Functional methionine synthase deficiency (cblE and cblG): clinical and biochemical heterogeneity

Functional methionine synthase deficiency is generally characterized by homocystinuria and hypomethioninemia in the absence of methylmalonic aciduria. Patients are divided into two classes, cblE and cblG, on the basis of complementation analysis. Presentation has usually been in the first 2 years of life, but one patient came to medical attention at age 21 years with symptoms initially diagnosed as multiple sclerosis. Common findings among 11 patients (4 with cblE and 7 with cblG) have included megaloblastic anemia (all patients) and various neurological deficits including developmental retardation (10 patients), cerebral atrophy (8 patients), hypotonia (7 patients), EEG abnormalities (6 patients), and nystagmus (5 patients). Hypertonia, seizures, blindness, and ataxia were less frequent. All patients have responded to therapy with cobalamin with resolution of anemia and biochemical abnormalities; neurological deficits resolved more slowly and in some cases incompletely. Hydroxycobalamin has been more effective than cyanocobalamin. Fibroblasts from patients with cblE (5 patients) and cblG (6 patients) all showed decreased intracellular levels of methylcobalamin (MeCbl) and decreased incorporation of label from 5-methyltetrahydrofolate into macromolecules, suggesting decreased activity of the MeCbl-dependent enzyme methionine synthase. Methionine synthase specific activity in extracts of all cblE fibroblasts was normal or near-normal under standard reducing conditions; synthase specific activity in extracts of 5 cblG patients was low but was high in a 6th patient measured in another laboratory. Thus, there is heterogeneity among patients with functional methionine synthase deficiency both in clinical presentation and in the results of biochemical studies of cultured cells.     

Human methionine synthase: cDNA cloning and identification of mutations in patients of the cblG complementation group of folate/cobalamin disorders

Methionine synthase catalyzes the remethylation of homocysteine to methionine in a methylcobalamin-dependent reaction. We used specific regions of homology within the methionine synthase sequences of several lower organisms to clone a human methionine synthase cDNA by a combination of RT-PCR and inverse PCR. The enzyme is 1265 amino acids in length and contains the seven residue structure-based sequence fingerprint identified for cobalamin-containing enzymes. The gene was localized to chromosome 1q43 by the FISH technique. We have identified one missense mutation and a 3 bp deletion in patients of the cblG complementation group of inherited homocysteine/folate disorders by SSCP and sequence analysis, as well as an amino acid substitution present in high frequency in the general population. We discuss the possibility that a mild deficiency of methionine synthase activity could be associated with mild hyperhomocysteinemia, a risk factor for cardiovascular disease and possibly neural tube defects.      

cblE type of homocystinuria due to methionine synthase reductase deficiency: functional correction by minigene expression

The cblE type of homocystinuria is a rare autosomal recessive disorder caused by impaired reductive activation of methionine synthase. Although earlier biochemical studies proposed that the methionine synthase enzyme might be activated by two different reducing systems, mutations were reported in only the methionine synthase reductase gene (MTRR) in cblE patients. The pathogenicity of MTRR mutations, however, has not yet been tested functionally. We report on nine patients of European origin affected by the cblE type of homocystinuria. They presented between 2 weeks and 3 years of age (median age 4 weeks) with anemia, which was macrocytic in only three patients, and with neurological involvement in all but two cases. Bone marrow examination performed in seven patients showed megaloblastic changes in all but one of them. All patients exhibited moderate to severe hyperhomocysteinemia (median plasma total homocysteine [Hcy] 92 mumol/L, range 44-169), while clearly reduced methionine was observed only in four cases. Pathogenic mutations were identified in both parental alleles of the MTRR gene in all patients. Five known (c.903+469T>C, c.1361C>T, c.1459G>A, c.1557-4_1557+3del7, and c.1622_1623dupTA) and three novel mutations (c.7A>T, c.1573C>T, and c.1953-6_1953-2del5) were detected. Importantly, transfection of fibroblasts of cblE patients with a wild-type MTRR minigene expression construct resulted in a significant approximately four-fold increase of methionine synthesis, indicating correction of the enzyme defect. Our study shows a link between a milder predominantly hematological presentation and homozygosity for the c.1361C>T mutation, but no other obvious genotype-phenotype correlation. The identification of mutations in the MTRR gene, together with restoration of methionine synthesis following MTRR minigene expression in cblE cells confirms that this disease is caused by defects in the MTRR gene.     

