Arcobacter spp. in raw milk from vending machines in Piedmont and occurrence of virulence genes in isolates.

Arcobacter spp. has been recognized as an emerging foodborne pathogen and a hazard to human health. In the dairy chain, it has been isolated from different sources, nevertheless data on Arcobacter occurrence in raw milk provided by vending machines are few. This study aimed to identify potentially pathogenic Arcobacter spp. in raw milk intended for human consumption sold through vending machines located in Piedmont. In an 8-month period, 37 raw milk samples were collected from 24 dairy farms: 12 (32,4%) were collected directly in farm from bulk tank milk and 25 (67,6%) from vending machines. Eight (21,6%) out of the 37 milk samples and 7 (29,2%) out of the 24 dairy farms were positive for Arcobacter spp. by culture examination. Four (16%) out of the 25 samples from vending machines and 4 (33,3%) out of the 12 samples from bulk tank milk were positive. All 8 isolates were identified as A. butzleri both by MALDI-TOF MS and multiplex end-point PCR. According to the detection of virulence genes, a total of four Patho-types were highlighted: 5 isolates in P-type 1 and only one isolate for each of the P-types 2-3-4. A. butzleri isolates carrying encoding virulence factors genes were isolated from raw milk intended for human consumption: these findings strengthen the compulsory consumption after boiling as required by current legislation and suggest the need of enlarging the analytical investigations to other microorganisms not yet included in the food safety criteria.

Modulation of in-membrane receptor clustering upon binding of multivalent ligands.

In living cells and biomimetic systems alike, multivalent ligands in solution can induce clustering of membrane receptors. The link between the receptor clustering and the ligand binding remains, however, poorly defined. Using minimalist divalent ligands, we develop a model that allows quantifying the modulation of receptor clustering by binding of ligands with any number of binding sites. The ligands, with weak binding affinity for the receptor and with binding sites held together by flexible linkers, lead to nearly quantitative clustering upon binding in a wide range of experimental conditions, showing that efficient modulation of receptor clustering does not require pre-organization or large binding affinities per binding site. Simulations show that, in the presence of ligands with five or more binding sites, an on/off clustering response follows a very small change in receptor density in the membrane, which is consistent with the highly cooperative behavior of multivalent biomolecular systems.

MLPA-based evidence for sequence gain: pitfalls in confirmation and necessity for exclusion of false positives.

Multiplex ligation-dependent probe amplification (MLPA) has become a standard method for identifying copy number mutations in diagnostic and research settings. The occurrence of false-positive deletion findings and the underlying causes are well recognized, whereas false-positive duplication/amplification findings have not been appreciated so far. We here present three pertinent cases which were only identified on extended, nonstandard secondary analyses. We also offer and experimentally validate a potential explanation. Our findings imply that MLPA data indicating gain of genomic sequence require validation on an independent sample or by an independent method.

Mitochondrial DNA polymorphisms/haplogroups in hereditary spastic paraplegia.

Mitochondrial dysfunction could contribute to the development of spastic paraplegia. Among others, two of the genes implicated in hereditary spastic paraplegia encoded mitochondrial proteins and some of the clinical features frequently found in these patients resemble those observed in patients with mitochondrial DNA (mtDNA) mutations. We investigated the association between common mtDNA polymorphisms and spastic paraplegia. The ten mtDNA polymorphisms that defined the common European haplogroups were determined in 424 patients, 19% with a complicated phenotype. A rare haplogroup was associated with the disease in patients without a SPG3A, SPG4, or SPG7 mutation. Allele 10398G was more frequent among patients with a pure versus complicated phenotype. This mtDNA polymorphism was previously associated with the risk of developing other neurodegenerative diseases. In conclusion, some mtDNA polymorphisms could contribute to the development of spastic paraplegia or act as modifiers of the phenotype.

Spinocerebellar ataxia type 15: diagnostic assessment, frequency, and phenotypic features.

