Mutation Update and Review of Severe Methylenetetrahydrofolate Reductase Deficiency

Severe 5,10‐methylenetetrahydrofolate reductase (MTHFR) deficiency is caused by mutations in the MTHFR gene and results in hyperhomocysteinemia and varying severity of disease, ranging from neonatal lethal to adult onset. Including those described here, 109 MTHFR mutations have been reported in 171 families, consisting of 70 missense mutations, 17 that primarily affect splicing, 11 nonsense mutations, seven small deletions, two no‐stop mutations, one small duplication, and one large duplication. Only 36% of mutations recur in unrelated families, indicating that most are “private.” The most common mutation is c.1530A>G (numbered from NM_005957.4, p.Lys510 = ) causing a splicing defect, found in 13 families; the most common missense mutation is c.1129C>T (p.Arg377Cys) identified in 10 families. To increase disease understanding, we report enzymatic activity, detected mutations, and clinical onset information (early, <1 year; or late, >1 year) for all published patients available, demonstrating that patients with early onset have less residual enzyme activity than those presenting later. We also review animal models, diagnostic approaches, clinical presentations, and treatment options. This is the first large review of mutations in MTHFR, highlighting the wide spectrum of disease‐causing mutations.     

Insights into the Regulatory Domain of Cystathionine Beta‐Synthase: Characterization of Six Variant Proteins

Cystathionine beta‐synthase (CBS) catalyzes the formation of cystathionine from homocysteine and serine. CBS is allosterically activated by S‐adenosylmethionine (SAM), which binds to its C‐terminal regulatory domain. Mutations in this domain lead to variants with high residual activity but lacking SAM activation. We characterized six C‐terminal CBS variants (p.P427L, p.D444N, p.V449G, p.S500L, p.K523Sfs*18, and p.L540Q). To understand the effect of C‐terminal mutations on the functional/structural properties of CBS, we performed dynamic light scattering, differential scanning fluorimetry, limited proteolysis, enzymatic characterization, and determination of SAM‐binding affinity. Kinetic data confirm that the enzymatic function of these variants is not impaired. Although lacking SAM activation, the p.P427L and p.S500L were able to bind SAM at a lower extent than the wild type (WT), confirming that SAM binding and activation can be two independent events. At the structural level, the C‐terminal variants presented various effects, either showing catalytic core instability and increased susceptibility toward aggregation or presenting with similar or higher stability than the WT. Our study highlights as the common feature to the C‐terminal variants an impaired binding of SAM and no increase in enzymatic activity with physiological concentrations of the activator, suggesting the loss of regulation by SAM as a potential pathogenic mechanism.     

Methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms and age of onset in schizophrenia: A combined analysis of independent samples

Methylenetetrahydrofolate reductase (MTHFR) is involved in the one‐carbon cycle, which is of importance for nucleotide synthesis and methylation of DNA, membranes, proteins and lipids. The MTHFR gene includes two common polymorphisms (rs1801133 or C677T; rs1801131 or A1298C) which both alter enzyme activity. The T‐allele of the C677T polymorphism has recently been associated with earlier age at onset of schizophrenia. In the present study we examined the association between the MTHFR C677T and A1298C polymorphisms and age at onset of schizophrenia in twelve samples consisting of 3,213 unrelated schizophrenia patients, including the original Scandinavian sample. There was no consistent relationship between MTHFR C677T, A1298C or combined 677T/1298C carriers and age of onset in schizophrenia when the results of each study were combined using meta‐analysis. The present results suggest that the investigated MTHFR polymorphisms do not influence age of onset in schizophrenia. © 2011 Wiley‐Liss, Inc.     

