Elevated cerebrospinal fluid sphingomyelin levels in prodromal Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, but still without known disease mechanism, proper treatment and efficient diagnostic tools for an early stage diagnosis. There is increasing evidence that lipids, especially cholesterol and sphingolipids, may play a role in pathological processes that occur in the AD brain even in very early stages of the disease. However, lipid changes in cerebrospinal fluid (CSF) of individuals with AD have not been well studied. In previous work, we developed a reproducible and sensitive nano-HPLC-MS method for CSF phospholipids screening and conducted a pilot study to find potential phospholipid changes in CSF from individuals with AD dementia. We observed a slight increase (24%) of sphingomyelin (SM) in CSF samples from patients with probable AD compared to non-demented controls. The goal of this work was to validate our findings and to analyze how SM CSF levels change in different stages of AD from prodromal to mild and moderate AD. We found significantly increased SM levels (50.4±11.2%, p=0.003) in the CSF from individuals with prodromal AD compared to cognitively normal controls, but no change in CSF SM levels between mild and moderate AD groups and cognitively normal controls. These results suggest that alterations in the SM metabolism may contribute to early pathological processes leading to AD.

Nano-HPLC-MS analysis of phospholipids in cerebrospinal fluid of Alzheimer's disease patients--a pilot study.

There is emerging evidence that lipids play an important role in many neurodegenerative processes, for example in Alzheimer's disease (AD). Although different lipid alterations in the AD brain have been reported, there have only been very few investigations of lipid changes in the cerebrospinal fluid (CSF). Recent developments in mass spectrometry (MS) have enabled fast and sensitive detection of lipid species in different biological matrixes. In this study we developed an on-line HPLC-MS method for phospholipid profiling in the CSF based on nano-HPLC separation using an Amide column and detection with electrospray (ESI) quadrupole-time of flight (QTOF) MS. We achieved good separation, reproducibility, and sensitivity in monitoring of the major phospholipid classes, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), and sphingomyelin (SM) in CSF. To emphasize the applicability of the method, a pilot study was performed on a group of CSF samples (N = 16) from individuals with probable AD and non-demented controls. We observed a statistically significant increase of SM levels (24.3 ± 2.4%) in CSF from probable AD individuals vs. controls. Our findings indicate that SM levels in the CSF could potentially provide a new lead in AD biomarker research, and show the potential of the method for disease-associated CSF phospholipid screening.

Haplotype and AGG interspersion analysis of FMR1 alleles in a Croatian population: no founder effect detected in patients with fragile X syndrome.

Several studies have suggested that fragile X syndrome (FRAXA), the most common inherited form of mental retardation, originated from a limited number of founder chromosomes. The aim of this study is to assess the genetic origin of fragile X syndrome in a Croatian population. We performed a haplotype analysis of the polymorphic loci DXS548 and FRAXAC1 in 18 unrelated fragile X and 56 control chromosomes. The AGG interspersion pattern of the FMR1 CGG repeat region was analyzed by sequencing. This is the first report on haplotype and AGG interspersion analysis of the fragile X syndrome gene in a Croatian population-the only eastern European population of Slavic origin analyzed so far. Our findings are intriguing, because they show a distinct distribution of the DXS548 and FRAXAC1 alleles in our fragile X population compared to other European fragile X populations. The DXS548/FRAXAC1 haplotype 194/154 (7-3), which is common among normal populations, was found to be the most frequent haplotype in our fragile X population as well. The AGG interspersion analysis indicated that AGG loss rather than haplotype may determine FMR1 allele instability. Our results suggest that no common ancestral X chromosome is associated with fragile X syndrome in the Croatian population studied. Further analysis of the origin of fragile X syndrome among other Slavic populations will be necessary to better define its eastern European distribution.

Oxidative stress parameters in plasma of Huntington's disease patients, asymptomatic Huntington's disease gene carriers and healthy subjects : a cross-sectional study.

BACKGROUND: Animal data and postmortem studies suggest a role of oxidative stress in the Huntington's disease (HD), but in vivo human studies have been scarce. AIM: To assess the presence of oxidative stress in HD patients and its occurrence relative to clinical symptoms. METHODS: Oxidative stress markers were determined in plasma of HD patients (n = 19), asymptomatic HD gene carriers (with > 38 CAG repeats) (n = 11) and their respective sex and agematched healthy controls (n = 47 and n = 22) in a cross-sectional study. RESULTS: With adjustment for age and sex, HD patients had higher plasma lipid peroxidation (LP) levels (ratio 1.20, 95% CI 1.09 to 1.32, p < 0.001) and lower reduced glutathione (GSH) levels (ratio 0.72, CI 0.55 to 0.94, p = 0.011) than their age and sex-matched controls. Although considerably younger, HD gene carriers did not differ from HD patients regarding LP and GSH levels, and had higher plasma LP (ratio 1.16, CI 1.02 to 1.32, p = 0.016) and lower GSH than their matched controls (ratio 0.73, CI 0.5 to 1.05). They had higher LP (ratio 1.18, CI 1.02 to 1.34, p = 0.019) and lower GSH (ratio 0.75, CI 0.51 to 1.11) than the healthy subjects matched to HD patients. CONCLUSIONS: Oxidative stress is more pronounced in HD patients and asymptomatic HD gene carriers than in healthy subjects. Differences in plasma LP and GSH are in line with the brain findings in animal models of HD. Data suggest that oxidative stress occurs before the onset of the HD symptoms.

