Novel intragenic deletion within the FXN gene in a patient with typical phenotype of Friedreich ataxia: may be more prevalent than we think?

BACKGROUND: Friedreich ataxia is the most common inherited ataxia in Europe and is mainly caused by biallelic pathogenic expansions of the GAA trinucleotide repeat in intron 1 of the FXN gene that lead to a decrease in frataxin protein levels. Rarely, affected individuals carry either a large intragenic deletion or whole-gene deletion of FXN on one allele and a full-penetrance expanded GAA repeat on the other allele. CASE PRESENTATION: We report here a patient that presented the typical clinical features of FRDA and genetic analysis of FXN intron 1 led to the assumption that the patient carried the common biallelic expansion. Subsequently, parental sample testing led to the identification of a novel intragenic deletion involving the 5'UTR upstream region and exons 1 and 2 of the FXN gene by MLPA. CONCLUSIONS: With this case, we want to raise awareness about the potentially higher prevalence of intragenic deletions and underline the essential role of parental sample testing in providing accurate genetic counselling.

Relation of the Serum Levels of DKK-1 and Osteoprotegerin with Bone Mass in Tightly Controlled Rheumatoid Arthritis.

OBJECTIVE: To analyze the association between serum levels of osteoprotegerin (OPG) and Dickkopf-related protein 1 (DKK-1) and the annual percent change (Δ%) in bone mineral density (BMD) in patients with tightly controlled rheumatoid arthritis (RA). METHODS: Observational mixed-study. RA patients followed-up with a tight-control strategy were included. Bone densitometries were performed at baseline (T0) and follow-up (T1) and serum levels of OPG and DKK-1 were measured by ELISA also in T0 and T1; additional clinical variables included disease activity measures, and treatment for RA and osteoporosis. Descriptive bivariate and multivariate analyses, stratified by gender, were performed. RESULTS: We included 97 RA patients (70% female, with a mean age of 53 years, and 76% with low activity by DAS28); 95% were treated with DMARDs and 37% with anti-osteoporotic drugs. Mean time between T0 and T1 was 2.7 years. Most patients had their BMD improved. The mean Δ%BMD was +0.42% for lumbar spine, +0.15% for femoral neck and +0.91% for total femur. In men, baseline OPG was significantly associated with higher BMD loss (ß coefficient -0.64) at the femoral neck. In women, DKK-1 was associated with higher BMD loss at the femoral neck (ß coefficient -0.09), and total femur (ß coefficient -0.11); however, DKK-1 was associated with lower BMD loss at the lumbar spine (ß coefficient 0.06). CONCLUSION: In tightly controlled RA patients, we have found no evidence of bone loss. The role of DKK1 and OPG seems small and might be related to sex and location.

Association of APOA5 and APOC3 Genetic Polymorphisms With Severity of Hypertriglyceridemia in Patients With Cutaneous T-Cell Lymphoma Treated With Bexarotene.

