotein C-ll deficiency syndrome due to apo C-IIHamburg: clinical and biochemical features and Hphl restriction enzyme polymorphism

We have characterized the clinical and biochemical features of three siblings of a kindred with severe hypertriglyceridaemia due to apolipoprotein C-II (apo C-II) deficiency caused by the mutation described as apo C-IIHamburg. The clinical syndrome is characterized by recurrent pancreatitis in two of three affected individuals, with discrete hepatosplenomegaly in all three patients and cholelithiasis in one. Eruptive xanthomas and lipemia retinalis were absent. Plasma lipoproteins were characterized by fasting chylomicronaemia, reduced low density lipoproteins (LDL) and low high density lipoproteins (HDL). The marked hypertriglyceridaemia could be corrected promptly by infusion of normal plasma. Apolipoprotein C-II (apo C-II) levels in homozygotes were very low (0.01 mg dl-1), and mean apo C-II levels in heterozygotes were lower (2.08 +/- 0.11 mg dl-1) than in normal family members (3.38 +/- 0.75 mg dl-1). Lipoprotein lipase and hepatic triglyceride lipase activities in post-heparin plasma were normal. Zonal ultracentrifugation revealed a marked increase in triglyceride-rich lipoproteins and reduced LDL and HDL. LDL consisted of two fractions with higher hydrated density of the main fraction compared with normals with a trend to normalization on a fat-free diet. The molecular defect in the apo C-II Hamburg gene has been previously identified as a donor splice site mutation in the second intron. This leads to abnormal splicing of the apo C-II Hamburg mRNA and apo C-II deficiency in plasma. The mutation causes the loss of an HphI restriction enzyme site present in the normal apo C-II gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutation of the fumarase gene in two siblings with progressive encephalopathy and fumarase deficiency.

We report an inborn error of the tricarboxylic acid cycle, fumarase deficiency, in two siblings born to first cousin parents. They presented with progressive encephalopathy, dystonia, leucopenia, and neutropenia. Elevation of lactate in the cerebrospinal fluid and high fumarate excretion in the urine led us to investigate the activities of the respiratory chain and of the Krebs cycle, and to finally identify fumarase deficiency in these two children. The deficiency was profound and present in all tissues investigated, affecting the cytosolic and the mitochondrial fumarase isoenzymes to the same degree. Analysis of fumarase cDNA demonstrated that both patients were homozygous for a missense mutation, a G-955-->C transversion, predicting a Glu-319-->Gln substitution. This substitution occurred in a highly conserved region of the fumarase cDNA. Both parents exhibited half the expected fumarase activity in their lymphocytes and were found to be heterozygous for this substitution. The present study is to our knowledge the first molecular characterization of tricarboxylic acid deficiency, a rare inherited inborn error of metabolism in childhood.     

Human erythrocyte thiopurine methyltransferase: Radiochemical microassay and biochemical properties

A radiochemical micromethod for the determination of thiopurine methyltransferase (TPMT) activity in human red blood cells (RBC) is described. Both 6-mercaptopurine and 6-thioguanine were substrates for the TPMT activity in the human RBC: Apparent Michaelis-Menten (K_M) values for 6-mercaptopurine and 6-thioguanine were 3.2 × 10^?4 M and 2.0 × 10^?4 M, respectively. The apparent K_M value for S-adenosyl-l-methionine, a co-substrate for the reaction, was 1.7 × 10^?6 M. The pH optimum for the reaction was approximately 7.5. Blood samples from 73 randomly selected adult subjects had a mean activity of 10.2 ± 2.4 (mean ± S.D.) units/ml packed red blood cells. The range of activities was from 4.6 to 14.2 units/ml. The results of experiments in which partially purified human kidney TPMT was added to RBC lysates and of experiments in which “low” and “high” activity lysates were mixed gave no indication that individual variations in RBC TPMT activity were due to endogenous inhibitors or activators of the enzyme.     