Molecular basis for methionine synthase reductase deficiency in patients belonging to the cblE complementation group of disorders in folate/cobalamin metabolism.

Methionine synthase reductase (MSR) deficiency is an autosomal recessive disorder of folate/cobalamin metabolism leading to hyperhomocysteinemia, hypo- methioninemia and megaloblastic anemia. Deficiency in MSR activity occurs as the result of a defect in the MSR enzyme, which is required for the reductive activation of methionine synthase (MS). MS itself is responsible for the folate/cobalamin-dependent conversion of homo- cysteine to methionine. We have recently cloned the cDNA corresponding to the MSR protein, a novel member of the ferredoxin-NADP(+)reductase (FNR) family of electron transferases. We have used RT-PCR, heteroduplex, single-strand conformation poly- morphism (SSCP) and DNA sequence analyses to reveal 11 mutations in eight patients from seven families belonging to the cblE complementation group of patients of cobalamin metabolism that is defective in the MSR protein. The mutations include splicing defects leading to large insertions or deletions, as well as a number of smaller deletions and point mutations. Apart from an intronic substitution found in two unrelated patients, the mutations appear singular among individuals. Of the eleven, three are nonsense mutations, allowing for the identification of two patients for whom little if any MSR protein should be produced. The remaining eight involve point mutations or in-frame disruptions of the coding sequence and are distributed throughout the coding region, including proposed FMN, FAD and NADPH binding sites. These data demonstrate a unique requirement for MSR in the reductive activation of MS.     

Analysis of multiple mutations in the hALG6 gene in a patient with congenital disorder of glycosylation Ic

Congenital disorder of glycosylation Ic is caused by mutations in the hALG6 gene that encodes an alpha-1,3 glucosyltransferase. This enzyme is required for the addition of the first glucose residue to the lipid-linked oligosaccharide precursor for N-linked glycosylation. Here we describe the biochemical and molecular analysis of a patient with three mutations in the hALG6 gene. The maternal allele has an intronic G --> A mutation resulting in skipping of exon3 (IVS3 + 5G > A). This produces a nonfunctional enzyme as shown by its inability to restore normal glycosylation in a Saccharomyces cerevisiae strain lacking a functional ALG6. The paternal allele has two mutations. One is a deletion of three bases (895-897delATA) leading to an in-frame deletion of isoleucine 299 (delI299) located in a transmembrane domain. The second mutation on the same allele 911T > C causes a F304S change. When expressed in the ALG6 deficient yeast strain, this allele restores glycosylation but the mRNA is unstable or inefficiently transcribed, contributing to the impaired glycosylation in the patient.     

Ligase IV syndrome

Ligase IV (LIG4) syndrome belongs to the group of hereditary disorders associated with impaired DNA damage response mechanisms. Subjects affected with this rare autosomal recessive disease exhibit microcephaly, unusual facial features, growth retardation, developmental delay, skin anomalies, and are typically pancytopenic. The disease is characterized by pronounced radiosensitivity, genome instability, malignancy, immunodeficiency, and bone marrow abnormalities. LIG4 syndrome results from mutations in the DNA ligase IV gene encoding an enzyme that plays a pivotal role in repairing double strand DNA breaks and V(D)J recombination. Since LIG4 null-mutant mice are embryonic lethal and biallelic null mutations have not been described to date in LIG4-deficient patients, viability of the DNA ligase IV deficiency syndrome appears to require at least one allele with a hypomorphic mutation. Mutations R278H, Q280R, H282L, M249E located in the vicinity of the active site are typical hypomorphic because they do not affect ligase expression and retain residual albeit reduced activity of the enzyme at levels of 5–10% of that for the wild-type ligase. Carriers heterozygous for those mutations usually develop moderate defects in V(D)J recombination, mild immune abnormalities and malignancy. In contrast, mutations resided in OBD, i.e. in the C-terminal subdomain of the catalytic domain, and in XRCC4-binding domain more dramatically inhibit the ligase function and also greatly decrease its expression. A truncating mutation R580X and a frameshift mutation K424FS resulting in loss of the C-terminal XRCC4-binding domain have deleterious effect on both expression and function of LIG4 and represent a null allele.     

