Journal of Molecular Biomarkers & Diagnosis

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) has become an important tool to complement traditional methodologies used in the clinical chemistry laboratory. In early 1970s, majority of the mass spectrometric analyses of clinical samples utilized the application of gas chromatography/mass spectrometry (GC/MS) which is particularly suitable for analytes that are small molecules and volatile. However, many biomolecules have high molecular mass, are thermolabile and/ or extremely polar. Sample extraction and derivatization required for GC/MS analysis of clinical samples is usually extensive, complicated as well as laborious. Not only such sample preparation is difficult, quite often it is not possible to perform on certain analytes of interest. On the other hand, LC/MS can be easily performed on biological samples (primarily body fluids such as blood, urine, cerebrospinal fluid) which are often extremely hydrophilic and include compounds with a wide range of molecular masses such as amino acids, fatty acids, bile acids that are less than 500 Da as well as peptides, proteins, glycoproteins and oligonucleotides with high molecular masses.

Abstract

Background:Estrogens, acting through their cellular receptors namely alpha and beta, have a role in the development of systemic lupus erythematosus (SLE).

Objectives: To investigate whether polymorphisms of ESR1 and ESR2 genes is related to the susceptibility of juvenile (jSLE) and adult Systemic lupus erythematosus (aSLE) and to detect their association with clinical and laboratory characteristics of the disease.

Methods: Genomic DNA was extracted from 32 adult onset SLE (aSLE), 33 juvenile onset SLE (jSLE) and 60 age and gender matched controls. Genotyping of ESR1 and ESR2 was done using the restriction fragment length polymorphism (RFLP) and tetra primer ARM-PCR methods respectively.

Results: There was a statistically significant difference in the genetic polymorphisms of ESR2 between the two studied groups (aSLE, jSLE) and the control group as regards the homomutant AA genotype (OR:0.058, p value: 0.000) and the A allele(OR: 0.195, p value:0.007) in case of aSLE, and in homomutant AA genotype (OR:0.269, p value:0.017) and the A allele (OR: 0.397, p value: 0.003) in case of jSLE but the study could not find any statistically significant difference in the genetic polymorphisms of ESR1 between the control and the two groups.

Conclusion: This study revealed that ESR1 genetic polymorphism is not genetic risk or protective factor for neither aSLE nor jSLE susceptibility, but ESR2 genetic polymorphism is reported as protective factor for aSLE and jSLE among our studied population. Certain alleles are associated with certain clinical and laboratory parameters.

Introduction: Approximately 25% of non-small cell lung cancer (NSCLC) tumors contain mutations in KRAS. These tumors are insensitive to therapy directed against the epidermal growth factor receptor and appear to be resistant to adjuvant chemotherapy. The current study demonstrates the performance of the cobas® KRAS Mutation Test (cobas test), a TaqMelt polymerase chain reaction (PCR) assay designed to detect 19 mutations in codons 12, 13, and 61.

Methods: To reflect real-world testing conditions, the study used formalin-fixed, paraffin-embedded tissue (FFPET) NSCLC samples for the predominant mutations. DNA blends of cell lines and plasmids were used where FFPET samples were not available.

Results: In the limit of detection study, a correct mutation call rate of ≥95% was obtained with approximately 5% mutant sequences using 3.1-50.0 ng DNA per PCR reaction. Mutation levels as low as 2.4% consistently yielded correct mutation calls when 50.0 ng DNA was used for testing. The cobas test performance was compared to Sanger sequencing in a method correlation using two cobas reagent lots and 194 specimens. After resolution of discordant results using 454 sequencing, the positive, negative, and overall percent agreement for mutations in codons 12/13 and 61 between the cobas test and sequencing ranged from 97.0% to 100%. Mutation detection was 100% reproducible and showed greater specificity than Sanger sequencing. Test performance was not impaired by the presence of interfering substances or clinically relevant microbes, and inclusivity testing demonstrated the kit’s ability to detect rare mutations.

Conclusions: The cobas test is a robust, sensitive, and reproducible method for detecting KRAS mutations in FFPET tumor samples from NSCLC patients.

Background: Polycystic ovary syndrome (PCOS) is the most common metabolic abnormality such as changes in lipid profile, diabetes, hypertension and metabolic syndrome occurring in young women of reproductive age. Low vitamin D levels were found to be associated with the development of obesity and insulin resistance in women with PCOS. Variants on vitamin D receptor (VDR) gene have also been related to metabolic comorbidities in general population.

Aim: The aim of this case-control study was to investigate whether the VDR gene polymorphisms are associated with susceptibility to PCOS.

Methods: Women with PCOS and a control group, all aged 16-40 years, were enrolled. Genotyping of VDR Fok-I (rs2228570), VDR Apa-I (rs7975232) as well as GC (rs2282679), DHCR7 (rs12785878) SNPs between groups were determined by using direct sequencing. Serum 25-hydroxyvitamin D [25(OH)] levels were measured by ELISA.

Results: Mean serum 25(OH)D in the PCOS and control samples were 19.08 ± 7 and 23.27 ± 6.03 (p=0.048) which were significantly lower in PCOS patients compared with controls. CC genotype of the VDR Apa-I SNP was same frequent in PCOS (25.6%) and controls (25.6%) (OR: 0.9995; 95%CI: 0.528 to 1.8921; p=0.9987). The CC genotype was also significantly associated with lower E2 (p=0.031) and androstenedione levels (p=0.026). We observed a significant association of GC polymorphismwith 25(OH)D levels. PCOS women carrying the GG genotype (in GC genes) had significantly higher risk for vitamin D deficiency than women carrying the TT genotype.

Conclusions: In conclusion, data from this study indicate that vitamin D levels are lower, and vitamin D deficiency more frequent, in PCOS than in controls. The present findings suggest that the Apa-I, Fok-I polymorphism of the VDR gene is associated with PCOS and seems to modulate ovarian steroid secretion. Further studies are needed to better clarify the biological mechanisms by which the polymorphism influences PCOS risk.