JMH variants: serologic, clinical, and biochemical analyses in two cases

BACKGROUND: JMH is a high-frequency red cell blood group antigen that resides on a 76- to 80-kDa glycosylphosphatidylinositol-linked protein also known as CDw108. Antibodies with JMH specificity are often autoimmune and are usually, if not always, clinically benign. Some individuals with JMH-variant antigen produce alloantibodies to JMH, but little evidence concerning their clinical significance is available. This article reports on two patients who express a JMH-variant antigen and produced alloanti-JMH. STUDY DESIGN AND METHODS: Murine monoclonal antibodies and human antibodies to JMH were used in hemagglutination, radioimmunoassay, and Western blot testing of red cells from two JMH- variant patients; antiserum from one of these patients was also used in biochemical studies. In addition, in vivo survival of JMH-positive red cells was studied in the same patient. RESULTS: Biochemically, both examples of red cells with the JMH-variant phenotype expressed a JMH protein with a molecular weight similar to that of the normal JMH protein. For both patients, family studies suggested an autosomal recessive pattern of inheritance. Survival study demonstrated reduced in vivo red cell survival in one patient. CONCLUSION: JMH-variant phenotypes express a protein of normal molecular weight and are inherited in an autosomal recessive pattern. Furthermore, individuals with this phenotype can produce clinically significant antibodies.     

二例先天性角化不良症患儿临床特征和基因分析

目的 对2例先天性角化不良症(DC)患儿进行临床和基因分析.方法 2例患儿均表现为皮肤黏膜异常三联征(指/趾甲营养不良、黏膜白斑病、皮肤网状色素沉着)和骨髓衰竭.临床诊断为DC.提取患儿外周血DNA,PCR扩增DC的6个热点基因,包括DKC1、TERT、TERC、TINF2、NOP10、NHP2,进行DNA测序和基因分析.结果 2例患儿标本均克隆出TINF2基因中第6号外显子的c.845G→A(R282H)突变.结论 低龄患儿出现典型皮肤黏膜改变、骨髓衰竭时,应考虑到DC可能.早期行相关基因检测可避免误诊、漏诊.2例TINF2基因c.845G→A(R282H)突变为国内首次报道.     

Updated clinical and glycomic features of mannosyl-oligosaccharide glucosidase deficiency: Two case reports

BACKGROUNDMannosyl-oligosaccharide glucosidase (MOGS) deficiency is an extremely rare type of congenital disorder of glycosylation (CDG), with only 12 reported cases. Its clinical, genetic, and glycomic features are still expanding. Our aim is to update the novel clinical and glycosylation features of 2 previously reported patients with MOGS-CDG.CASE SUMMARYWe collected comprehensive clinical information, and conducted the immunoglobulin G1 glycosylation assay using nano-electrospray ionization source quadruple time-of-flight mass spectrometry. Novel dysmorphic features included an enlarged tongue, forwardly rotated earlobes, a birth mark, overlapped toes, and abnormal fat distribution. Novel imaging findings included pericardial effusion, a deep interarytenoid groove, mild congenital subglottic stenosis, and laryngomalacia. Novel laboratory findings included peripheral leukocytosis with neutrophil predominance, elevated C-reactive protein and creatine kinase, dyslipidemia, coagulopathy, complement 3 and complement 4 deficiencies, decreased proportions of T lymphocytes and natural killer cells, and increased serum interleukin 6. Glycosylation studies showed a significant increase of hypermannosylated glycopeptides (Glc3Man7GlcNAc2/N2H10 and Man5GlcNAc2/N2H5) and hypersialylated glycopeptides. A compensatory glycosylation pathway leading to an increase in Man5GlcNAc2/N2H5 was indicated with the glycosylation profile. CONCLUSIONWe confirmed abnormal glycomics in 1 patient, expanding the clinical and glycomic spectrum of MOGS-CDG. We also postulated a compensatory glycosylation pathway, leading to a possible serum biomarker for future diagnosis.     

Human erythrocyte thiol methyltransferase: radiochemical microassay and biochemical properties.