Importance: Hypertriglyceridemia is the most frequent and limiting adverse effect of bexarotene therapy in cutaneous T-cell lymphoma (CTCL). Despite standard prophylactic measures, there is a wide variability in the severity of this complication, which could be associated with both genetic and environmental factors. Objectives: To analyze the association between genetic polymorphisms of apolipoprotein genes APOA5, APOC3, and APOE and the severity of hypertriglyceridemia during bexarotene therapy and to optimize patient selection for bexarotene therapy based on adverse effect profile. Design, Setting, and Participants: This case series study was conducted in 12 university referral hospitals in Spain from September 17, 2014, to February 6, 2015. One hundred twenty-five patients with a confirmed diagnosis of CTCL who had received bexarotene therapy for at least 3 months were enrolled. Nine patients were excluded owing to missing analytic triglyceride level data, leaving a study group of 116 patients. Data on demographic and cardiovascular risk factor were collected, and a complete blood analysis, including lipid profile and genetic analysis from a saliva sample, was performed. Main Outcomes and Measures: Primary outcomes were the maximal triglyceride levels reported in association with the minor alleles of the polymorphisms studied. Results: Among 116 patients, the mean (SD) age was 61.2 (14.7) years, 69 (59.5%) were men, and 85 (73.2%) had mycosis fungoides, the most prevalent form of CTCL. During bexarotene therapy, 96 patients (82.7%) experienced hypertriglyceridemia, which was severe or extreme in 8 of these patients (8.3%). Patients who carried minor alleles of the polymorphisms did not show significant differences in baseline triglyceride concentrations. After bexarotene treatment, carriers of at least 1 of the 2 minor alleles of APOA5 c.-1131T>C and APOC3 c.*40C>G showed lower levels of triglycerides than noncarriers (mean [SD], 241.59 [169.91] vs 330.97 [169.03] mg/dL, respectively; P = .02). Conclusions and Relevance: These results indicate that the screening of APOA5 and APOC3 genotypes may be useful to estimate changes in triglyceride concentrations during bexarotene treatment in patients with CTCL and also to identify the best candidates for bexarotene therapy based on the expected adverse effect profile.

Measurement uncertainty and metrological traceability of whole blood cyclosporin A mass concentration results obtained by UHPLC-MS/MS.

BACKGROUND: Traceable and accurate results of cyclosporine A (CsA) mass concentrations in whole blood are required to ensure the monitoring of immunosuppressive therapy in transplant recipients. Metrological traceability and measurement uncertainty can allow ensuring reliability and comparability of these results over time and space. In this study, we provide a practical and detailed example of how the traceability and uncertainty of mass concentration of CsA results, obtained using an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) procedure, can be described and estimated. METHODS: Traceability was described mainly according to ISO 17511 and information obtained from certificates facilitated with the manufacturer's calibrators. Uncertainty estimation was performed using the bottom-up and top-down approaches. For the bottom-up approach, the most relevant sources of uncertainty were identified and later used to estimate the standard, combined and expanded uncertainties. For the top-down approach, expanded uncertainty was estimated directly using intralab quality control data mainly. RESULTS: Mass concentration of CsA results was traceable to the manufacturer's product calibrators used to calibrate the UHPLC-MS/MS procedure. The expanded uncertainties estimated by the bottom-up and top-down approaches were 7.4% and 7.2%, respectively. CONCLUSIONS: After performing the bottom-up and top-down approaches, we observed that their results were quite similar. This fact would confirm that the top-down approach could be sufficient for estimating uncertainty of CsA mass concentrations in whole blood results in clinical laboratories. Finally, we hope that this study can help and motivate clinical laboratories to describe metrological traceability and to perform measurement uncertainty studies based on the simpler top-down approach.

Development and validation of a measurement procedure based on ultra-high performance liquid chromatography-tandem mass spectrometry for simultaneous measurement of ß-lactam antibiotic concentration in human plasma.

BACKGROUND: The administration of ß-lactam antibiotics in continuous infusion could let optimize the pharmacokinetic/pharmacodynamic parameters, especially in the treatment of serious bacterial infections. In this context, and also due to variability in their plasmatic concentrations, therapeutic drug monitoring (TDM) may be useful to optimize dosing and, therefore, be useful for the clinicians. MATERIAL AND METHODS: We developed and validated a measurement procedure based on ultra-high performance liquid chromatography-tandem mass spectrometry for simultaneous measurement of amoxicillin, ampicillin, cloxacillin, piperacillin, cefepime, ceftazidime, cefuroxime, aztreonam and meropenem concentrations in plasma. The chromatographic separation was achieved using an Acquity®-UPLC® BEH™ (2.1×100mm id, 1.7µm) reverse-phase C18 column, with a water/acetonitrile linear gradient containing 0.1% formic acid at a 0.4mL/min flow rate. ß-Lactam antibiotics and their internal standards were detected by electrospray ionization mass spectrometry in multiple reaction monitoring mode. RESULTS: Chromatography run time was 7.0min and ß-lactam antibiotics eluted at retention times ranging between 1.08 and 1.91min. The lower limits of quantification were between 0.50 and 1.00mg/L. Coefficients of variation and relative bias absolute values were <13.3% and 14.7%, respectively. Recovery values ranged from 55.7% to 84.8%. Evaluation of the matrix effect showed ion enhancement for all antibiotics. No interferences or carry-over were observed. CONCLUSIONS: Our measurement procedure could be applied to daily clinical laboratory practice to measure the concentration of ß-lactam antibiotics in plasma, for instance in patients with bone and joint infections and critically ill patients.