Role of amniotic fluid homocysteine level and of fetal 5,10‐methylenetetrahydrafolate reductase genotype in the etiology of neural tube defects

A mutation in the gene 5,10‐methylenetetrahydrofolate reductase (MTHFR), leading to altered homocysteine metabolism, has been identified in parents and fetuses with fetal neural tube defects. We sought to determine which is of greater importance in fetal neural tube defect formation: the fetal MTHFR mutation or elevated amniotic fluid homocysteine level. We retrieved stored amniotic fluid from cases of isolated fetal neural tube defect diagnosed from 1988 to 1998 (n = 80), and from normal controls matched for race, month and year of amniocentesis, and maternal age. The presence or absence of the 677C→T mutation of MTHFR was determined and homocysteine levels were measured; cases and controls were compared. Significantly more cases than controls were heterozygous or homozygous for the 677C→T MTHFR mutation (44% vs 17%, P ≤ 0.001). Cases were also significantly more likely than controls to have an amniotic fluid homocysteine level above the 90th centile (>1.85 μmol per liter); 27% vs 10%, P = 0.02. Thirty one cases and 12 controls had an abnormal genotype; however, amniotic fluid homocysteine levels were not significantly different between these two groups (6/31, or 19% of cases had an elevated homocysteine compared to 1/12, or 8% of controls; P = 0.65). In contrast, 40 cases and 60 controls had a normal genotype; the neural tube defect cases had significantly higher homocysteine levels (13/40, or 32% of cases had an elevated homocysteine level compared to only 6/60, or 10% of controls; P = 0.008). Although both abnormal fetal MTHFR genotype and abnormal amniotic fluid homocysteine concentration are significantly associated with neural tube defects, the association with amniotic fluid homocysteine concentration is significant regardless of the fetal MTHFR genotype. The relationship between maternal and fetal homocysteine metabolism is complex. Am. J. Med. Genet. 90:12–16, 2000. © 2000 Wiley‐Liss, Inc.     

Increased Resistance to Malaria in Mice with Methylenetetrahydrofolate Reductase (Mthfr) Deficiency Suggests a Mechanism for Selection of the MTHFR 677C>T (c.665C>T) Variant

The polymorphism 677C>T (NM_005957.4:c.665C>T/p.Ala222Val, rs1801133:C>T) in methylenetetrahydrofolate reductase (MTHFR) results in mild enzymatic deficiency and increased risk for several complex traits including adverse reproductive outcomes, birth defects, and heart disease. Despite these deleterious effects, homozygosity is high (5%–15%) in many populations, and among the highest in Mediterranean regions, where malaria was historically endemic and may have conferred a selective advantage for other mutations. We infected Mthfr‐deficient (Mthfr^+/?) and MTHFR overexpressing (MTHFR^Tg) mice with Plasmodium berghei ANKA to induce cerebral malaria. Mthfr^+/? mice survived longer (P < 0.02, log‐rank test), and MTHFR^Tg mice died earlier (P < 0.05, log‐rank test) after infection compared with wild‐type littermates. Flow cytometry revealed increased lymphocyte populations and increased CCR4⁺ NK cells in spleen of Mthfr^+/? mice; MTHFR^Tg animals had decreased numbers of these NK cells. Interferon‐γ and interleukin‐10 immunoreactive proteins were increased and decreased, respectively, in brain of Mthfr^+/? mice compared with wild‐type. We suggest that mild MTHFR deficiency protects against malarial infection and that this phenomenon may have led to the high frequency of the 677C>T/c.665C>T variant in human populations.     

A Deficiency of Cysteine Impairs Fibrillin-1 Deposition: Implications for the Pathogenesis of Cystathionine β-Synthase Deficiency