Functional methionine synthase deficiency due to cblG disorder: A report of two patients and a review

Functional methionine synthase deficiency due to abnormal methylcobalamin metabolism causes megaloblastic anemia, moderate to severe developmental delay, lethargy, and anorexia in association with homocystinuria. Patients with this disorder of cobalamin metabolism can be classified into two separate groups, cblE or cblG, primarily on the basis of complementation analysis with cultured skin fibroblasts. We describe two unrelated boys, ages 3 and 5 years, with the cblG defect in methylcobalamin synthesis. Both children presented with severe developmental delay, lethargy, anorexia, and megaloblastic anemia. The diagnosis of homocystinuria was delayed in each case due to difficulties with detection of small amounts of homocystine in physiologic samples. The clinical course of cblG disease is favorably altered by treatment with intramuscular hydroxycobalamin. Megaloblastosis in the presence of adequate supplies of cobalamin and folate in the blood must alert the clinician to the possibility of functional methionine synthase deficiency and should prompt a careful search for associated biochemical hallmarks, including homocystinuria/emia. Am. J. Med. Genet. 71:384–390, 1997. © 1997 Wiley-Liss, Inc.     

Mutations in the regulatory domain of cystathionine beta synthase can functionally suppress patient-derived mutations in cis

Human cystathionine beta--synthase (CBS) is an S-adenosylmethionine-regulated enzyme that plays a key role in the metabolism of homocysteine. Mutations in CBS are known to cause homocystinuria, an inborn error in metabolism. We previously developed a yeast functional assay for CBS and used it to characterize mutations found in homocystinuric patients. We discovered that many patient-derived mutations are functionally suppressed by deletion of the C-terminal 142 amino acids, which contain a 53 amino acid motif known as the CBS domain. This domain is found in a wide variety of proteins of diverse biological function. Here we have used a genetic screen to identify missense mutations in the C-terminal region of CBS that can suppress the most common patient mutation, I278T. Seven suppressor mutations were identified, four of which map to the CBS domain. When combined in cis with another pathogenic mutation, V168M, six of seven of the suppressor mutations rescued the yeast phenotype. Enzyme activity analyses indicate that the suppressors restore activity from <2% to 17--64% of the wild-type levels. Analysis of the suppressor mutations in the absence of the pathogenic mutation shows that six of the seven suppressor alleles have lost enzymatic responsiveness to S-adenosylmethionine. Using homology modeling, we show that the suppressor mutations appear to map on one face of the CBS domain. Our results indicate that subtle changes to the C-terminus of CBS can restore activity to mutant proteins and provide a rationale for screening for compounds that can activate mutant CBS alleles.     

Identification of the active site residues in the nsP2 proteinase of Sindbis virus.

The nonstructural polyproteins of Sindbis virus are processed by a virus-encoded proteinase which is located in the C-terminal domain of nsP2. Here we have performed a mutagenic analysis to identify the active site residues of this proteinase. Substitution of other amino acids for either Cys-481 or His-558 completely abolished proteolytic processing of Sindbis virus polyproteins in vitro. Substitutions within this domain for a second cysteine conserved among alphaviruses, for four other conserved histidines, or for a conserved serine did not affect the activity of the enzyme. These results suggest that nsP2 is a papain-like proteinase whose catalytic dyad is composed of Cys-481 and His-558. Since an asparagine residue has been implicated in the active site of papain, we changed the four conserved asparagine residues in the C-terminal half of nsP2 and found that all could be substituted without total loss of activity. Among papain-like proteinases, the residue following the catalytic histidine is alanine or glycine in the plant and animal enzymes, and the presence of Trp-559 in alphaviruses is unusual. A mutant enzyme containing Ala-559 was completely inactive, implying that Trp-559 is essential for a functional proteinase. All of these mutations were introduced into a full-length clone of Sindbis virus from which infectious RNA could be transcribed in vitro, and the effects of these changes on viability were tested. In all cases it was found that mutations which abolished proteolytic activity were lethal, whether or not these mutations were in the catalytic residues, indicating that proteolysis of the nonstructural polyprotein is essential for Sindbis replication.     

