Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

BACKGROUND: Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity. METHODOLOGY/PRINCIPAL FINDINGS: We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models. CONCLUSIONS/SIGNIFICANCE: These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity

BRIP1 (BACH1) variants and familial breast cancer risk: a case-control study

<p>Abstract</p> <p>Background</p> <p>Inactivating and truncating mutations of the nuclear BRCA1-interacting protein 1 (BRIP1) have been shown to be the major cause of Fanconi anaemia and, due to subsequent alterations of BRCA1 function, predispose to breast cancer (BC).</p> <p>Methods</p> <p>We investigated the effect of BRIP1 -64G>A and Pro919Ser on familial BC risk by means of TaqMan allelic discrimination, analysing <it>BRCA1/BRCA2 </it>mutation-negative index patients of 571 German BC families and 712 control individuals.</p> <p>Results</p> <p>No significant differences in genotype frequencies between BC cases and controls for BRIP1 -64G>A and Pro919Ser were observed.</p> <p>Conclusion</p> <p>We found no effect of the putatively functional BRIP1 variants -64G>A and Pro919Ser on the risk of familial BC.</p

BACH1 Ser919Pro variant and breast cancer risk

BACKGROUND: BACH1 (BRCA1-associated C-terminal helicase 1; also known as BRCA1-interacting protein 1, BRIP1) is a helicase protein that interacts in vivo with BRCA1, the protein product of one of the major genes for hereditary predisposition to breast cancer. Previously, two BACH1 germ line missense mutations have been identified in early-onset breast cancer patients with and without family history of breast and ovarian cancer. In this study, we aimed to evaluate whether there are BACH1 genetic variants that contribute to breast cancer risk in Finland. METHODS: The BACH1 gene was screened for germ line alterations among probands from 43 Finnish BRCA1/2 negative breast cancer families. Recently, one of the observed common variants, Ser-allele of the Ser919Pro polymorphism, was suggested to associate with an increased breast cancer risk, and was here evaluated in an independent, large series of 888 unselected breast cancer patients and in 736 healthy controls. RESULTS: Six BACH1 germ line alterations were observed in the mutation analysis, but none of these were found to associate with the cancer phenotype. The Val193Ile variant that was seen in only one family was further screened in an independent series of 346 familial breast cancer cases and 183 healthy controls, but no additional carriers were observed. Individuals with the BACH1 Ser919-allele were not found to have an increased breast cancer risk when the Pro/Ser heterozygotes (OR 0.90; 95% CI 0.70–1.16; p = 0.427) or Ser/Ser homozygotes (OR 1.02; 95% CI 0.76–1.35; p = 0.91) were compared to Pro/Pro homozygotes, and there was no association of the variant with any breast tumor characteristics, age at cancer diagnosis, family history of cancer, or survival. CONCLUSION: Our results suggest that the BACH1 Ser919 is not a breast cancer predisposition allele in the Finnish study population. Together with previous studies, our results also indicate that although some rare germ line variants in BACH1 may contribute to breast cancer development, the contribution of BACH1 germline alterations to familial breast cancer seems marginal

Mutation analysis of the Fanconi anaemia A gene in breast tumours with loss of heterozygosity at 16q24.3

The recently identified Fanconi anaemia A (FAA) gene is located on chromosomal band 16q24.3 within a region that has been frequently reported to show loss of heterozygosity (LOH) in breast cancer. FAA mutation analysis of 19 breast tumours with specific LOH at 16q24.3 was performed. Single-stranded conformational polymorphism (SSCP) analysis on cDNA and genomic DNA, and Southern blotting failed to identify any tumour-specific mutations. Five polymorphisms were identified, but frequencies of occurrence did not deviate from those in a normal control population. Therefore, the FAA gene is not the gene targeted by LOH at 16q24.3 in breast cancer. Another tumour suppressor gene in this chromosomal region remains to be identified. © 1999 Cancer Research Campaig