Screening for fragile X syndrome: results from a school for mentally retarded children.

UNLABELLED: Fragile X syndrome is the most common inherited form of familial mental retardation. The purpose of this study was to identify yet unrecognized fragile X individuals and to estimate the frequency of both the FRAXA and FRAXE forms of the disease in a population of mentally retarded children attending a special school in Croatia. The results are reported of molecular screening of 114 children with mild to severe mental retardation. Three individuals (2.6%) with the FRAXA form of the fragile X syndrome and one boy (0.9%) with FRAXE mental retardation were detected; a total of four newly diagnosed fragile X families were identified. Closer clinical examination revealed that behavioural and speech disturbances were clearly present among all fragile X cases (both FRAXA and FRAXE), indicating that these features could be additional diagnostic criteria for the preselection of individuals at risk. CONCLUSION: Fragile X screening among mentally retarded children attending a special school should be highly encouraged to reveal the cause of mental retardation and to detect yet unrecognized fragile X individuals. The frequency of fragile X syndrome in a such population in Croatia was found to correlate with similar results from previous studies. However, since at the time of diagnosis all affected families had a second or even a third child born, earlier diagnosis should be considered to provide greater benefit to fragile X families.

A simple and rapid analysis of triplet repeat diseases by expand long PCR.

It is now known that 15 monogenic, mostly neurological, disorders are caused by the same type of mutations that occur in trinucleotide repeat sequences in certain genes. Since they share a nonspecific and variable phenotype, the accurate diagnosis could be made only by DNA analysis. We developed an Expand Long PCR assay that provides more reliable molecular diagnosis of such disorders. Its main characteristics are robust amplification of expanded alleles, simplicity, low cost and speed. We suggest the use of Expand Long PCR for routine molecular diagnosis of triplet repeat diseases, and present such analysis of the fragile X syndrome, myotonic dystrophy and Huntington's disease.

DNA analysis of the fragile X syndrome in an at risk pediatric population in croatia: simple clinical preselection criteria can considerably improve the cost-effectiveness of fragile X screening studies.

Advances in understanding the molecular basis of the fragile X syndrome, the most common cause of inherited mental retardation, have elicited new prospects for population-based studies identifying affected individuals and fragile X families, thus contributing in prevention of the disease. In comparison with numerous fragile X screening studies where unselected groups of individuals with mental retardation, developmental delay, learning disability or autistic-like behaviour had been observed, we performed fragile X analysis on clinically preselected individuals. The group we studied consisted of 108 children with mental retardation of unknown cause or positive family history who had at least one physical and/or behavioural characteristic often observed among fragile X individuals. A relative high frequency of the fragile X positive cases (13% overall, 17.3% in males) was detected, suggesting that simple preselection criteria can considerably increase the proportion of fragile X-positive cases, and therefore, improve the cost-effectiveness of fragile X testing. Retrospective clinical analysis using a simplified six-item fragile X checklist confirmed that scoring criteria can be used to additionally preselect individuals at risk. Our results also indicate that this syndrome is underdiagnosed in Croatia and that a further effort must be made to detect unrecognised cases.

Expand Long PCR for fragile X mutation detection.

Fragile X mutation detection by DNA analysis enables accurate diagnosis of the fragile X syndrome. The mutation involves the expansion of CGG repeats in the FMR1 gene and has been primarily detected by the Southern blotting method. In this study we present a novel, efficient and reliable PCR protocol that is more convenient for routine diagnosis of the fragile X syndrome. This method is based on the use of the Expand Long PCR System, which enables the amplification of normal, premutated and full-mutated alleles, and therefore provides complete CGG repeat analysis of the FMR1 gene. Normal alleles were easily detected by ethidium bromide staining of the agarose gels, suggesting that this assay could be used as a screening test for a large number of referrals. The amplified premutations and full mutations were identified by hybridization with a digoxigenin-labeled 5'-(CGG)5-3' probe, followed by chemiluminescent detection. The accuracy of our Expand Long PCR protocol was confirmed by Southern blot analysis, illustrating that the Expand Long PCR results concur with those of Southern blotting. In this paper we propose a new strategy for molecular diagnosis of the fragile X syndrome in which our Expand Long PCR assay is used as the first screening test for fragile X mutation detection.