BACKGROUND: To guide time- and cost-efficient analyses of the increasing number of autosomal-dominant spinocerebellar ataxia genes (SCAs), more information about frequency distributions, phenotypic characteristics and optimal diagnostic strategies is warranted. OBJECTIVE: To assess the prevalence and phenotypic spectrum of SCA15 and to confirm multiplex ligation-dependent probe amplification (MLPA) as a robust and efficient strategy for routine molecular diagnosis. METHODS: Fifty-six German SCA families negative for common repeat expansions were screened for ITPR1 deletions by MLPA. Samples with conspicuous MLPA data were additionally assessed by high-density single nucleotide polymorphism (SNP) array to confirm MLPA results and further determine the size of deletions. The phenotype of patients harbouring ITPR1 deletions was characterised by standardised clinical, electrophysiological and imaging assessment. RESULTS: SCA15 accounted for 8.9% (5/56) of SCA families negative for common SCA repeat expansions. All deletions detected by MLPA were confirmed by SNP array. One of the ITPR1 deletions preserved exons 1 and 2 in the 5' prime UTR of the ITPR1 gene. All patients with SCA15 (n=10) presented with slowly progressive cerebellar ataxia and vermal cerebellar atrophy, while clinical and electrophysiological signs of extracerebellar affection were mild and more variable. CONCLUSIONS: SCA15 is the most common non-trinucleotide repeat SCA in Central Europe. Screening for ITPR1 deletions should be considered in patients with slowly progressive SCA, vermal cerebellar atrophy and prominent tremor after excluding common SCA repeat expansions. Promoter and exon 2 of ITPR1 may be preserved from the deletion in some cases of SCA15.

Mutational spectrum of the SPG4 (SPAST) and SPG3A (ATL1) genes in Spanish patients with hereditary spastic paraplegia.

BACKGROUND: Hereditary Spastic Paraplegias (HSP) are characterized by progressive spasticity and weakness of the lower limbs. At least 45 loci have been identified in families with autosomal dominant (AD), autosomal recessive (AR), or X-linked hereditary patterns. Mutations in the SPAST (SPG4) and ATL1 (SPG3A) genes would account for about 50% of the ADHSP cases. METHODS: We defined the SPAST and ATL1 mutational spectrum in a total of 370 unrelated HSP index cases from Spain (83% with a pure phenotype). RESULTS: We found 50 SPAST mutations (including two large deletions) in 54 patients and 7 ATL1 mutations in 11 patients. A total of 33 of the SPAST and 3 of the ATL1 were new mutations. A total of 141 (31%) were familial cases, and we found a higher frequency of mutation carriers among these compared to apparently sporadic cases (38% vs. 5%). Five of the SPAST mutations were predicted to affect the pre-mRNA splicing, and in 4 of them we demonstrated this effect at the cDNA level. In addition to large deletions, splicing, frameshifting, and missense mutations, we also found a nucleotide change in the stop codon that would result in a larger ORF. CONCLUSIONS: In a large cohort of Spanish patients with spastic paraplegia, SPAST and ATL1 mutations were found in 15% of the cases. These mutations were more frequent in familial cases (compared to sporadic), and were associated with heterogeneous clinical manifestations.

Unique spectrum of SPAST variants in Estonian HSP patients: presence of benign missense changes but lack of exonic rearrangements.

BACKGROUND: Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous disorder that can be an autosomal-dominant, autosomal-recessive, or X-linked disease. The most common autosomal-dominant form of the disease derives from mutations in the SPAST gene. METHODS: The aim of this study was to analyze 49 patients diagnosed with HSP from the Estonian population for sequence variants of the SPAST gene and to describe the associated phenotypes. Healthy control individuals (n = 100) with no family history of HSP were also analyzed. All patient samples were screened using denaturing high performance liquid chromatography (DHPLC) and multiplex ligation-dependent probe amplification (MLPA) assay. Samples with abnormal DHPLC and MLPA profiles were sequenced, with the same regions sequenced in control samples. RESULTS: Sequence variants of SPAST were identified in 19/49 HSP patients (38.8%), twelve among them had pathogenic mutations. Within the latter group there was one sporadic case. Eight patients had pure, and four - complex HSP. The twelve variants were identified: seven pathogenic (c.1174-1G>C, c.1185delA, c.1276C>T, c.1352_1356delGAGAA, c.1378C>A, c.1518_1519insTC, c.1841_1842insA) and five non-pathogenic (c.131C>T, c.484G>A, c.685A>G, c.1245+202delG, c.1245+215G>C). Only 2 of these mutations had previously been described (c.131C>T, c.1245+202delG). Three mutations, c.1174-1G>C, c.1276 C>T, c.1378C>A, showed intrafamilial segregation. CONCLUSION: This study identified new variants of the SPAST gene which included benign missense variants and short insertions/deletions. No large rearrangements were found. Based on these data, 7 new pathogenic variants of HSP are associated with clinical phenotypes.

Mutational screening of the mortalin gene (HSPA9) in Parkinson's disease.