A radiochemical microassay for the measurement of thiol methyltransferase (TMT) activity in human red blood cell (RBC) membranes has been developed. Both 2-mercaptoethanol and dithiothreitol were used as substrates for the enzyme. The pH optimum of the reaction was approximately 9.0 when glycine NaOH was used as a buffer. The apparent Michaelis-Menten (KM) value for the methyl donor for the reaction, S-adenosyl-L-methionine, was 43 mumol/l. Human RBC TMT activity was neither activated nor inhibited by Ca2+, Mg2+, or tropolone, but the enzyme was inhibited by SKF 525A and by reagents that react with sulfhydryl groups. The mean TMT activity in blood from 289 randomly selected adult white subjects was 10.93 +/- 3.22 units per mg protein (mean +/- S.D.). The activity was the same in samples from men and women. The results of experiments in which TMT activity was measured in mextures of RBC membranes with relatively "low" and relatively "high" activities provided no evidence that individual variations in the enzyme activity were due to variations in endogenous TMT activators or inhibitors.     

Mouse thiopurine methyltransferase pharmacogenetics: biochemical studies and recombinant inbred strains

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of 6-mercaptopurine and other heterocyclic and aromatic thiol compounds. In humans, TPMT activity is controlled by a common genetic polymorphism. C57BL/6J (B6) and AKR/J (AK) inbred mice have low hepatic and renal TPMT activities, whereas DBA/2J (D2) mice have high enzyme activities. Low TPMT activity is inherited in these mice as an autosomal recessive trait. The properties of TPMT in liver homogenates from B6, AK and D2 mice were compared in order to study the biochemical basis for inherited differences in TPMT activity among these strains. Biochemical and physical properties of hepatic TPMT were very similar in all three strains. Apparent Michaelis (Km) constants for 6-mercaptopurine were 0.98, 0.75 and 1.1 mM for B6, AK and D2 mice, respectively. Apparent Km values for S-adenosyl-L-methionine, the methyl donor for the reaction, were 2.2, 1.5 and 3.0 microM for B6, AK and D2 mice. IC50 values for inhibition by 3,4-dimethoxy-5-hydroxybenzoic acid were 0.83, 1.0 and 1.2 microM, whereas IC50 values for inhibition by S-adenosyl-L-homocysteine were 5.4, 6.6 and 5.8 microM for B6, AK and D2 mice, respectively. Half-life and slope values for thermal inactivation of hepatic TPMT were similar among B6, AK and D2 mice. No differences among strains in Rf values of the enzyme activity after electrophoresis were detected. Ion exchange chromatography with an NaCl gradient showed a major peak of TPMT activity that eluted with 51 to 56 mM NaCl for all three strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Short-chain acyl-coenzyme A dehydrogenase deficiency. Clinical and biochemical studies in two patients.

We describe two patients with short-chain acyl-coenzyme A (CoA) dehydrogenase (SCADH) deficiency. Neonate I excreted large amounts of ethylmalonate and methylsuccinate; ethylmalonate excretion increased after a medium-chain triglyceride load. Neonate II died postnatally and excreted ethylmalonate, butyrate, 3-hydroxybutyrate, adipate, and lactate. Both neonates' fibroblasts catabolized [1-14C]butyrate poorly (29-64% of control). Neonate I had moderately decreased [1-14C]octanoate catabolism (43-60% of control), while neonate II oxidized this substrate normally; both catabolized radiolabeled palmitate, succinate, and/or leucine normally. Cell sonicates from neonates I and II dehydrogenated [2,3-3H]butyryl-CoA poorly (41 and 53% of control) and [2,3-3H]octanoyl-CoA more effectively (59 and 95% of control). Mitochondrial acyl-CoA dehydrogenase (ADH) activities with butyryl- and octanoyl-CoAs were 37 and 56% of control in neonate I, and 47 and 81% of control in neonate II, respectively. Monospecific medium-chain ADH (MCADH) antisera inhibited MCADH activity towards both butyryl- and octanoyl-CoAs, revealing SCADH activities to be 1 and 11% of control for neonates I and II, respectively. Fibroblast SCADH and MCADH activities were normal in an adult female with muscular SCADH deficiency.     

Cobalamin deficiency presenting with thrombotic microangiopathy (TMA) features: A systematic review

Introduction

Cobalamin deficiency may result in hematologic characteristics similar to thrombotic microangiopathy (TMA). To facilitate diagnosis, we reviewed reported cases of acquired cobalamin deficiency presenting with TMA features (c.def-TMA).

