Cerebrotendinous xanthomatosis without neurological involvement.

BACKGROUND: Cerebrotendinous xanthomatosis (CTX) is an autosomal recessively inherited inborn error of metabolism. Neurological symptoms are considered to be a clinical hallmark of untreated adult patients. We describe a 'milder CTX phenotype', without neurological involvement. METHODS: We performed a retrospective patient file study in 79 genetically confirmed Dutch patients with CTX (55 patients aged ≥ 21 years) to study the clinical heterogeneity of CTX. We studied the frequency of adult patients with CTX without neurological involvement at diagnosis, in our Dutch cohort, and included a family from South Africa and patients from Italy, USA, Chile and Asia from the literature. RESULTS: In total, we describe 19 adult patients with CTX from 16 independent families, without neurological symptoms at diagnosis. A relatively small percentage (21%, n = 4) had a history of cataract. The majority, 84% (n = 16), presented with tendon xanthomas as the sole or predominant feature. The majority of patients showed increased plasma cholesterol levels. No correlation was found between this 'milder phenotype', the cholestanol levels and the CYP27A1 genotype. In addition, we describe three novel mutations in the CYP27A1 gene. CONCLUSIONS: This study shows the clinical heterogeneity of CTX, highlighting the existence of a 'milder phenotype', that is without neurological involvement at diagnosis. Adult patients with CTX may present with tendon xanthomas as the sole or predominant feature, mimicking familial hypercholesterolemia. It is important to realize that the absence of neurological symptoms does not rule out the development of future neurological symptoms. As CTX is a treatable disorder, early diagnosis and initiation of treatment when additional clinical signs occur is therefore essential.

A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase.

Hyperhomocysteinaemia has been identified as a risk factor for cerebrovascular, peripheral vascular and coronary heart disease. Elevated levels of plasma homocysteine can result from genetic or nutrient-related disturbances in the trans-sulphuration or re-methylation pathways for homocysteine metabolism. 5, 10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulatory form of folate and carbon donor for the re-methylation of homocysteine to methionine. Reduced MTHFR activity with a thermolabile enzyme has been reported in patients with coronary and peripheral artery disease. We have identified a common mutation in MTHFR which alters a highly-conserved amino acid; the substitution occurs at a frequency of approximately 38% of unselected chromosomes. The mutation in the heterozygous or homozygous state correlates with reduced enzyme activity and increased thermolability in lymphocyte extracts; in vitro expression of a mutagenized cDNA containing the mutation confirms its effect on thermolability of MTHFR. Finally, individuals homozygous for the mutation have significantly elevated plasma homocysteine levels. This mutation in MTHFR may represent an important genetic risk factor in vascular disease.

Clinical imaging and neuropathological correlations in an unusual case of cerebrotendinous xanthomatosis.

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive lipid storage disorder due to a deficiency of the mitochondrial enzyme sterol 27-hydroxylase (CYP 27) with reduced or no chenodeoxycholic synthesis. This deficiency leads to an accumulation of cholestanol in different sites such as the eye lens, central nervous system or tendons. We report a 64-year-old female patient with a progressive gait disorder associated with cognitive decline since the age of 59. The patient had no mental retardation, cataract or chronic diarrhea. Her family reported increasing behavioral modifications 10 years previously. Clinical examination revealed a spastic paraplegia and bilateral xanthomas on the Achilles tendons. Cerebral magnetic resonance imaging (MRI) revealed diffuse hyperintense T2 abnormalities in the pyramidal tracts from the internal capsules to the cerebral peduncles also Technetium-99m-ECD brain SPECT showed a severe cerebellar hypoperfusion. Serum cholestanol analysis was 7 µmol/l (N). After 2 years, she was bedridden and died of aspiration pneumonia. The neuropathological study confirmed the CTX diagnosis and the sequencing analysis revealed that she was compound heterozygous for two mutations in the CYP27A1 gene: 1435 C > T (exon 7) on one allele and a new mutation, 1017 G > C (exon 5) on the other. The interest of the present case is to report neuropathology findings strongly correlated with the MRI and SPECT abnormalities.

Defective cystathionine beta-synthase regulation by S-adenosylmethionine in a partially pyridoxine responsive homocystinuria patient.

