Michael Linnebank

Polymorphisms in glutathione S-transferase omega-1 and AD, vascular dementia, and stroke.

Background: Glutathione S-transferase omega-1 (GSTO1) protects from oxidative stress, a risk factor for Alzheimer disease (AD), vascular dementia (VaD), and stroke. Polymorphisms in GSTO1 might influence the function of the protein and thus the risk of AD, VaD, and stroke. Methods: The GSTO1 gene was screened for variations. The effect of the detected polymorphisms on the risk of AD, VaD, and stroke was evaluated. CSF levels of cholesterol and plasma homocysteine levels were compared according to the GSTO1 genotype. Results: Two missense polymorphisms in exon 4 of GSTO1 (Ala140Asp and Glu155ΔGlu) were detected and tested for their association with AD, VaD, and stroke. The Asp/Asp and Ala/Asp genotypes increased the risk of stroke (p = 0.003, OR = 2.1), and the Asp/Asp genotype increased the risk of VaD (p = 0.02, OR = 2.2). GSTO1 polymorphisms did not influence the risk of AD, but the Asp allele influenced the age at onset (p = 0.05). In nondemented probands CSF levels of cholesterol were increased in carriers of the Asp/Asp genotype (p = 0.004); however, in patients with manifest dementia the authors found decreased CSF levels of cholesterol in carriers of the Asp/Asp genotype (p = 0.028). Serum homocysteine levels in stroke patients were higher in carriers of at least one Asp allele (p = 0.011). Conclusion: The GSTO1 Asp allele may be a genetic risk factor for cerebrovascular diseases, and might influence the course of Alzheimer disease, even though effects vary in different studies.

The 677T genotype of the common MTHFR thermolabile variant and fasting homocysteine in childhood venous thrombosis

Abstract Controlled data on the association of MTHFR genotypes, hyperhomocysteinaemia and their interaction with factor V G1691A with childhood thrombosis are not yet available. Therefore we conducted a case-control study comparing 141 childhood patients with venous thrombosis with 345 healthy controls. The MTHFR C677T genotypes, FV G1691A and prothrombin G20210A were evaluated; in addition, fasting homocysteine concentrations were measured in a subgroup of 60 children and 80 healthy controls. 10.4% of the healthy control population showed the MTHFR TT genotype, 34.2% the CT genotype and 55.4% the CC variant. MTHFR genotypes account for fasting homocysteine concentrations in healthy controls (CC: 5.5 μmol/l (4–7.2); CT: 7 μmol/l (3.9–9.8); TT: 12.1 μmol/l (7.7–13.3)) with an upper age-specific 95th percentile of 8.3 μmol/l. The following frequencies (patients versus controls), odds ratios (OR) and 95% confidence intervals (CI) were found for single defects: MTHFR 677TT genotype (10.6% vs. 10.4%; OR/CI: 1.02/0.54–1.93; P= 0.99) and CT genotype (43.8% vs. 34.2%; OR/CI: 2.12/1.42–3.16; P= 0.0000). A combination of FV G1691A mutation and MTHFR 677CT genotype was found in 9.9% of patients and in 2.9% of the controls (OR/CI: 3.8/1.64–8.75; P= 0.027). Fasting homocysteine median (range) concentrations in the patient group were significantly higher than in the controls (7 μmol/l (3–23) vs. 5.5 μmol/l (3–8.4); P= 0.0004), and homocysteine concentrations >8.3 μmol/l were found in 40% of patients vs. 2.5% of the controls (OR/CI: 22/2.64–183; P= 0.0003). Conclusion Data of this childhood case-control study suggest that mildly elevated fasting homocysteine concentrations >8.3 μmol/l and the CT genotype of the MTHFR C677T variant are significant risk factors for venous vascular occlusion in children.

Structure of the human argininosuccinate synthetase gene and an improved system for molecular diagnostics in patients with classical and mild citrullinemia

Abstract. Deficiency of argininosuccinate synthetase (ASS) causes citrullinemia, an autosomal recessive inherited defect of the urea cycle. Most patients described so far have presented with the classical form of the disease. There are also patients with a mild form of citrullinemia in whom the exact molecular basis and clinical relevance are uncertain. Mutations in the human ASS gene have not yet been described in mildly affected or asymptomatic patients with citrullinemia. The genomic sequence of the human ASS gene is not precisely known making mutation analysis difficult. Here, the entire genomic DNA sequence and mutations in the ASS gene of patients with the classical and mild form of the disease are described. The mutations c.1168G→A (G390R) and IVS13+5 G→A and the novel mutation c.323G→T (R108L) have been found to be associated with classical citrullinemia, whereas the novel mutations c.535T→G (W179R), and c.1085G→T (G362V) have been detected on alleles of the mildly affected patients. Thus, mutations found in the human ASS gene of asymptomatic children with biochemical abnormalities and in some cases enzymatically proven citrullinemia have allowed us to classify these cases as ASS-deficient patients. The elucidation of the structure of the human ASS gene has made possible the use of intronic primers for molecular analysis of patients with mild disease and the classical form, and provides another option for prenatal diagnostics in affected families with the severe type.