Cystathionine β-synthase (CBS) deficiency is an inborn error of amino acid metabolism that has pleiotropic manifestations and is commonly called “homocystinuria.” The features include skeletal, ocular, and vascular defects, some of which are reminiscent of those found in Marfan syndrome (MFS). Because of the spectrum of clinical effects, the pathogenesis of homocystinuria has long been thought to involve the extracellular matrix (ECM), and the condition has been classified as a heritable disorder of connective tissue. Because of the superficial similarities with MFS, we and others (Pyeritz, in McKusicks Heritable Disorders of Connective Tissue, St. Louis, Mosby–Year Book Inc., 5th ed., pp 137–178, 1993; Pyeritz, in Principles and Practice of Medical Genetics, New York, Churchill Livingstone, 3rd ed., pp 1027–1066, 1997; Mudd, Levy, and Skovby, in The Metabolic and Molecular Bases of Inherited Disease, New York, McGraw-Hill Publishing Co., 7th ed., pp 1279–1327, 1995) have speculated how CBS deficiency might affect fibrillin-1, the protein altered in MFS. For example, the altered plasma concentrations of homocysteine and/or cysteine in patients with CBS deficiency may hinder fibrillin-1 synthesis, deposition, or both. When arterial smooth muscle cells were cultured under conditions of cysteine deficiency, fibrillin-1 deposition into the ECM was greatly diminished as revealed by immunocytochemistry. Excessive homocysteine, in contrast, had little, if any, effect on fibrillin-1 deposition. When cysteine concentrations were returned to normal, the smooth muscle cells began to accumulate a matrix rich in fibrillin-1. Type I collagen, the major matrix component synthesized by these smooth muscle cells, was not reduced by low cysteine concentrations nor high homocysteine concentrations. These results demonstrate that a deficiency of cysteine and subsequent inhibition of fibrillin-1 accumulation in CBS deficient patients may be at least partly responsible for their phenotype, and suggest that maintenance of normal plasma cyst(e)ine levels may be an important therapeutic goal.     

Propylene Polymerization with rac‐SiMe2(2‐Me‐4‐PhInd)2ZrMe2/MAO: Polymer Characterization and Kinetic Models

Poly(propylene)s were prepared with metallocene catalyst rac‐SiMe₂(2‐Me‐4‐PhInd)₂ZrMe₂/MAO (rac‐dimethylsilylbis(2‐methyl‐4‐phenylindenyl)dimethylzirconium/methylaluminoxane) in heptane solution at temperatures from 50 to 80 °C with varying concentrations of monomer, hydrogen, triisobutylaluminium (TIBA) and MAO. Polymer molar mass depended on the monomer, MAO, TIBA, and hydrogen concentrations and on polymerization temperature. The isotacticity was very high (mmmm > 95%), and only a slight decrease was detected at high temperatures. Regio selectivity was also high; the total amount of 2,1‐ and 3,1‐insertions was less than 0.4 mol‐%. Lowering the monomer concentration and raising the temperature increased the amount of 3,1 defects over the amount of 2,1 defects. End‐group analysis by ¹³C NMR spectroscopy revealed isobutyl and allyl end‐groups. Chain transfer to aluminium and β‐CH₃ elimination were concluded to be the dominating chain‐termination mechanisms. The importance of β‐CH₃ elimination increased with temperature. Hydrogen addition changed both the initiation and termination mechanisms as indicated by the presence of propyl, butyl and 2,3‐dimethylbutyl end‐groups. According to modeling studies, the molar mass follows a first‐order relationship with propylene and hydrogen concentrations, and a half‐order relationship with MAO concentration. Arrhenius‐type activation energy coefficients were 125 kJ · mol^?1 for β‐CH₃ elimination, 66 kJ · mol^?1 for chain transfer to aluminium, and 53 kJ · mol^?1 for chain transfer to hydrogen. A value of 45 kJ · mol^?1 was used for the propagation.

     

Insights into the Ring‐Expansion Polymerization of Thiiranes with 2,4‐Thiazolidinedione

The behavior of the ring‐expansion homopolymerization of 2 (phenoxymethyl)thiirane (PMT) and propylene sulfide (PS), respectively, with thiazolidine‐2,4‐dione (TZD) as a cyclic initiator is investigated. The polymerizations show steadily growing molar masses with increasing monomer conversions. In addition, reversible merging reactions between rings are observed, with up to six merged macrocycles formed. The degree of merging is strongly dependent on the initial monomer concentration, whereas temperature has only a small impact. Under optimized conditions, ring‐poly(PMT) polymer with values of M _n up to 50 250 g mol^?1 and dispersities down to 1.11 can be synthesized. DSC and ESI‐MS measurements of the novel ring‐poly(PS) prove the formation of ring polymer having topological purity above 95%.

