Discovery of phosphatidylcholines and sphingomyelins as biomarkers for ovarian endometriosis.

BACKGROUND: Current non-invasive diagnostic methods for endometriosis lack sensitivity and specificity. In search for new diagnostic biomarkers for ovarian endometriosis, we used a hypothesis-generating targeted metabolomics approach. METHODS: In a case-control study, we collected plasma of study participants and analysed their metabolic profiles. We selected a group of 40 patients with ovarian endometriosis who underwent laparoscopic surgery and a control group of 52 healthy women who underwent sterilization at the University Clinical Centre Ljubljana, Slovenia. Over 140 targeted analytes included glycerophospholipids, sphingolipids and acylcarnitines. The analytes were quantified by electrospray ionization tandem mass spectrometry. For assessing the strength of association between the metabolite or metabolite ratios and the disease, we used crude and adjusted odds ratios. A stepwise logistic regression procedure was used for selecting the best combination of biomarkers. RESULTS: Eight lipid metabolites were identified as endometriosis-associated biomarkers due to elevated levels in patients compared with controls. A model containing hydroxysphingomyelin SMOH C16:1 and the ratio between phosphatidylcholine PCaa C36:2 to ether-phospholipid PCae C34:2, adjusted for the effect of age and the BMI, resulted in a sensitivity of 90.0%, a specificity of 84.3% and a ratio of the positive likelihood ratio to the negative likelihood ratio of 48.3. CONCLUSIONS: Our results suggest that endometriosis is associated with elevated levels of sphingomyelins and phosphatidylcholines, which might contribute to the suppression of apoptosis and affect lipid-associated signalling pathways. Our findings suggest novel potential routes for therapy by specifically blocking highly up-regulated isoforms of phosphpolipase A2 and lysophosphatidylcholine acyltransferase 4.

Aldo-keto reductases AKR1C1, AKR1C2 and AKR1C3 may enhance progesterone metabolism in ovarian endometriosis.

Endometriosis is a very common disease that is characterized by increased formation of estradiol and disturbed progesterone action. This latter is usually explained by a lack of progesterone receptor B (PR-B) expression, while the role of pre-receptor metabolism of progesterone is not yet fully understood. In normal endometrium, progesterone is metabolized by reductive 20α-hydroxysteroid dehydrogenases (20α-HSDs), 3α/ß-HSDs and 5α/ß-reductases. The aldo-keto reductases 1C1 and 1C3 (AKR1C1 and AKR1C3) are the major reductive 20α-HSDs, while the oxidative reaction is catalyzed by 17ß-HSD type 2 (HSD17B2). Also, 3α-HSD and 3ß-HSD activities have been associated with the AKR1C isozymes. Additionally, 5α-reductase types 1 and 2 (SRD5A1, SRD5A2) and 5ß-reductase (AKR1D1) are responsible for the formation of 5α- and 5ß-reduced pregnanes. In this study, we examined the expression of PR-AB and the progesterone metabolizing enzymes in 31 specimens of ovarian endometriosis and 28 specimens of normal endometrium. Real-time PCR analysis revealed significantly decreased mRNA levels of PR-AB, HSD17B2 and SRD5A2, significantly increased mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1, and negligible mRNA levels of AKR1D1. Immunohistochemistry staining of endometriotic tissue compared to control endometrium showed significantly lower PR-B levels in epithelial cells and no significant differences in stromal cells, there were no significant differences in the expression of AKR1C3 and significantly higher AKR1C2 levels were seen only in stromal cells. Our expression analysis data at the mRNA level and partially at the cellular level thus suggest enhanced metabolism of progesterone by SRD5A1 and the 20α-HSD and 3α/ß-HSD activities of AKR1C1, AKR1C2 and AKR1C3.

Combinations of genetic changes in the human cAMP-responsive element modulator gene: a clue towards understanding some forms of male infertility?

The cAMP-responsive element modulator (CREM) gene plays a pivotal role in the mouse spermatogenesis, but its role in the human infertility has not been fully established. We performed a mutation screening in 13 Slovenian men with round spermatid arrest and in six controls. Eleven genetic changes have been identified in the human CREM gene, three novel single-nucleotide polymorphisms [within the promoters P1, P3 and intervening sequence 1 (IVS1)], one insertion (IVS2) and one non-sense mutation (exon gamma). Some infertile patients seem to accumulate potentially harmful genetic changes. We identified a patient with no CREM immunoreactive protein that was homozygous for the nucleotide changes in all promoters, IVS 1, 2, 6, and was heterozygous for the mutation in exon gamma. Interestingly, insertion in IVS2 (IVS2-58_55insT) results in a four-fold decrease in binding of nuclear proteins. Computer predictions suggested the presence of a potential novel CREM promoter, however, random amplification of cDNA ends from the human testis cDNA library was not successful in confirming a novel transcription start site of the CREM gene. Screening of a larger number of patients and controls is required to elucidate whether the observed combinations of genetic changes in the CREM gene can explain some forms of male infertility.

Mutations in the hMLH1 gene in Slovenian patients with gastric carcinoma.