Clinical, enzymatic, and molecular genetic characterization of a biochemical variant type of argininosuccinic aciduria: prenatal and postnatal diagnosis in five unrelated families

A biochemical variant of argininosuccinate lyase deficiency, found in five individuals, is introduced. In comparison to classical patients, the variant cases of argininosuccinate lyase deficiency were characterized by residual enzyme activity as measured by the incorporation of [14C]citrulline into proteins. The five patients of different ethnic backgrounds presented with relatively mild clinical symptoms, variable age of onset, marked argininosuccinic aciduria and severe, but not complete, deficiency of argininosuccinate lyase. [14C]Citrulline incorporation into proteins, which is completely blocked in classical argininosuccinic aciduria, was only partially reduced in fibroblasts of these patients. Further investigation showed that previous standard conditions of the assay were not optimal. Higher concentrations of citrulline in the incubation medium strongly stimulated 14C incorporation in normal cells, but not in the patients; as a result, the relative incorporation level in the patients dropped to 6–28% compared to 18–75% of normal in the original procedure. Prenatal diagnosis was successfully performed in three of the families. Affected pregnancies were indicated by (partial) deficiency of [14C]citrulline incorporation in chorionic villi and/or increased levels of argininosuccinate in amniotic fluid. Analysis of the ASL gene in the five patients revealed a considerable allelic heterogeneity. Three novel mutations—R385C (2 patients), V178M and R379C—were detected in homozygous states, whereas one patient was compound heterozygous for the known mutations R193Q and Q286R. In conclusion, there are patients of different ethnic backgrounds who are characterized by residual activity of argininosuccinate lyase and who present with less severe clinical courses. In addition, we present an improved biochemical assay for accurate prenatal and postnatal diagnosis.

Cardiomyopathy in congenital disorders of glycosylation

Congenital disorders of glycosylation are a group of inherited metabolic multisystem disorders characterized by defects in the glycosylation of proteins and lipids. In most cases, neuromuscular disease is present. The purpose of this study was to characterize the cardiological aspects in this disorder. From the literature, we identified six children with congenital disorders of glycosylation associated with cardiac disease. We then screened for cardiovascular manifestations 20 patients diagnosed with congenital disorders of glycosylation at our own institution. Of the 6 patients identified in the literature, 4 had hypertrophic cardiomyopathy, while in the other 2 the cardiac diagnosis was unclear. The mean age at cardiac diagnosis was 5 months, with a range from 34 weeks to 24 months. Of the patients, five had died at a mean age of 3.5 months, with a range from 1.5 to 6 months, with one documented cardiac death. Three of our 20 patients (15%) had coexistent cardiomyopathy, and in three additional patients presenting with cardiomyopathy we made the diagnosis of a congenital disorder of glycosylation. In our cohort, dilated cardiomyopathy was found in two-thirds of the patients, with hypertrophic cardiomyopathy in the other third. The mean age at cardiac diagnosis was 19 months, with a range from 0.5 to 84 months. Of these patients, two died in infancy at a mean age of 4 months, specifically at 1.5 and 7 months, due to cardiac disease, with one dying suddenly. The remaining four patients are alive with minor to severe cardiac dysfunction. We conclude that congenital disorders of glycosylation have to be considered in the differential diagnosis of children presenting with cardiomyopathy, and that all patients with congenital disorders of glycosylation should be screened for an associated cardiomyopathy. Cardiac involvement contributes significantly to morbidity and mortality, and probably to sudden cardiac death in this disorder.

Lack of genetic dispositions to hyperhomocysteinemia in Alzheimer disease

Recent reports suggested a link of homocysteine (Hcy) metabolism and Alzheimer disease (AD) [Kruman et al., 2002; Seshadri et al., 2002]. Impairment of Hcy metabolism might be due to nutritional or genetic factors, for example, a lack of folate, vitamin B6 or B12, or due to genetic variants of genes coding for enzymes involved in Hcy metabolism. We aimed to investigate whether the functional polymorphisms methylenetetrahydrofolate reductase (MTHFR) c.677C>T (A222V) and c.1298A>C (E429A), methionine synthase (MS c.2756A>G (D919G), and cystathionine b-synthase (CBS) c.833T>C (I278T) and 844ins68 (tandem repeat affecting RNA splicing) are of relevance for AD. This study includes 162 German AD patients (72 9 years; m/f 52/110) who were diagnosed at the Department of Psychiatry according to the Diagnostic and Statistical Manual of Mental Disorders IV, supported by clinical examination, detailed structured interviews, neuropsychological testing, and MRI-studies. Age of onset was recorded at the initial visit as theagewhensymptomswerefirst recognizedby thepatients themselves or by those in their personal environment. As controls, 169 healthy individuals (71 7 years, m/f 74/95) were recruited from the local population in collaboration of the town census bureau and the board of data protection, NorthrhineWestfalia, Germany. All individuals were unrelated and of Caucasian ancestry. All participants or their statutory agents gave informedwritten consent. The studywas approved by the local ethics committee. Analysis of the five polymorphisms was performed as published previously [Linnebank et al., 2004]. In addition, the apolipoprotein E (APOE) e4 allele was analyzed as internal control. This allele has repeatedly been proven as a risk factor for both the development and an earlier onset of sporadic AD [Schellenberg, 1995]. Logistic regression analysis was used to examine the simultaneous effects of variants of genes involved inHcymetabolism and the APOE e4 allele on the risk for AD. Age and sex were included as covariates. The allele frequencies between the investigated groups were compared using w-tests and the Fisher exact test when appropriate. Kaplan–Meier survival analysis was used to compare the age of onset in AD patients depending on the different polymorphisms. Significance was set at two-sided alpha <0.05. Hardy–Weinberg equilibrium was confirmed for all populations. The presence of the APOE e4 allele (0.61 in patients vs. 0.28 in controls) was confirmed as a risk factor for AD (w1⁄4 16.3, df1⁄4 1, P< 0.001, respectively) and for an earlier onset of AD (log rank1⁄4 5.38, df1⁄4 1; P1⁄4 0.020) supporting the representative character of our study population. In contrast,

