The first patient diagnosed with cytochrome c oxidase deficient Leigh syndrome: progress report.

Mutations in SURF1, an assembly gene for cytochrome c oxidase (COX), the fourth complex of the oxidative phosphorylation system, are most frequently encountered in patients with COX deficiency. We describe a patient with Leigh syndrome harbouring a mutation in SURF1 who was reported decades ago with a tissue-specific cytochrome c oxidase deficiency.

Mutation detection in four candidate genes (OXA1L, MRS2L, YME1L and MIPEP) for combined deficiencies in the oxidative phosphorylation system.

Mitochondria are the main energy-producing organelles of the cell. Five complexes embedded in the mitochondrial inner membrane, together constituting the oxidative phosphorylation (OXPHOS) system, comprise the final steps in cellular energy production. Many patients with a mitochondrial defect suffer from a so-called combined deficiency, meaning that the enzymatic activities of two or more complexes of the OXPHOS system are decreased. Numerous mutations have been described in nuclear genes that are involved in the functioning of a single complex of the OXPHOS system. However, little attention has been paid to patients with a deficiency of more than one complex of this particular system. In this study we have investigated four nuclear genes (OXA1L, MRS2L, YME1L and MIPEP) that might be involved in the pathology of combined enzymatic deficiencies of the OXPHOS system. Based on the results of yeast knockouts of these four proteins, we have sequenced the open reading frame of OXA1L in eight patients with an enzymatic deficiency of complexes I and IV. MRS2L, YME1L and MIPEP have been sequenced in three patients with a combined defect of complexes III and IV. No mutations were detected in these patients, showing that at least in these patients the OXPHOS system deficiency cannot be explained by a mutation in these four genes.

A second common variant in the methylenetetrahydrofolate reductase (MTHFR) gene and its relationship to MTHFR enzyme activity, homocysteine, and cardiovascular disease risk.

Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinemia, an independent and graded risk factor for cardiovascular disease (CVD). We examined the relationship of two polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, the 677C-->T and 1298A-->C variants, to MTHFR activity, homocysteine concentrations, and risk of CVD in a population of 190 vascular disease patients and 601 apparently healthy controls. The mean specific and residual MTHFR activities were significantly lower in 677CT and 677TT individuals (both P<0.001). The 1298A-->C mutation alone showed no effect on MTHFR activities. However, when the 677C-->T genotype was taken into account, the 1298A-->C mutation also caused a significant decrease in MTHFR activities, which was observed in both the homozygous 1298CC (P<0.001) and the heterozygous 1298AC states (P=0.005). Both the 677TT as the 677CT genotypes were associated with significantly higher fasting and postload homocysteine levels than 677CC (P<0.001 and P=0.003, respectively). The 1298A-->C mutation had no effect on fasting or postload homocysteine levels. Since homocysteine itself is considered to be positively associated with the risk of CVD, these findings indicate that the 1298A-->C mutation cannot be considered a major risk factor for CVD.

The molecular basis of Dutch infantile nephropathic cystinosis.

Infantile nephropathic cystinosis, an inborn error of metabolism with an autosomal recessive inheritance pattern, is characterized by lysosomal storage of the amino acid cystine due to an impaired transport of cystine out of the lysosomes. Initial clinical features consist of the renal Fanconi syndrome and crystals in the cornea. Oral therapy with cysteamine lowers the intracellular cystine content. Recently, the gene coding for the integral membrane protein cystinosin, which is responsible for membrane transport of cystine (CTNS), was cloned. Mutation analysis of the CTNS gene of Caucasian patients revealed a common 57-kb deletion, and several other mutations spread throughout the entire gene. In the present study, we developed an improved screening method for the detection of the common 57-kb deletion. By use of this method we detected the 57-kb deletion in 59% of the examined Dutch alleles. The remaining alleles were screened for other mutations by genomic sequencing of the different exons, revealing three previously described mutations. Furthermore, we studied a possible genotype-phenotype relation of the homozygous deleted patients, which could not be demonstrated in our study population. Next to biochemical determination of cystine in leukocytes or fibroblasts, molecular genetic analysis enables prenatal diagnosis and facilitates identification of carriers.

A 31 bp VNTR in the cystathionine beta-synthase (CBS) gene is associated with reduced CBS activity and elevated post-load homocysteine levels.

Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinaemia, an independent and graded risk factor for cardiovascular disease (CVD). Although heterozygosity for cystathionine beta-synthase (CBS) deficiency has been excluded as a major genetic cause of mild hyperhomocysteinaemia in vascular disease, mutations in (non-)coding DNA sequences may lead to a mildly decreased CBS expression and, consequently, to elevated plasma homocysteine levels. We assessed the association between a 31 bp VNTR, that spans the exon 13-intron 13 boundary of the CBS gene, and fasting, post-methionine load and increase upon methionine load plasma homocysteine levels in 190 patients with arterial occlusive disease, and in 381 controls. The 31 bp VNTR consists of 16, 17, 18, 19 or 21 repeat units and shows a significant increase in plasma homocysteine concentrations with an increasing number of repeat elements, in particular after methionine loading. In 26 vascular disease patients the relationship between this 31 bp VNTR and CBS enzyme activity in cultured fibroblasts was studied. The CBS enzyme activity decreased with increasing number of repeat units of the 31 bp VNTR. RT-PCR experiments showed evidence of alternative splicing at the exon 13-intron 13 splice junction site. The 31 bp VNTR in the CBS gene is associated with post-methionine load hyperhomocysteinaemia that may predispose individuals to an increased risk of cardiovascular diseases.

Is mutated serine hydroxymethyltransferase (SHMT) involved in the etiology of neural tube defects?

Neural tube defects (NTD) arise in the first weeks of pregnancy due to a combination of environmental and genetic factors. In mothers of children with NTD elevated homocysteine (Hcy) levels and decreased plasma folate levels were observed, which suggests a defect in the folate-dependent Hcy metabolism. Therefore, mutations in genes coding for enzymes of this metabolism could be involved in NTD. Serine hydroxymethyltransferase (SHMT) catalyzes the reversible reaction of serine and tetrahydrofolate (THF) to glycine and 5,10-methylene THF. Two different isoforms of SHMT are known, one is present in the cytosol (cSHMT) and the other in the mitochondrion (mSHMT). Theoretically, mutated SHMT could lead to elevated Hcy levels and to an altered distribution of the different folate derivatives and might therefore become a risk factor for NTD. This study concerns the molecular genetic analysis of genes coding for both isoforms of the SHMT enzyme by single-stranded conformation polymorphism analysis. Several mutations as well as polymorphisms were found in both genes. The relevance of two variations, the 1420 C>T mutation of the cytosolic isoform and the 4-bp deletion of the mitochondrial isoform (delTCTT 1721-1724), to NTD risk was tested in a study group, which consisted of 109 NTD patients, 120 mothers of children with NTD, and 420 controls. Neither of the two polymorphisms led to an increased risk of NTD. In mothers with the 1420 CC genotype, significant increased Hcy levels are present. Also, significantly decreased red blood cell folate and plasma folate levels were present in individuals with the 1420 CC genotype. Probably, the 1420 C>T polymorphism causes a shift in distribution of the different folate derivatives. The 4-bp deletion of the mSHMT gene did not lead to altered Hcy or folate levels. So far, the results of this study provide no direct evidence for a role of defective SHMT functioning in NTD. Still, the influence of the 1420 C>T polymorphism of the cSHMT gene on the folate-related risk of NTD needs further investigation.

Folate, homocysteine and neural tube defects: an overview.

Folate administration substantially reduces the risk on neural tube detects (NTD). The interest for studying a disturbed homocysteine (Hcy) metabolism in relation to NTD was raised by the observation of elevated blood Hcy levels in mothers of a NTD child. This observation resulted in the examination of enzymes involved in the folate-dependent Hcy metabolism. Thus far, this has led to the identification of the first and likely a second genetic risk factor for NTD. The C677T and A1298C mutations in the methylenetetrahydrofolate reductase (MTHFR) gene are associated with an increased risk of NTD and cause elevated Hcy concentrations. These levels can be normalized by additional folate intake. Thus, a dysfunctional MTHFR partly explains the observed elevated Hcy levels in women with NTD pregnancies and also, in part, the protective effect of folate on NTD. Although the MTHFR polymorphisms are only moderate risk factors, population-wide they may account for an important part of the observed NTD prevalence.

The homocysteine distribution: (mis)judging the burden.

The nonfasting plasma total homocysteine (P-tHcy) concentration was measured in a random sample of 3025 Dutch adults aged 20-65 years (main study). The positively skewed distribution had a geometric mean of 13.9 micromol/L in men and 12.6 micromol/L in women. Blood of the main study was not cooled or centrifuged immediately after drawing. A stability study (n = 26) indicated that this could have resulted in a small (0.4 micromol/L) overestimation of the means. A comparative study (n = 88), and a reproduction of these results in an entirely different population (n = 213), showed a systematic difference in P-tHcy concentration of -2.4 micromol/L between our laboratory (Nijmegen, the Netherlands) and that in Bergen, Norway. With the information of the additional studies we provided precise and valid data of the Dutch P-tHcy distribution, from which we conclude the status in the Netherlands is worse than in other European countries. Furthermore, we showed that comparison of P-tHcy data is complicated unless the interlaboratory differences are known. @ 2001 Elsevier Science Inc.