Expression and characterization of mutations in human very long-chain acyl-CoA dehydrogenase using a prokaryotic system

Very long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first enzymatic step in the mitochondrial beta-oxidation of fatty acids 14-20 carbons in length. More than 100 cases of VLCAD deficiency have been reported with the disease varying from a severe, often fatal neonatal form to a mild adult-onset form. VLCAD is distinguished from matrix-soluble acyl-CoA dehydrogenases by its unique C-terminal domain, homodimeric structure, and localization to the inner mitochondrial membrane. We have for the first time expressed and purified VLCAD using a bacterial system. Recombinant VLCAD had similar biochemical properties to those reported for native VLCAD and the bacterial system was used to study six previously described disease-causing missense mutations including the two most common mild mutations (T220M, V243A), a mutation leading to the severe disease phenotype (R429W), and three mutations in the C-terminal domain (A450P, L462P, and R573W). Of particular interest was the finding that the A450P and L462P bacterial extracts had normal or increased amounts of VLCAD antigen and activity. In the pure form L462P had roughly 30% of wild-type activity while A450P was normal. Using computer modeling both mutations were mapped to a predicted charged surface of VLCAD that we postulate interacts with the mitochondrial membrane. In a membrane pull down assay both mutants showed greatly reduced mitochondrial membrane association, suggesting a mechanism for the disease in these patients. In summary, the bacterial expression system developed here will significantly advance our understanding of both the clinical aspects of VLCAD deficiency and the basic biochemistry of the enzyme.     

Both amino acid changes in nsP1 of Sindbis virusLM21 contribute to and are required for efficient expression of the mutant phenotype

SVLM21 is a mutant of Sindbis virus which in contrast to the standard virus, SVSTD, is able to replicate in Aedes albopictus mosquito cells deprived of methionine. Previously, by making use of the infectious Toto plasmids, we had constructed recombinant viruses containing various SVLM21 sequences, and were thereby able to map the mutations associated with the SVLM21 phenotype to the gene for the nonstructural protein nsP1. Two mutations were found in the nsP1 gene of SVLM21. These led to predicted amino acid changes at residue 87 from Arg to Leu, and at residue 88 from Ser to Cys. In the work presented here, we assess the relative contributions of these two mutations to the SVLM21 phenotype using site-directed mutagenesis to obtain virus encoding only the change to Leu at residue 87 of nsP1 (SVMS319), or only the change to Cys at residue 88 (SVMS321). In addition we show that SVLM10, which was isolated during the selection procedure for SVLM21, encodes only the change at residue 88. In addition to its ability to grow in methionine-deprived mosquito cells, SVLM21 differs from SVSTD in two other respects: (1) it shows an increased sensitivity to neplanocin A (NPA) and (2) it generates increased levels of methyltransferase in infected cells. Whether we looked at resistance to low methionine, sensitivity to NPA, or levels of methyltransferase generated, SVMS319, SVMS321, and SVLM10 all expressed only a partial SVLM21 phenotype. Furthermore we were not able in these experiments to distinguish between these three viruses. We conclude therefore that both amino acid changes, i.e., at residues 87 and 88, are required to produce the full SVLM21 phenotype, and that both changes contribute equally.     

The role of cystathionine beta-synthase in homocysteine metabolism

Cystathionine beta-synthase (CBS) is the first enzyme in the transsulfuration pathway, catalyzing the conversion of serine and homocysteine to cystathionine and water. The enzyme contains three functional domains. The middle domain contains the catalytic core, which is responsible for the pyridoxal phosphate-catalyzed reaction. The C-terminal domain contains a negative regulatory region that is responsible for allosteric activation of the enzyme by S-adenosylmethionine. The N-terminal domain contains heme, and this domain regulates the enzyme in response to redox conditions. Besides its canonical reaction, CBS can catalyze alternative reactions that produce hydrogen sulfide, a novel neuromodulator in the brain. Mutations in human CBS result in homocystinuria, an autosomal recessive disorder characterized by defects in a variety of different organ systems. The most common CBS allele is 833T>C (I278T), which is associated with pyridoxine-responsive homocystinuria. A complementation system in S. cerevisiae has been developed for analysis of human CBS mutations. Using this system, it has been discovered that deletion of the C-terminal domain of CBS can suppress the functional defects of many patient-derived mutations. This finding suggests it may be possible to develop drugs that interact with the C-terminal domain of CBS to treat elevated homocysteine in humans.     