Mortalin is a mitochondrial chaperone of the heat shock protein 70 family. Mortalin plays a central role in mitochondrial biogenesis through its capacity to direct the import of nuclear-encoded proteins into the mitochondria. As mitochondrial dysfunction has been involved in Parkinson's disease (PD), changes in mortalin function and expression could manifest as a higher risk of developing PD. In agreement with this, mortalin expression was decreased in the mitochondrial fraction of neurons from the substantia nigra of PD patients. We hypothesised that DNA variants in the mortalin gene (HSPA9) could contribute to the risk of developing PD. We analysed the 17 HSPA9 coding exons in 330 PD patients and 250 controls. In addition to several polymorphisms, found in patients and controls, three variants were found in 3 patients but none of the controls: two missense (R126 > W and P509 > S) and a 17 bp insertion in intron 8 (predicted to affect RNA splicing). Our study suggests that putative mutations in the mortalin, although rare, could contribute to the risk of developing PD.

Mutational screening of the mitochondrial transcription factors B1 and B2 (TFB1M and TFB2M) in Parkinson's disease.

Mitochondrial dysfunction has been implicated in Parkinson's disease (PD). The nuclear encoded transcription factors A, B1 and B2 are essential for mitochondrial DNA replication. Sequence variants at the genes encoding TFAM, TFB1M and TFB2M could contribute to the risk of developing PD. Here, we searched for TFB1M and TFB2M nucleotide variants in a cohort of PD-patients (n=300) and healthy controls (n=200) from Spain. Single strand conformation analysis and direct sequencing were used to determine the variation at all the coding exons of the two genes. In addition to previously reported polymorphisms, we found several rare variants in patients and controls. Allele frequencies for all the nucleotide changes did not differ between patients and controls. Our work suggests that DNA variants in TFB1M and TFB2M did not contribute to the risk for PD in our population.

Mitochondrial transcription factor A (TFAM) gene variation and risk of late-onset Alzheimer's disease.

Impaired mitochondrial function and an increased number of mutations in mitochondrial DNA (mtDNA) has been found in brains of patients with late-onset Alzheimer's disease (LOAD). The TFAM-gene encodes the mitochondrial transcription factor A, a protein that controls the transcription, replication, damage sensing, and repair of mtDNA. TFAM is on human chromosome region 10q21.1, where a locus for LOAD has been mapped. Our objective was to determine the role of TFAM-gene variation in the risk of LOAD. The seven TFAM coding exons were analysed through single strand conformation analysis and direct sequencing in a cohort of Spanish LOAD-patients and healthy controls. We found four common polymorphisms, two in the flanquing intronic and two in the coding sequences. Polymorphism rs1937 (+35 G/C) was the only missense change (S12T). Genotyping of this polymorphism in 300 LOAD-patients and 183 healthy controls showed a significantly higher frequency of GG-homozygotes in the patients (92% vs. 86%; p=0.04; OR=1.91, 95%CI=1.02-3.50). This suggests that S12 is a risk factor for LOAD in our population. In conclusion, rare variants (mutations) in the TFAM gene were not found in LOAD-patients, but the S12T polymorphism was a moderate risk factor for LOAD in our population.

Mitochondrial transcription factor A (TFAM) gene variation in Parkinson's disease.

Mitochondrial function is necessary to supply the energy required for cell metabolism. Mutations/polymorphisms in mitochondrial DNA (mtDNA) have been implicated in Parkinson's disease (PD). The mitochondrial transcription factor A (TFAM) controls the transcription of mtDNA and regulates the mtDNA-copy number, thus being important for maintaining ATP production. TFAM dysfunction may also be involved in PD, and TFAM gene mutations/polymorphisms could contribute to the risk of developing PD. We searched for gene variants in the seven TFAM-exons in a total of 250 PD-patients. We found five common polymorphisms, and only one was a missense change (S12T in exon 1). Genotype and allele frequencies did not differ between patients and healthy controls (n=225) for the five polymorphisms. Our work suggests that TFAM-variants did not contribute to the risk of developing PD.

No association between Parkinson's disease and three polymorphisms in the eNOS, nNOS, and iNOS genes.

Nitric oxide synthases (NOS) and mitochondrial DNA-polymorphisms have been associated with the risk of developing Parkinson's disease (PD). In this report, we genotyped 450 PD-patients and 200 controls for three polymorphisms in the endothelial, inducible and neuronal NOS-genes, and for the T4336C and A10398G mitochondrial DNA-polymorphisms. None of the eNOS (intron 4 VNTR), iNOS (exon 22 A/G), or nNOS (exon 29T/C) were significantly associated with PD. Mitochondrial 4336C increased the PD-risk among women (OR=6.13), while the 10398G had a protective effect (OR=0.52). We did not find significantly interactions between the NOS and mitochondrial polymorphisms in the risk for PD in our population.