     

Mechanistic Insights into the Shear‐Induced β‐Form Crystal Formation of iPP

The conformation of a helical type polymer chain in the crystalline phase prefers the state with lower energy. The molecular simulation of the melting behaviors of α and β isotactic poly(propylene) (iPP) crystals with ultrafast heating shows that the ratio of right‐handed and left‐handed helix stays at 50% in α‐iPP and changes from 100% to 50% in β‐iPP before and after melting. Till date, the formation of β form crystal in iPP has not been understood well together with the mechanism of the crystallization process. One of the approaches to produce β‐iPP is to shear the melt. The influence of shear flow on the crystallization behavior of iPP was investigated via ex situ and in situ X‐ray measurements. It was found that preceding or simultaneously initiated formation of β‐iPP together with α‐iPP occurs under proper shear conditions. The β‐rich iPP sample could no longer form β crystal at 135 °C after heating to the β‐melting temperature with the melting of β‐iPP and the retaining of oriented α‐iPP, which is contrary to the accepted formation process of β‐iPP. A mechanism was proposed to understand the shear‐induced β crystal in iPP, considering the mesophase and stereocomplex type helical conformation of the polymer chains.

     

PLP and MALDI‐ToF Determination of Propagation Rate Coefficients of Fast‐Polymerizing Acrylates with Heterocyclic Side‐Chains: Tetrahydrofurfuryl Acrylate and (R)‐α‐Acryloyloxy‐β,β‐dimethyl‐γ‐butyrolactone

The PLP technique in combination with MALDI‐ToF‐MS was used to determine the Arrhenius plots for the propagation rate coefficients of THFA and ADBL, which polymerize much faster than alkyl acrylates. It is demonstrated that this is not due to higher propagation rate coefficients. It is shown that the temperature at which the PLP experiment brakes down increases in the order ADBL > THFA > alkyl acrylates, indicating a high extent of transfer to polymer for these monomers. Although k_p decreases in the same order, the decrease of the corresponding overall polymerization rate is much larger. Therefore, the high polymerization rates of ADBL and THFA cannot be accounted for by fast propagation rates but more likely are linked to a low rate of termination.

     

Molecular Genetic Characterization of 151 Mut‐Type Methylmalonic Aciduria Patients and Identification of 41 Novel Mutations in MUT

Isolated methylmalonic aciduria (MMA) is an autosomal‐recessive disorder of propionate metabolism that is most commonly caused by mutations in the methylmalonyl‐CoA mutase (MUT) gene (mut‐type MMA). We investigated a cohort of 151 patients, classifying 114 patients as mut⁰ and 32 as mut^? (five not defined). As per the definition, mut^? patients showed a higher propionate incorporation ratio in vitro, which was correlated to a considerably later age of onset compared with mut⁰ patients. In all patients, we found a total of 110 different mutations, of which 41 were novel. While the missense alleles p.Asn219Tyr, p.Arg369His, and p.Arg694Trp recurred in >10 alleles, 47 mutations were identified only once, suggesting many patients carry private mutations. Deficient alleles in the mut^? subclass were almost exclusively caused by missense mutations, found disproportionately in the C‐terminal cofactor binding domain. On the contrary, only half of the mut⁰ mutations were of the missense type. Western blot analysis revealed reduced MUT protein for all 34 cell lines (27 mut⁰, seven mut^?) tested, suggesting protein instability as a major mechanism of deficiency in mut‐type MMA. This large‐scale evaluation helps to characterize the landscape of MUT mutations and their relationship to dysfunction and disease.     