Semiautomated DNA mutation analysis using a robotic workstation and molecular beacons.

BACKGROUND: Our increasing knowledge of the genetic basis of inheritable diseases requires the development of automated reliable methods for high-throughput analyses. METHODS: We investigated the combination of semiautomated DNA extraction from blood using a robotic workstation, followed by automated mutation detection using highly specific fluorescent DNA probes, so-called molecular beacons, which can discriminate between alleles with as little as one single-base mutation. We designed two molecular beacons, one recognizing the wild-type allele and the other the mutant allele, to determine genotypes in a single reaction. To evaluate this procedure, we examined the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, which is associated with an increased risk for cardiovascular disease and neural tube defects. DNA was isolated from 10 microL of fresh EDTA-blood samples by use of a robotic workstation. The DNA samples were analyzed using molecular beacons as well as conventional methods. RESULTS: Both methods were compared, and no differences were found between outcomes of genotyping. CONCLUSIONS: The described assay enables robust and automated extraction of DNA and analysis of up to 96 samples (10 microL of blood per sample) within 5 h. This is superior to conventional methods and makes it suitable for high-throughput analyses.

Betaine-homocysteine methyltransferase (BHMT): genomic sequencing and relevance to hyperhomocysteinemia and vascular disease in humans.

Elevated homocysteine levels have been associated with arteriosclerosis and thrombosis. Hyperhomocysteinemia is caused by altered functioning of enzymes of its metabolism due to either inherited or acquired factors. Betaine-homocysteine methyltransferase (BHMT) serves, next to methionine synthase, as a facilitator of methyl group donation for remethylation of homocysteine into methionine, and reduced functioning of BHMT could theoretically result in elevated homocysteine levels. Recently, the genomic sequence of the BHMT gene was published. Mutation analysis may reveal mutations of the BHMT gene that could lead to hyperhomocysteinemia. In the present study we performed genomic sequencing of the BHMT gene of 16 vascular patients with hyperhomocysteinemia and detected three mutations in the coding region of this gene. The first was an amino acid substitution of glycine to serine (G199S), which was found only in the heterozygous state. The second mutation was a substitution of glutamine to arginine (Q239R), and the last mutation was an amino acid substitution of glutamine to histidine (Q406H). The latter was also found only in the heterozygous state. The relevance of these mutations was tested in a study group, which consists of 190 cases with vascular disease and 601 controls. The influence of these three mutations on homocysteine levels was investigated. None of the three mutations led to significantly changed homocysteine levels. In addition, no differences in genotype distribution between cases and controls were found. So far, our results provide no evidence for a role of defective BHMT functioning in hyperhomocysteinemia or subsequently in vascular disease.

Automated extraction and amplification of DNA from whole blood using a robotic workstation and an integrated thermocycler.

Growing knowledge of the genetic basis of inheritable diseases has resulted in a rapidly increasing demand for DNA mutation analysis. Current methods are reliable and suitable for low-throughput mutation analyses, but are unable to cope with the increasing demand for genetic analyses, necessitating the development of new, fully automated and reliable methods. We developed a semi-automated method for DNA mutation analysis by integrating a thermocycler into a robotic pipetting workstation. DNA was extracted from 84 samples of 10 microl of EDTA-treated whole blood using magnetic beads within 2 h. Directly after isolation, the DNA was automatically transferred to an integrated thermocycler for amplification. Our semi-automated method proved to be reliable and robust, showing unambiguously interpretable PCR signals without occurrence of contamination. It is also faster than conventional manual methods. Only a brief manual intervention is required to remove and refit the seal of the PCR plate. This semi-automated assay is a step forward in the development of fully automated assays for DNA mutation analysis.

Nuclear genes and oxidative phosphorylation disorders: a review.

UNLABELLED: Knowledge concerning the approximately 70 human nuclear genes creating the essential building-blocks of the five multi-protein subunit complexes of the oxidative phosphorylation (OXPHOS) system has been expanded greatly in the past few years. However, knowledge concerning the numerous human genes involved in the regulation of transcription, translation, post-translational modification, mitochondrial signalling, import, quality control, folding and assembly of the OXPHOS system is still rather scanty. It may be expected that this scenario, by the application of direct (candidate gene identification by comparison between known genes in lower species and the human expressed sequence tag database) and indirect genetic strategies (the chromosome transfer technique, linkage analysis and positional cloning) will rapidly change. By now, a limited number of structural and non-structural nuclear gene defects have been found. CONCLUSION: This review summarises the state of our current knowledge of nuclear gene mutations in oxidative phosphorylation disorders.