Analysis of SLX4/FANCP in non-BRCA1/2-mutated breast cancer families

<p>Abstract</p> <p>Background</p> <p>Genes that, when mutated, cause Fanconi anemia or greatly increase breast cancer risk encode for proteins that converge on a homology-directed DNA damage repair process. Mutations in the <it>SLX4 </it>gene, which encodes for a scaffold protein involved in the repair of interstrand cross-links, have recently been identified in unclassified Fanconi anemia patients. A mutation analysis of <it>SLX4 </it>in German or Byelorussian familial cases of breast cancer without detected mutations in <it>BRCA1 </it>or <it>BRCA2 </it>has been completed, with globally negative results.</p> <p>Methods</p> <p>The genomic region of <it>SLX4</it>, comprising all exons and exon-intron boundaries, was sequenced in 94 Spanish familial breast cancer cases that match a criterion indicating the potential presence of a highly-penetrant germline mutation, following exclusion of <it>BRCA1 </it>or <it>BRCA2 </it>mutations.</p> <p>Results</p> <p>This mutational analysis revealed extensive genetic variation of <it>SLX4</it>, with 21 novel single nucleotide variants; however, none could be linked to a clear alteration of the protein function. Nonetheless, genotyping 10 variants (nine novel, all missense amino acid changes) in a set of controls (138 women and 146 men) did not detect seven of them.</p> <p>Conclusions</p> <p>Overall, while the results of this study do not identify clearly pathogenic mutations of <it>SLX4 </it>contributing to breast cancer risk, further genetic analysis, combined with functional assays of the identified rare variants, may be warranted to conclusively assess the potential link with the disease.</p

Developmentally Regulated Sphingolipid Degradation in Leishmania major

Leishmania parasites alternate between extracellular promastigotes in sandflies and intracellular amastigotes in mammals. These protozoans acquire sphingolipids (SLs) through de novo synthesis (to produce inositol phosphorylceramide) and salvage (to obtain sphingomyelin from the host). A single ISCL (Inositol phosphoSphingolipid phospholipase C-Like) enzyme is responsible for the degradation of both inositol phosphorylceramide (the IPC hydrolase or IPCase activity) and sphingomyelin (the SMase activity). Recent studies of a L. major ISCL-null mutant (iscl−) indicate that SL degradation is required for promastigote survival in stationary phase, especially under acidic pH. ISCL is also essential for L. major proliferation in mammals. To further understand the role of ISCL in Leishmania growth and virulence, we introduced a sole IPCase or a sole SMase into the iscl− mutant. Results showed that restoration of IPCase only complemented the acid resistance defect in iscl− promastigotes and improved their survival in macrophages, but failed to recover virulence in mice. In contrast, a sole SMase fully restored parasite infectivity in mice but was unable to reverse the promastigote defects in iscl−. These findings suggest that SL degradation in Leishmania possesses separate roles in different stages: while the IPCase activity is important for promastigote survival and acid tolerance, the SMase activity is required for amastigote proliferation in mammals. Consistent with these findings, ISCL was preferentially expressed in stationary phase promastigotes and amastigotes. Together, our results indicate that SL degradation by Leishmania is critical for parasites to establish and sustain infection in the mammalian host

Functional Role of the Polymorphic 647 T/C Variant of ENT1 (SLC29A1) and Its Association with Alcohol Withdrawal Seizures

Adenosine is involved in several neurological and behavioral disorders including alcoholism. In cultured cell and animal studies, type 1 equilibrative nucleoside transporter (ENT1, slc29a1), which regulates adenosine levels, is known to regulate ethanol sensitivity and preference. Interestingly, in humans, the ENT1 (SLC29A1) gene contains a non-synonymous single nucleotide polymorphism (647 T/C; rs45573936) that might be involved in the functional change of ENT1. Our functional analysis showed that prolonged ethanol exposure increased adenosine uptake activity of mutant cells (ENT1-216Thr) compared to wild-type (ENT1-216Ile) transfected cells, which might result in reduced extracellular adenosine levels. We found that mice lacking ENT1 displayed increased propensity to ethanol withdrawal seizures compared to wild-type littermates. We further investigated a possible association of the 647C variant with alcoholism and the history of alcohol withdrawal seizures in subjects of European ancestry recruited from two independent sites. Analyses of the combined data set showed an association of the 647C variant and alcohol dependence with withdrawal seizures at the nominally significant level. Together with the functional data, our findings suggest a potential contribution of a genetic variant of ENT1 to the development of alcoholism with increased risk of alcohol withdrawal-induced seizures in humans