Genetic analysis of carbamoylphosphate synthetase I and ornithine transcarbamylase deficiency using fibroblasts

Abstract Deficiencies of carbamoylphosphate synthetase or of ornithine transcarbamylase, two urea cycle enzymes located within mitochondria, often present as severe neonatal hyperammonaemic crises and have a poor prognosis. While genetic analysis of the X-chromosomal transmitted ornithine transcarbamylase deficiency (OTC) is performed by exon-wise mutation screening of genomic DNA in most cases, identification of mutations in the autosomal inherited carbamoylphosphate synthetase (CPS 1) deficiency requires analysis of transcripts due to the unknown genomic structure. We tested the hypothesis that CPS 1 and OTC are expressed at low levels in fibroblasts and indeed were able to amplify full-length cDNA from that source. Using a reverse transcriptase polymerase chain reaction based procedure we completely characterised the genetic background in five patients and identified three novel mutations and a novel polymorphism of the CPS 1 gene (deletion/insertion 2170delGCTCinsCCA, nonsense mutation 2359C>T, missense mutation 3161T > G and Thr1406Asn, respectively), as well as the missense mutations 482A > G and 994T > A of the OTC gene. Conclusion Cultured fibroblasts are an easily accessible source for genetic analysis of inborn errors of urea cycle enzymes which are functionally expressed only in liver and gut.

Isolated thrombosis due to the cystathionine β-synthase mutation c.833T>C (I278T)

Hereditary homocystinuria due to cystathionine β-synthase (CBS) deficiency is a rare disease (about 1:20 000 in Germany) often complicated by thromboembolism. Single mutations, which affect the C-terminal region of the CBS enzyme, lead to isolated thrombosis without further symptoms typical for homocystinuria such as atherosclerosis, psychomotor retardation, and dislocation of the ocular lenses. In this study, DNA samples of patients with stroke (n=225) and sinus thrombosis (n=46) were screened for the most common homocystinuria mutation, CBS I278T. In each group one homozygous patient was identified. Thus, not only C-terminal mutations but also the most common mutation in classical homocystinuria, CBS I278T, can lead to isolated thrombophilic events. These data support the hypothesis that homocystinuria is an underdiagnosed disease.

Hereditary elevation of angiotensin converting enzyme suggesting neurosarcoidosis

We report a 49-year-old woman with arterial hypertension, transitory neurologic symptoms, and suspected neurosarcoidosis based on elevated plasma and CSF angiotensin converting enzyme (ACE) levels. Sarcoidosis is a multisystem disease of unknown etiology, histopathologically characterized by the formation of noncaseating granulomas. CNS involvement occurs in about 5% of patients, and neurosarcoidosis can present without systemic manifestation.1,2⇓ The most common clinical manifestations of neurosarcoidosis are cranial nerve palsies and CNS parenchymal disease.1,2⇓ MRI typically reveals diffuse meningeal involvement, mass lesions, or diffuse white matter lesions.1,2⇓ Plasma levels of ACE (enzyme commission 3.4.15.1) are increased in 70 to 80% of patients with systemic sarcoidosis. In patients with neurosarcoidosis, plasma ACE levels are increased in about 24%, and …

Genetic association study of the P-type ATPase ATP13A2 in late-onset Parkinson's disease.

A role of ATP13A2 in early‐onset Parkinsonism (EOP) has been proposed. Conversely, the contribution of this ATPase to late‐onset Parkinsons disease (PD) remains unexplored. We therefore conducted a case–control association study in this age‐of‐onset group with PD. The initial sample was of German origin and consisted of 220 patients with late‐onset PD (mean age of onset 60.1 years) and 232 age‐matched unrelated controls. Five single nucleotide polymorphisms (SNPs) covering ATP13A2 and its common haplotypes were genotyped. The overall association results in this sample were negative. Interestingly, gender stratification gave a positive result for SNP rs11203280 (PUNC = 0.016) in men. This result could not be reproduced in a replication sample of German and Serbian origin composed of 161 patients with late‐onset PD (mean age of onset 51.7 years) and 150 age‐ and ethnic‐matched controls. In conclusion, we found no consistent evidence for an association between ATP13A2 and late‐onset PD.