SURFEIT-1 gene analysis and two-dimensional blue native gel electrophoresis in cytochrome c oxidase deficiency.

Leigh syndrome, a progressive, often fatal, neurodegenerative disorder, is frequently associated with a deficiency in the activity of cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain. In contrast to NADH:ubiquinone oxidoreductase and succinate dehydrogenase deficiencies, no mutations in nuclear genes encoding COX subunits have been identified thus far. Very recently, however, a Leigh syndrome complementation group has been identified which showed mutations in the SURFEIT-1 (SURF-1) gene. The results of a mutational detection study in 16 new randomly selected COX-deficient patients revealed a new mutation (C688T) in 2 patients and the earlier reported 845delCT mutation in 2 additional patients. In addition, we evaluated the diagnostic value of two-dimensional blue native gel electrophoresis. We show that this technique reveals distinct patterns of both fully and partially assembled COX complexes and is thereby capable of discrimination between COX-deficient SURF-1 and non-SURF-1-mutated patients.

The molecular basis of cystathionine beta-synthase deficiency in Dutch patients with homocystinuria: effect of CBS genotype on biochemical and clinical phenotype and on response to treatment.

Homocystinuria due to cystathionine beta-synthase (CBS) deficiency, inherited as an autosomal recessive trait, is the most prevalent inborn error of methionine metabolism. Its diverse clinical expression may include ectopia lentis, skeletal abnormalities, mental retardation, and premature arteriosclerosis and thrombosis. This variability is likely caused by considerable genetic heterogeneity. We investigated the molecular basis of CBS deficiency in 29 Dutch patients from 21 unrelated pedigrees and studied the possibility of a genotype-phenotype relationship with regard to biochemical and clinical expression and response to homocysteine-lowering treatment. Clinical symptoms and biochemical parameters were recorded at diagnosis and during long-term follow-up. Of 10 different mutations detected in the CBS gene, 833T-->C (I278T) was predominant, present in 23 (55%) of 42 independent alleles. At diagnosis, homozygotes for this mutation (n=12) tended to have higher homocysteine levels than those seen in patients with other genotypes (n=17), but similar clinical manifestations. During follow-up, I278T homozygotes responded more efficiently to homocysteine-lowering treatment. After 378 patient-years of treatment, only 2 vascular events were recorded; without treatment, at least 30 would have been expected (P<.01). This intervention in Dutch patients significantly reduces the risk of cardiovascular disease and other sequelae of classical homocystinuria syndrome.

Molecular genetic analysis of human folate receptors in neural tube defects.

Neural tube defects (NTDs) are the most common congenital malformations and are considered to have a multifactorial origin, having both genetic and environmental components. Periconceptional folate administration reduces the recurrence and occurrence risk by 70-100%. Recently we discovered the first genetic risk factors for NTDs: the 677 C-->T and the 1298 A-->C mutations in the methylenetetrahydrofolate reductase gene explaining at the most 35-50% of the protective effect of folate. In this study we further explored the genetic component of NTDs by analysing the coding region, including the intron-exon boundaries and signal sequences of the folate receptor genes by SSCP analysis. Among 39 patients with spina bifida (SB), 47 mothers with a child with SB, and 10 controls, no polymorphism was present in the folate receptor alpha (FR-alpha) gene or in the folate receptor beta (FR-beta) gene.

Identification of four novel mutations in severe methylenetetrahydrofolate reductase deficiency.

Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is an inborn error of folate metabolism, and is inherited as an autosomal recessive trait. MTHFR is a key enzyme in folate-dependent remethylation of homocysteine, and reduces 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Patients with this severe enzymatic deficiency are biochemically characterised by homocystinuria and hypomethioninaemia, and may suffer from neurological abnormalities, mental retardation and premature vascular disease. Here we report the molecular basis of severe MTHFR deficiency in four unrelated families from Turkish/Greek ancestry. By use of reverse-transcriptase (RT)-PCR, subsequently followed by direct sequencing analysis, we were able to identify four novel mutations in the MTHFR gene: two missense (983A-->G; 1027T-->G) and two nonsense (1084C-->T; 1711C-->T) mutations. Furthermore, a splice variant containing a premature termination codon, was observed in one patient, probably as a secondary effect of the 1027T-->G missense mutation. The ongoing identification and characterisation of mutations in the MTHFR gene will provide further insight into the heterogeneity of the clinical phenotype in severe MTHFR deficiency.