Alterations of multiple oncogenes and tumor suppressor genes, together with genetic instability, are responsible for carcinogenesis in gastric cancer. The microsatellite mutator phenotype is the cause of many somatic frameshift and point mutations in non-coding repetitive sequences and in coding regions associated with cell proliferation and apoptosis. Genetic mutations in hMLH1 and transcriptional silencing of its promoter by hypermethylation lead to the inactivation of the mismatch repair system. In our study, we screened for mutations the hMLH1 gene in patients expressing the microsatellite instability genotype by using single-strand conformational polymorphism analysis and direct sequencing. Seven changes were identified; of these, three (A92P, E433Q, and K618A) were germline mutations and the other four (IVS5 453 + 79 A > G, I219V, 1039 - 7 del (T)(n), and IVS15 1668 - 19 A > G) germline polymorphisms. A92P and E433Q are novel, previously unidentified mutations. In addition, we found a rather complex distribution of mutations and polymorphisms in individual patients and in two cases also a methylated hMLH1 promoter.

Characterization of the mouse lanosterol 14alpha-demethylase (CYP51), a new member of the evolutionarily most conserved cytochrome P450 family.

Genes encoding sterol 14alpha-demethylases in eukaryotes and in Mycobacterium belong to the CYP51 family which is evolutionary the most conserved gene family within the cytochrome P450 superfamily. We have characterized a new member of this family, the mouse lanosterol 14alpha-demethylase, with the aim to study the in vivo role of this gene in spermatogenesis in mammals. The amino acid sequence of mouse Cyp51 is 96% identical to rat and 91% to human. Comparison of all known CYP51 proteins by the neighbor-joining method suggests that fungal and animal CYP51 genes arose from a common ancestral gene (98.3% probability) and interestingly, that plant and bacterial CYP51 genes share a common progenitor (88.8% probability). This suggests that the first CYP51 gene may have arisen in eukaryotes and has been transferred horizontally from plants to Mycobacterium. The mouse CYP51 gene is approximately 17-kb long and contains 10 exons. Transcription starts at several locations within the CpG island, which is characteristic for the TATA-less housekeeping genes. The mouse 5'-untranslated region (800 bp) contains putative cAMP-responsive elements (CRE), sterol regulatory elements (SRE) and GC-boxes at positions similar to human and rat, suggesting an evolutionary conserved mechanism of CYP51 transcriptional regulation in mammals. The mouse Cyp51 gene resides on chromosome 5, region A2, close to the centromere. No signals outside this region were detected as well as no evidence of processed pseudogenes using long PCR was found. This indicates that the mouse genome most likely lacks CYP51 processed pseudogenes.

K5 D328E: a novel missense mutation in the linker 12 domain of keratin 5 associated with epidermolysis bullosa simplex (Weber-Cockayne).

A novel missense mutation was detected in the L12 region of keratin 5 (K5) in a Slovene family diagnosed with a Weber-Cockayne variant of epidermolysis bullosa simplex (EBS). Direct sequencing identified a heterozygous GAC to GAA substitution altering codon 328 of K5 from Asp to Glu in all affected family members, while no mutation was observed either in the healthy individual or the 50 unrelated control samples. Asp(328) of K5 (position 12 in the L12 domain) is remarkably conserved among all type II keratins. K5 L12:D12E is the third mutation found to affect this residue in K5-related EBS, indicating the importance of Asp(328) for K5 structure and the dramatic effect that fine changes can have on keratin intermediate filament integrity.

Mutational analysis of 30 Slovenian cystic fibrosis patients compared to known Slovenian and European CF mutation spectra.

More than 800 mutations have been indentified in the CFTR gene. This vast mutation diversity makes the search for molecular defects in cystic fibrosis difficult. Out of 100 Slovenian CF families, we have screened 30, using DGGE and SSCP as mutation detection techniques, while the remaining 70 have been studied previously. Together our and the previous studies have been able to indentify 18 CF mutations which cover 77.6% of the CF alleles in those families. The relative frequency of deltaF508 is 62.7% which is significantly higher than the average reported for the Mediterranean South European region (51.6%). At the same time, significant differences in mutation frequencies were found for the G542X, R1162X, W1282X, N1303K and 3905insT mutations. Several, otherwise rare mutations have been detected, such as: I148T, Q552X, 457TAT-->G, R1006H, 2907delTT, 3667ins4, A559T and G576A. An interesting fact is that A559T was so far found mostly in CF patients of African-American origin. These results imply that a high heterogeneity of CF mutations occurs within the small population of Slovenia, consisting only of 2 million inhabitants. In view of the spectrum and frequencies of detected mutations, Slovenian population expresses characteristics of Mediterranean and central European countries, and at the same time shows also distinctive differences and unique region specific CF mutations (Q685X, D192G, S4X).

Screen of 55 Slovenian haemophilia A patients: identification of 2 novel mutations (S-1R and IVS23+1G-->A) and discussion of mutation spectrum. Mutation in brief no. 241. Online.

Using polymerase chain reaction, single-stranded conformational polymorphism (SSCP), TaqI restriction analysis and direct sequencing, exons 1, 7, 8, 9, 12, 13, 14, 18, 22, 23, 24, and 26 of the factor VIII gene were screened for point mutations in 55 Slovenian haemophilia A patients. In eighteen patients eleven different mutations were found; one (in six patients) in exon 26, one (in two patients) in exon 24, two in exon 23, one in intron 23, one in exon 18, one in exon 12, one in exon 8, two (1 + 1 in two patients) in exon 7 and one in exon 1. Of the mutations detected one has recently been reported by us (Q602X), and two are novel; S-1R in exon 1 and IVS23+1G-->A in intron 23.