Clinical diversity of MYH7‐related cardiomyopathies: Insights into genotype–phenotype correlations

MYH7‐related disease (MRD) is the most common hereditary primary cardiomyopathy (CM), with pathogenic MYH7 variants accounting for approximately 40% of familial hypertrophic CMs. MRDs may also present as skeletal myopathies, with or without CM. Since pathogenic MYH7 variants result in highly variable clinical phenotypes, from mild to fatal forms of cardiac and skeletal myopathies, genotype–phenotype correlations are not always apparent, and translation of the genetic findings to clinical practice can be complicated. Data on genotype–phenotype correlations can help facilitate more specific and personalized decisions on treatment strategies, surveillance, and genetic counseling. We present a series of six MRD pedigrees with rare genotypes, encompassing various clinical presentations and inheritance patterns. This study provides new insights into the spectrum of MRD that is directly translatable to clinical practice.     

Clinical and Molecular Characterization of X‐linked Hyper‐IgM Syndrome Patients in China

X‐linked Hyper‐IgM syndrome (XHIM) is caused by mutations of CD154 gene also known as CD40 ligand (CD40L). CD40L is expressed in activated T cells and interacts with CD40 receptor expressed on B lymphocytes and dendritic cells. Affected patients present cellular and humoral immune defects, with infections by intracellular, opportunistic and extracellular pathogens. In the present study, we investigate molecular defects involved in the XHIM in five patients and identified five distinct CD40L mutations, three of which had not been previously described. P1 harboured a novel p.L193P mutation, which abolished the expression of CD40L. P2 had a frameshift deletion in exon 3 (p.E108fsX19), which also decreased the protein expression. P3 demonstrated p.E54X change in exon 2. P4 harboured the p.Q186X change in the exon 5. P5 demonstrated p. E142X change in exon 5. Mutations in P3, P4 and P5 all led to the production of premature CD40L protein. Two of the five genetically defined patients received umbilical cord blood stem cell transplantation from unrelated donor and achieved clinical remission, and the expression of CD40L on the peripheral blood mononuclear cells restored. These mutations reflect the heterogeneity of CD40L gene, indicating the need for accurate and reliable molecular testing in patients suspected of XHIM.     

Cystathionine β‐synthase mutations: effect of mutation topology on folding and activity

Misfolding of mutant enzymes may play an important role in the pathogenesis of cystathionine β‐synthase (CBS) deficiency. We examined properties of a series of 27 mutant variants, which together represent 70% of known alleles observed in patients with homocystinuria due to CBS deficiency. The median amount of SDS‐soluble mutant CBS polypeptides in the pellet after centrifugation of bacterial extracts was increased by 50% compared to the wild type. Moreover, mutants formed on average only 12% of tetramers and their median activity reached only 3% of the wild‐type enzyme. In contrast to the wild‐type CBS about half of mutants were not activated by S‐adenosylmethionine. Expression at 18°C substantially increased the activity of five mutants in parallel with increasing the amounts of tetramers. We further analyzed the role of solvent accessibility of mutants as a determinant of their folding and activity. Buried mutations formed on average less tetramers and exhibited 23 times lower activity than the solvent exposed mutations. In summary, our results show that topology of mutations predicts in part the behavior of mutant CBS, and that misfolding may be an important and frequent pathogenic mechanism in CBS deficiency. Hum Mutat 31:1–11, 2010. © 2010 Wiley‐Liss, Inc.     

Novel Genetic,Clinical, and Pathomechanistic Insights into TFG‐Associated Hereditary Spastic Paraplegia

Hereditary spastic paraplegias (HSPs) are genetically and clinically heterogeneous axonopathies primarily affecting upper motor neurons and, in complex forms, additional neurons. Here, we report two families with distinct recessive mutations in TFG, previously suggested to cause HSP based on findings in a single small family with complex HSP. The first carried a homozygous c.317G>A (p.R106H) variant and presented with pure HSP. The second carried the same homozygous c.316C>T (p.R106C) variant previously reported and displayed a similarly complex phenotype including optic atrophy. Haplotyping and bisulfate sequencing revealed evidence for a c.316C>T founder allele, as well as for a c.316_317 mutation hotspot. Expression of mutant TFG proteins in cultured neurons revealed mitochondrial fragmentation, the extent of which correlated with clinical severity. Our findings confirm the causal nature of bi‐allelic TFG mutations for HSP, broaden the clinical and mutational spectra, and suggest mitochondrial impairment to represent a pathomechanistic link to other neurodegenerative conditions.     