Molecular genetic analysis of the gene encoding the trifunctional enzyme MTHFD (methylenetetrahydrofolate-dehydrogenase, methenyltetrahydrofolate-cyclohydrolase, formyltetrahydrofolate synthetase) in patients with neural tube defects.

It is now well recognized that periconceptional folic acid or folic acid containing multivitamin supplementation reduces the risk of neural tube defects (NTDs). Recently we were able to show that homozygosity for a thermolabile variant of the enzyme methylenetetrahydrofolate reductase is associated with an increased risk for spina bifida in patients recruited from the Dutch population. However, this genetic risk factor could not account for all folic acid preventable NTDs. In an attempt to identify additional folate related enzymes that contribute to NTD etiology we now studied the methylenetetrahydrofolate dehydrogenase gene on chromosome 14q24 which encodes a single protein with three catalytic properties important in the folate metabolism. The cDNA sequence of 38 familial and 79 sporadic patients was screened for the presence of mutations by single strand conformation polymorphism (SSCP) analysis followed by sequencing. Two amino acid substitutions were identified. The first one (R293H) was detected in a patient with familial spina bifida and not in 300 control individuals. The mutation was inherited from the unaffected maternal grandmother and was also present in two younger brothers of the index patient, one of them displaying spina bifida occulta and the other being unaffected. The second change turned out to be an amino acid polymorphism (R653Q) that was present in both patients and controls with similar frequencies. Our results so far provide no evidence for a major role of the methylenetetrahydrofolate-dehydrogenase (MTHFD) gene in NTD etiology. However, the identification of a mutation in one family suggests that this gene can act as a risk factor for human NTD.

A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects?

Recently, we showed that homozygosity for the common 677(C-->T) mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, causing thermolability of the enzyme, is a risk factor for neural-tube defects (NTDs). We now report on another mutation in the same gene, the 1298(A-->C) mutation, which changes a glutamate into an alanine residue. This mutation destroys an MboII recognition site and has an allele frequency of .33. This 1298(A-->C) mutation results in decreased MTHFR activity (one-way analysis of variance [ANOVA] P < .0001), which is more pronounced in the homozygous than heterozygous state. Neither the homozygous nor the heterozygous state is associated with higher plasma homocysteine (Hcy) or a lower plasma folate concentration-phenomena that are evident with homozygosity for the 677(C-->T) mutation. However, there appears to be an interaction between these two common mutations. When compared with heterozygosity for either the 677(C-->T) or 1298(A-->C) mutations, the combined heterozygosity for the 1298(A-->C) and 677(C-->T) mutations was associated with reduced MTHFR specific activity (ANOVA P < .0001), higher Hcy, and decreased plasma folate levels (ANOVA P <.03). Thus, combined heterozygosity for both MTHFR mutations results in similar features as observed in homozygotes for the 677(C-->T) mutation. This combined heterozygosity was observed in 28% (n =86) of the NTD patients compared with 20% (n =403) among controls, resulting in an odds ratio of 2.04 (95% confidence interval: .9-4.7). These data suggest that the combined heterozygosity for the two MTHFR common mutations accounts for a proportion of folate-related NTDs, which is not explained by homozygosity for the 677(C-->T) mutation, and can be an additional genetic risk factor for NTDs.

Homozygous cystathionine beta-synthase deficiency, combined with factor V Leiden or thermolabile methylenetetrahydrofolate reductase in the risk of venous thrombosis.

Severe hyperhomocysteinemia in its most frequent form, is caused by a homozygous enzymatic deficiency of cystathionine beta-synthase (CBS). A major complication in CBS deficiency is deep venous thrombosis or pulmonary embolism. A recent report by Mandel et al (N Engl J Med 334:763, 1996) postulated factor V Leiden (FVL) to be an absolute prerequisite for the development of thromboembolism in patients with severe hyperhomocysteinemia. We studied 24 patients with homocystinuria caused by homozygous CBS deficiency from 18 unrelated kindreds for FVL and for the 677C-->T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene and investigated their possible interaction in the risk of venous thrombosis. Thrombotic complications were diagnosed in six patients, of whom only one was a carrier of FVL. On the contrary, thermolabile MTHFR caused by the 677C-->T mutation, was frequently observed among homocystinuria patients, especially among those with thromboembolic complications: three of six homocystinuria patients who had suffered from a thromboembolic event had thermolabile MTHFR. These data indicate that FVL is not an absolute prerequisite and probably not even a major determinant of venous thrombosis in homocystinuria, but, interestingly, thermolabile MTHFR may constitute a significant risk factor for thromboembolic complications in this inborn error of methionine metabolism.