We determined the molecular basis of cystathionine beta-synthase (CBS) deficiency in a partially pyridoxine-responsive homocystinuria patient. Direct sequencing of the entire CBS cDNA revealed the presence of a homozygous G1330A transition. This mutation causes an amino acid change from aspartic acid to asparagine (D444N) in the regulatory domain of the protein and abolishes a TaqI restriction site at DNA level. Despite the homozygous mutation, CBS activities in extracts of cultured fibroblasts of this patient were not in the homozygous but in the heterozygous range. Furthermore, we observed no stimulation of CBS activity by S-adenosylmethionine, contrary to a threefold stimulation in control fibroblast extract. The mutation was introduced in an E. coli expression system and CBS activities were measured after addition of different S-adenosylmethionine concentrations (0-200 microM). Again, we observed a defective stimulation of CBS activity by S-adenosylmethionine in the mutated construct, whereas the normal construct showed a threefold stimulation in activity. These data suggest that this D444N mutation interferes in S-adenosylmethionine regulation of CBS. Furthermore, it indicates the importance of S-adenosylmethionine regulation of the transsulfuration pathway in homocysteine homeostasis in humans.

Detection of Staphylococcus aureus in cystic fibrosis patients using breath VOC profiles.

Staphylococcus aureus (S. aureus) is a common bacterium infecting children with cystic fibrosis (CF). Since current detection methods are difficult to perform in children, there is need for an alternative. This proof of concept study investigates whether breath profiles can discriminate between S. aureus infected and non-infected CF patients based on volatile organic compounds (VOCs). We collected exhaled breath of CF patients with and without S. aureus airways infections in which VOCs were identified using gas chromatography-mass spectrometry. We classified these VOC profiles with sparse partial least squares discriminant analysis. Multivariate breath VOC profiles discriminated infected from non-infected CF patients with high sensitivity (100%) and specificity (80%). We identified the nine compounds most important for this discrimination. We successfully detected S. aureus infection in CF patients, using breath VOC profiles. Nine highlighted compounds can be used as a focus point in further biomarker identification research. The results show considerable potential for non-invasive diagnosis of airway infections.

Identification of Pseudomonas aeruginosa and Aspergillus fumigatus mono- and co-cultures based on volatile biomarker combinations.

Volatile organic compound (VOC) analysis in exhaled breath is proposed as a non-invasive method to detect respiratory infections in cystic fibrosis patients. Since polymicrobial infections are common, we assessed whether we could distinguish Pseudomonas aeruginosa and Aspergillus fumigatus mono- and co-cultures using the VOC emissions. We took headspace samples of P. aeruginosa, A. fumigatus and co-cultures at 16, 24 and 48 h after inoculation, in which VOCs were identified by thermal desorption combined with gas chromatography - mass spectrometry. Using multivariate analysis by Partial Least Squares Discriminant Analysis we found distinct VOC biomarker combinations for mono- and co-cultures at each sampling time point, showing that there is an interaction between the two pathogens, with P. aeruginosa dominating the co-culture at 48 h. Furthermore, time-independent VOC biomarker combinations were also obtained to predict correct identification of P. aeruginosa and A. fumigatus in mono-culture and in co-culture. This study shows that the VOC combinations in P. aeruginosa and A. fumigatus co-microbial environment are different from those released by these pathogens in mono-culture. Using advanced data analysis techniques such as PLS-DA, time-independent pathogen specific biomarker combinations can be generated that may help to detect mixed respiratory infections in exhaled breath of cystic fibrosis patients.

Phase I clinical and pharmacogenetic trial of irinotecan and raltitrexed administered every 21 days to patients with cancer.

PURPOSE: Irinotecan and raltitrexed display schedule-dependent synergy in vitro, which supports the clinical investigation of the combination. Functional polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene result in intracellular redistribution of folate derivatives, which may affect raltitrexed-associated cytotoxicity. PATIENTS AND METHODS: Patients with a range of solid cancers and good performance status received irinotecan as a 90-minute infusion on day 1 and raltitrexed as a 15-minute infusion on day 2, repeated every 21 days. Samples were collected for MTHFR C677T genotyping and fasting plasma homocysteine during the first cycle. RESULTS: Thirty-nine assessable patients received 127 cycles of therapy. Irinotecan doses ranged from 100 to 350 mg/m(2), and raltitrexed, 1.0 to 4.0 mg/m(2). Raltitrexed doses of more than 3.0 mg/m(2) were not tolerated and were associated with dose-limiting asthenia, diarrhea, and AST/ALT elevation. Irinotecan/raltitrexed doses of 350/3.0 mg/m(2) were well-tolerated; principal toxicities included neutropenia, diarrhea, and fatigue. Two partial responses were observed in patients with pretreated gastroesophageal cancers. Homozygotes with the MTHFR 677 TT polymorphism incurred significantly less raltitrexed-associated toxicity than those with either wild-type or heterozygous genotypes (P = .05). No significant differences were noted in plasma homocysteine values between the genotypic subtypes, and plasma homocysteine levels did not predict the risk of toxicity. CONCLUSION: Irinotecan and raltitrexed doses of 350 and 3.0 mg/m(2) are recommended for further study on a day 1, 2 schedule every 21 days. Efficacy results suggest that trials in upper and lower gastrointestinal malignancies are warranted. MTHFR C677T genotypes may be predictive of clinical raltitrexed toxicity.