Photoresponsive New Polysiloxanes with 4‐Substituted Azobenzene Side‐Groups. Synthesis,Characterization and Kinetics of the Reversible Trans‐Cis‐Trans Isomerization

New polydimethylsiloxanes with p‐substituted azobenzene side‐groups were synthesized. Thin films and solutions exhibit a photochemical trans‐cis isomerization of the azobenzene groups, followed by their cis‐trans thermal relaxation in the dark. In films, relaxation rates were found to be 100–1 000 times slower than the rates of photoisomerization, the former being very sensitive to the electron‐acceptor character of the substituents. in solution, the rates of cis‐trans relaxation are lower than those obtained for the solid state. This is ascribed to the dipolar intramolecular interactions between cis chromophores, which are favored in solution.

     

Four novel mutations in the cystathionine β‐synthase gene: Effect of a second linked mutation on the severity of the homocystinuric phenotype

Homocystinuria due to cystathionine β‐synthase (CBS) deficiency is frequently caused by missense mutations. In this article, we report four novel missense mutations in the CBS gene: 172C→T (R58W) linked in cis with A114V; 376A→G (M126V); 904G→A (E302K); and 1006C→T (R336C). The CBS activity of the corresponding mutant enzymes expressed in Escherichia coli was greatly diminished, confirming the pathogenicity of these mutations. Western analysis showed that the R58W+A114V and M126V mutant enzymes were unstable in E. coli, while the E302K subunits were partially degraded to shorter products. Using site‐directed mutagenesis we found that CBS containing either the R58W or A114V as the only mutations demonstrated 18% and 46% of normal activity, respectively. Both mutant forms of CBS were stable in E. coli. When these two mutations were expressed in cis, the resultant mutant protein exhibited activity 1.3% that of a control. All these in vitro results were in good agreement with the clinical manifestation in these patients. The Italian patient 2241, an A114V+R58W/M126V compound heterozygote, exhibited severe pyridoxine nonresponsive homocystinuria, while another Italian patient 2242, with an A114V/E302K genotype, responded to pyridoxine treatment and had a much milder phenotype. The third patient 3064, an English compound heterozygote for two severe mutations R336C and G307S, was B6 nonresponsive. This report of a ninth homocystinuric allele carrying two mutations in cis raises the possibility that double mutant alleles may be underestimated in homocystinuric patients. In this context, a search for additional mutations in cis may sometimes be necessary to establish a good genotype‐phenotype relationship. Hum Mutat 13:453–457, 1999. © 1999 Wiley‐Liss, Inc.     

A New Insight into the Mechanism of 1,3‐Dienes Cationic Polymerization I: Polymerization of 1,3‐Pentadiene with tBuCl/TiCl4 Initiating System: Kinetic and Mechanistic Study

The cationic polymerization of 1,3‐pentadiene using a tert‐butyl chloride (^tBuCl)/TiCl₄ initiating system in CH₂Cl₂ at different reaction conditions is reported. It is shown that the reaction rate increases with the increase of the ^tBuCl/TiCl₄ molar ratio, while the molecular weight distribution becomes narrower. Well‐defined oligo(1,3‐pentadiene)s ( ≤ 3500 g mol^?1; / ≤ 3.0) are obtained at high ^tBuCl/TiCl₄ molar ratio (340) and low temperature (–78 °C). ¹H and ¹³C NMR spectroscopy studies reveal the presence of tert‐butyl head and –CH₂–Cl end groups. The number‐average functionalities (F_ns) at the α‐ and ω‐ends are calculated to be F_n(^tBu) > 1 and F_n(Cl) < 1, respectively. The general mechanism of 1,3‐pentadiene polymerization is proposed.