多巴反应性肌张力障碍GCH1基因突变分析

目的探讨多巴反应性肌张力障碍（dopa responsive dystonia,DRD）三磷酸鸟苷环化水解酶Ⅰ基因（guanosinetriphosphate cyclohydrolaseⅠ,GCH1）的突变。方法应用聚合酶链反应、DNA直接测序和限制性内切酶酶切技术对6例散发DRD患者进行GCH1基因的突变分析,对100名健康对照者进行GCH1基因的PCR和限制性内切酶酶切分析。结果1例患者检测出GCH1基因一个新的点突变151（G→A）,为起始密码子突变,导致所编码的起始氨基酸由蛋氨酸变为异亮氨酸（M1I）而不能起始翻译。100名健康对照者等位基因无此突变。结论发现了GCH1基因一个新的杂合型点突变151（G→A）,我国散发DRD患者中存在GCH1基因突变。     

Identification of a novel in-frame de novo mutation in SPTAN1 in intellectual disability and pontocerebellar atrophy

Heterozygous in-frame mutations (p.E2207del and p.R2308_M2309dup) in the α-II subunit of spectrin (SPTAN1) were recently identified in two patients with intellectual disability (ID), infantile spasms (IS), hypomyelination, and brain atrophy. These mutations affected the C-terminal domain of the protein, which contains the nucleation site of the α/β spectrin heterodimer. By screening SPTAN1 in 95 patients with idiopathic ID, we found a de novo in-frame mutation (p.Q2202del) in the same C-terminal domain in a patient with mild generalized epilepsy and pontocerebellar atrophy, but without IS, hypomyelination, or other brain structural defects, allowing us to define the core phenotype associated with these C-terminal SPTAN1 mutations. We also found a de novo missense variant (p.R566P) of unclear clinical significance in a patient with non-syndromic ID. These two mutations induced different patterns of aggregation between spectrin subunits in transfected neuronal cell lines, providing a paradigm for the classification of candidate variants.     

Monitoring the emergence of hepatitis B virus polymerase gene variants during lamivudine therapy using the LightCycler

Treatment of chronic hepatitis B virus (HBV) infection with lamivudine is associated with the appearance in the circulation of HBV variants with mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the polymerase gene. Fluorometric real-time PCR with the LightCycler assay was used for the detection of resistant variants. Differences in the hybridization melting curve kinetics of probes bound to the sequences encoding the wild-type or the mutant YMDD motifs (YIDD or YVDD in which the methionine residue is altered to an isoleucine or a valine, respectively) distinguished the single-base changes responsible for the resistance phenotype. The LightCycler probe hybridization assay was applied to 40 serum specimens from 19 patients, and the results were correlated with the nucleotide sequences determined for the corresponding PCR products. All three variants could be identified in the specimens. PCR clones obtained from four patients early in the course and prior to lamivudine therapy were investigated for the appearance of YIDD and YVDD variants with the LightCycler assay. In one patient, a transient appearance of the YIDD variant was observed 6 weeks into therapy. Subsequently, after 11 months of lamivudine therapy, the YVDD variant emerged in that patient.     

Mutation pattern in the Bruton's tyrosine kinase gene in 26 unrelated patients with X-linked agammaglobulinemia

Mutation pattern was characterized in the Bruton's tyrosine kinase gene (BTK) in 26 patients with X-linked agammaglobulinemia, the first described immunoglobulin deficiency, and was related to BTK expression. A total of 24 different mutations were identified. Most BTK mutations were found to result in premature termination of the translation product. Mutations were detected in most BTK exons with a predominance of frameshift and nonsense mutations in the 5′ end of the gene and missense mutations in its 3′ part, corresponding to the catalytic domain of the enzyme. Nonsense and frameshift mutations were associated with diminished levels of BTK mRNA expression, except for a frameshift mutation in exon 17 and two nonsense mutations in exon 2, indicating that these cases are not confined to penultimate exons. One amino acid substitution (R28H) was found in the pleckstrin homology domain's residue, which is mutated in mice bearing the X-linked immunodeficiency phenotype; another substitution (R307G) was identified in the src homology domain 2. All remaining amino acid substitutions were found in the catalytic domain of Btk. Hum Mutat 9:418–425, 1997. © 1997 Wiley-Liss, Inc.