The effect of dantrolene sodium in Very Long Chain Acyl-CoA Dehydrogenase Deficiency.

We present a patient, who experienced recurrent episodes of rhabdomyolysis. Her beneficial response to treatment with dantrolene sodium was previously reported. Adult onset Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) deficiency has been diagnosed only recently. In adults, VLCAD deficiency results in recurrent fasting-, exercise-, or infection-induced muscle stiffness, muscle pain and myoglobinuria caused by rhabdomyolysis. This case illustrates for the first time the beneficial effect of dantrolene in VLCAD deficiency. We discuss the therapeutic mechanism of dantrolene sodium and its possible role as additional treatment modality for patients with VLCAD deficiency.

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.

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.

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.

The human and mouse methylenetetrahydrofolate reductase (MTHFR) genes: genomic organization, mRNA structure and linkage to the CLCN6 gene.

Methylenetetrahydrofolate reductase (MTHFR), a pivotal enzyme in folate metabolism, regulates the proportional distribution of one-carbon moieties between cellular methylation reactions and nucleic acid synthesis. The organization of the MTHFR gene and the structure of its mRNA were characterized in human and mouse. There are three mRNA transcripts of 2.8, 7.2 and 9.8 kb in human and two of 3.2 and 7.5 kb in mouse. Northern blot analysis revealed that human MTHFR MRNA is only present at low abundance in most tissues tested. Five kilobases of sequence flanking the 3' end of the human gene were isolated, and polyadenylation sites were defined by 3' RACE. The shorter 2.8 kb transcript and the two larger 7.2 and 9.8 kb transcripts utilize different polyadenylation signal sequences, 629 and 4937 bp downstream of the stop codon, respectively. The two mRNA species in mouse also result from differential polyadenylation. Approximately 7 and 3.5 kb upstream of the human and mouse genes, respectively, were isolated and sequenced. Transcription start sites in human MTHFR were mapped using 5' RACE. The 2.8 and 7.2 kb mRNAs originate from one of two transcription start sites that are 206 and 243 bp upstream of the ATG initiation codon, whereas transcription of the 9.8 kb mRNA is initiated at a start site located 2.8 kb upstream of the translation start codon. The putative MTHFR promoter does not have a TATA box but contains CpG islands and multiple potential Sp1 binding sites. The MTHFR gene was finely mapped to interval 16 of chromosome 1p36.3, a region deleted in many tumors, by establishing a close linkage to CLCN6, a putative chloride channel gene. A novel CA-repeat polymorphism identified within intron 2 of the CLCN6 gene may be useful in assessing loss of heterozygosity in such tumors. The multiple MTHFR mRNA species identified in this report may reflect an underlying complex set of gene regulatory mechanisms acting through an alternative transcription start site and/or polyadenylation signal sequence utilization.

Detection of a novel deletion in the cystathionine beta-synthase (CBS) gene using an improved genomic DNA based method.

We elucidated the intron-exon boundaries of the 15 coding exons of the human cystathionine beta-synthase (CBS) gene in order to establish an improved method based on PCR and direct sequencing for detection of CBS mutations. Using this method we identified the pathogenic mutations in two Danish siblings with CBS deficiency. Patients were compound heterozygotes: we detected the 833T-->C mutation and a novel 22 bp deletion of exon 4 (493-514del) that introduces a frameshift and a stop codon immediately after the deletion. The deletion resulted in no detectable mRNA from this allele, as assessed by sequencing of cDNA. The established method represents an improvement of the existing method based on sequencing of cDNA because it permits the detection of mutations within the entire coding region of the CBS gene from a peripheral blood sample, including splice mutations and mutations resulting in the lack or a reduced amount of transcript.

The 894 G > T variant of endothelial nitric oxide synthase (eNOS) increases the risk of recurrent venous thrombosis through interaction with elevated homocysteine levels.