Whipple's disease: clinical, biochemical, and histopathologic features and assessment of treatment in 29 patients

Whipple's disease is a chronic systemic illness, the optimal treatment of which remains poorly defined. In our analysis of a 30-year, 29-patient experience with Whipple's disease at the Mayo Clinic, the frequent initial manifestations of diarrhea, weight loss, arthritis, and lymphadenopathy correlated with findings reported previously by other investigators. Antibiotic therapy yielded rapid symptomatic and biochemical improvement, and histologic changes in the small bowel occurred subsequently. Despite antimicrobial therapy, relapses in patients with Whipple's disease are common, and the central nervous system is considered the most serious site of involvement for recurrence. Administration of an antibiotic agent that is able to cross the blood-brain barrier may be more important in preventing relapse than prolonged duration of initial antimicrobial therapy.     

10例遗传性凝血因子Ⅶ缺陷症分子发病机制与临床特性分析

目的探讨10例遗传性凝血因子Ⅶ(coagulation factor Ⅶ，FⅦ)缺陷症基因突变类型与临床特性。方法 检测凝血指标以明确诊断；用DNA直接测序法对先证者及其家庭成员FⅦ基因的全部外显子及其侧翼、5’和3’非翻译区进行分析，寻找基因突变；将含插入或缺失突变序列克隆人pMD18-T TA克隆载体中，对所得两条染色体相应序列分别测序，以确定突变在染色体上的分布。应用限制性内切酶对先证者及家系成员相应基因片段进行酶切分析，无酶切位点改变的基因片段用等位基因特异的PER(ASPCR)方法，证实测序所发现的突变。结果 在10例遗传性凝血因子Ⅶ缺陷症患者中发现8种类型的基因突变，其中6390T→C(Phe40Cys)，9482G→T(Argl52Leu)，和11487．9delC3种突变为国际首次报道；6种突变发生在催化区；除一种缺失突变外，其余均为点突变；所有的基因突变都来自先证者的父亲和(或)母亲。Thr359Met和Arg304Trp突变分别在4个及2个无亲缘关系的家系中重复出现。2例Thr359Met纯合突变(FⅦ：C分别为2％和3％)及1例Argl52Leu、11487-9delC及Arg304Trp复合杂合突变(FⅦ：C为1％)临床表型为重型；2例双重杂合突变(His348Gln和Thr359Met，Agr304Trp和Arg304Gln)临床表型分别为中型和无症状(FⅦ：C分别为3．4％和10％)。4例杂合突变(Phe40Cys伴有Arg353Gln杂合多态性、Thr359Met、Arg152Gln、-55C→T)FⅦ：C分别为1％、3．0％、1％、5．5％，临床表型为中型或轻型；1例-323P0／P10、73G→A及Arg353Gln复合杂合多态性(FⅦ：C为1％)的临床表型为轻型。结论 在10例遗传性凝血因子Ⅶ缺陷症患者中发现了8种类型的FⅦ基因突变，其中3种为新突变。中国汉族人中存在导致FⅦ基因缺陷的突变热点。纯合及双杂合FⅦ基因突变FⅦ：C较低，临床出血常较重；特定的杂合突变可有临床出血倾向，FⅦ基因突变与FⅦ：C及临床表型无明显的相关性。     

Tubulointerstitial nephritis and uveitis syndrome in two siblings

Two Japanese sisters with persistent uveitis showed significant increased levels of urinary beta-2 microglobulin. A percutaneous renal biopsy performed in the younger sister revealed tubulointerstitial nephritis (TIN) with helper/inducer T cell infiltrates. Also, abnormal 67-gallium accumulation in the kidneys, suggesting TIN, was observed in the other one at the same time. Although patients with the syndrome of tubulointerstitial nephritis and uveitis (TINU) have been reported to date, its occurrence in siblings has rarely been seen. Both of them shared same human leukocyte antigen (HLA) DR6, suggesting the potential association between HLA-DR6 and TINU.