BACKGROUND: Venous thrombosis is a multicausal disease involving both genetic as well as acquired risk factors. Hyperhomocysteinemia is associated with a 2-fold increased risk of recurrent venous thrombosis (RVT). Recently, the 894 G > T variant of endothelial nitric oxide synthase (eNOS) was postulated to be associated with hyperhomocysteinemia. OBJECTIVES: We hypothesized an interrelation of hyperhomocysteinemia, the eNOS 894 G > T variant and RVT risk. METHODS: The eNOS 894 G > T variant was studied in 170 cases with a history of RVT and 433 controls from the general population. RESULTS: The eNOS 894 TT genotype may increase RVT risk [odds ratio (OR) 1.3 (0.7-2.6)], but no association of the eNOS 894 G > T variant with elevated homocysteine was found in controls. Interestingly, in RVT cases the coexistence of both the 894 TT genotype and elevated tHcy levels (> 90th percentile) was more frequently present than in controls, which led to a substantially increased risk of recurrent venous thrombosis [fasting tHcy OR 5.3 (1.1-24.1), postload tHcy OR 6.5 (1.6-29.5)]. CONCLUSION: The results of the present study demonstrate that the eNOS 894 G > T variation interacts with elevated tHcy levels, leading to an increased risk of recurrent thrombotic events. This interaction points in the direction of S-nitrosation as a mechanism by which homocysteine exerts its detrimental effects on the hemostatic system.

The 677C-->T mutation in the methylenetetrahydrofolate reductase gene: associations with plasma total homocysteine levels and risk of coronary atherosclerotic disease.

Homozygosity for a 677C-->T mutation at the locus that codes for 5,10-methylenetetrahydrofolate reductase (MTHFR), a folate-dependent crucial enzyme in homocysteine metabolism, may render the enzyme thermolabile and less active and has been associated with increased levels of plasma total homocysteine (tHcy). We assessed whether this mutation was associated with increased risk of coronary atherosclerosis and plasma levels of tHcy and furthermore studied whether folate status would modify the associations. Data were collected from subjects with substantial coronary atherosclerosis (> or = 90% occlusion in one and > or = 40% occlusion in a second coronary artery, referred to as cases, n = 131) or virtually no coronary narrowing (referred to as coronary controls, n = 87) and from a population-based control group (n = 100), all residing in the Rotterdam area, The Netherlands. Both males and females, aged 25-65 years were studied. The frequency of homozygosity for the mutation (+/+) in cases (10.0%) did not significantly differ statistically from that observed in coronary controls (11.5%, P = 0.71), population-based controls (7.0%, P = 0.43), or combined control groups (9.1%, P = 0.80). In the overall group (as well as in the three subgroups), plasma tHcy levels, fasting and to a lesser extent after a methionine-loading test, were higher in +/+ subjects than in homozygous normal subjects (-/-), whereas heterozygous subjects (+/-) had intermediate levels (Ptrend = 0.001). The +/+ subjects with erythrocyte folate levels < 790 nmol/l (population median) had a 77%, (95% CI, 27-144%) higher geometric mean fasting tHcy (21.4, micromol/l) than those with higher erythrocyte folate (12.1 micromol/l). The odds ratio (OR) of coronary atherosclerosis for +/+ subjects, with +/- and -/- subjects as the reference group, in analyses with combined control groups, was 1.1 (95% CI, 0.5-2.4). The ORs were 2.2 (95% CI, 0.7-6.8) and 0.6 (95% CI, 0.2-1.7) among subjects with low and high folate levels, respectively. Our study indicates that homozygosity for the 677C-->T MTHFR mutation, especially in combination with low folate status, predisposes to high plasma levels of fasting tHcy. However, homozygosity for this mutation, whether or not in combination with low folate status, was not associated with increased risk of coronary artery disease.

The methionine synthase reductase (MTRR) A66G polymorphism is a novel genetic determinant of plasma homocysteine concentrations.

Epidemiological evidence has revealed that an elevated plasma homocysteine level (hyperhomocysteinemia) confers an increased risk of cardiovascular disease and neural tube defects. Hyperhomocysteinemia is caused by both nutritional (e.g. folate, vitamins B(6) and B(12)) and genetic factors, including functional polymorphisms of key enzymes involved in homocysteine metabolism. One such enzyme, methionine synthase reductase (MTRR), maintains adequate levels of methylcob(III)alamin, the activated cofactor for methionine synthase, which catalyzes the remethylation of homocysteine to methionine. A common MTRR polymorphism, i.e. a 66 A-->G substitution specifying an isoleucine to methionine substitution (I22M), was recently identified. To assess the influence of this polymorphism on total plasma homocysteine (tHcy), we undertook a genotype/phenotype analysis in a study population of 601 Northern-Irish men, aged 30--49, for which biochemical and genetic data relevant to folate/homocysteine metabolism had already been acquired. The 66AA genotype has a frequency of 29% in this population. We established that there was a significant influence of MTRR genotype on tHcy ranking (P=0.004) and that the 66AA genotype contributes to a moderate increase in tHcy levels across the distribution [OR 1.59 (95% CI: 1.10--2.25) for the 66AA genotype to be in the upper half of the tHcy distribution, P=0.03]. The homocysteine-elevating effect of the 66AA genotype is independent of serum folate, vitamin B(12) and vitamin B(6) levels. Based on published estimates of the enhanced cardiovascular disease risk conferred by defined increments of plasma tHcy, we estimate that 66AA homozygotes have, on average, an approximately 4% increase in cardiovascular disease risk compared to 66GG homozygotes. This study provides the first evidence that the MTRR A66G polymorphism significantly influences the circulating tHcy concentration.

Thermolabile methylenetetrahydrofolate reductase and factor V Leiden in the risk of deep-vein thrombosis.

Mild hyperhomocysteinemia is an established risk factor for both arteriosclerosis and thrombosis, and may be caused by genetic and environmental factors. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the cofactor for the methylation of homocysteine to methionine. Individuals with the thermolabile variant of MTHFR have decreased MTHFR activities, resulting in elevated plasma homocysteine concentrations. A homozygous 677C-->T transition in the MTHFR gene has recently been identified as the cause of reduced enzyme activity and thermolability of the protein. We studied the frequency of the homozygous mutant (+/+) genotype in 471 patients with deep-vein thrombosis and 474 healthy controls enrolled in The Leiden Thrombophilia Study (LETS), its interaction with factor V Leiden, and assessed the association between the MTHFR genotypes and plasma homocysteine concentration. Homozygosity for the 677C-->T polymorphism was observed in 47 (10%) patients, and in 47 (9.9%) controls (OR 1.01 [95% CI: 0.7-1.5]). No modified risk of the (+/+) genotype was observed in carriers of factor V Leiden. Our data suggest that, although the homozygous mutant genotype is associated with elevated plasma homocysteine concentrations, this homozygous mutation itself is not a genetic risk factor for deep-vein thrombosis, irrespective of factor V Leiden genotype.

Sequence analysis of the coding region of human methionine synthase: relevance to hyperhomocysteinaemia in neural-tube defects and vascular disease.

Elevated homocysteine (Hcy) levels are observed in two apparently unrelated diseases: neural-tube defects (NTD) and premature vascular disease. Defective human methionine synthase (MS) could result in elevated Hcy levels. We sequenced the coding region of MS in 8 hyperhomocysteinaemic patients (4 NTD patients and 4 patients with pregnancies complicated by spiral arterial disease, SAD). We identified only one mutation resulting in an amino acid substitution: an A-->G transition at bp 2756, converting an aspartic acid (D919) into a glycine (G). We screened genomic DNA for the presence of this mutation in 56 NTD patients, 69 mothers of children with NTD, 108 SAD patients and 364 controls. There was no increased prevalence of the GG and AG genotypes in NTD patients, their mothers or SAD patients. The D919G mutation does not seem to be a risk factor for NTD or vascular disease. We then examined the mean Hcy levels for each MS genotype. There was no correlation between GG- or AG-genotype and Hcy levels. The D919G mutation is thus a fairly prevalent, and probably benign polymorphism. This study, though limited, provides no evidence for a major involvement of MS in the aetiology of homocysteine-related diseases such as NTD or vascular disease.

Primary Carnitine (OCTN2) Deficiency Without Neonatal Carnitine Deficiency.

Although the diagnosis of a primary carnitine deficiency is usually based on a very low level of free and total carnitine (free carnitine: 1-5 µM, normal 20-55 µM) (Longo et al. 2006), we detected a patient via newborn screening with a total carnitine level 67 % of the normal value. At the age of 1 year, after interruption of carnitine supplementation for a 4-week period the carnitine profile was assessed and the free carnitine level had dropped to 10.4 µmol/l (normal: 20-55 µM) and total carnitine level had dropped to 12.7 µmol/l (normal: 25-65 µM). Transient carnitine deficiency was not likely anymore and DNA mutation analysis of the OCTN2 (SLC22A5) gene showed a homozygous c.136C>T (p.P46S) mutation, confirming the diagnosis of primary carnitine deficiency. We would like to emphasize that neonates with a primary carnitine deficiency might present with relatively high levels of total carnitine due to placental carnitine transfer, and also draw the attention to the importance of regular follow-up and the significance of genetic diagnostics in patients with a nonclassical presentation.