Effect of Osteoprotegerin and Dickkopf-Related Protein 1 on Radiological Progression in Tightly Controlled Rheumatoid Arthritis.

OBJECTIVE: To analyze the association between circulating osteoprotegerin (OPG) and Dickkopf-related protein 1 (DKK-1) and radiological progression in patients with tightly controlled rheumatoid arthritis (RA). METHODS: Serum levels of OPG and DKK-1 were measured in 97 RA patients who were treated according to a treat-to-target strategy (T2T) aimed at remission (DAS28<2.6). Radiologic joint damage progression was assessed by changes in the total Sharp-van der Heijde score (SHS) on serial radiographs of the hands and feet. The independent association between these biomarker levels and the structural damage endpoint was examined using regression analysis. RESULTS: The mean age of the 97 RA patients (68 women) at the time of the study was 54 ± 14 years, and the median disease duration was 1.6 ± 1.5 years. Most patients were seropositive for either RF or ACPA, and the large majority (76%) were in remission or had low disease activity. After a median follow-up time of 3.3 ± 1.5 years (range, 1-7.5 yrs.), the mean total SHS annual progression was 0.88 ± 2.20 units. Fifty-two percent of the patients had no progression (defined as a total SHS of zero). The mean serum OPG level did not change significantly over the study period (from 3.9 ± 1.8 to 4.07 ± 2.23 pmol/L), whereas the mean serum DKK-1 level decreased, although not significantly (from 29.9 ± 10.9 to 23.6 ± 18.8 pmol/L). In the multivariate analysis, the predictive factors increasing the likelihood of total SHS progression were age (OR per year = 1.10; p = 0.003) and a high mean C-reactive protein level over the study period (OR = 1.29; p = 0.005). Circulating OPG showed a protective effect reducing the likelihood of joint space narrowing by 60% (95% CI: 0.38-0.94) and the total SHS progression by 48% (95% CI: 0.28-0.83). The DKK-1 levels were not associated with radiological progression. CONCLUSION: In patients with tightly controlled RA, serum OPG was inversely associated with progression of joint destruction. This biomarker may be useful in combination with other risk factors to improve prediction in patients in clinical remission or low disease activity state.

Measurement of ceftazidime concentration in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry. Application to critically ill patients and patients with osteoarticular infections.

Ceftazidime is an antibiotic belonging to the third generation of the cephalosporin family. It is indicated in the treatment of serious, simple or mixed bacterial infections, and its administration in continuous or intermittent infusion allows optimization of the concentration of antibiotic to keep it above the minimum inhibitory concentration. We developed and validated a chromatographic method by ultra-performance liquid chromatography-tandem mass spectrometry to measure ceftazidime concentration in human plasma. Following extraction with acetonitrile and 1,2-dichloroethane, the chromatographic separation was achieved using an Acquity ® UPLC ® BEH(TM) (2.1 × 100 mm i.d., 1.7 µm) reverse-phase C18 column, with a water-acetonitrile linear gradient containing 0.1% formic acid at a 0.4 mL/min flow rate. Ceftazidime and its internal standard (cefotaxime) were detected by electrospray ionization mass spectrometry in positive ion multiple reaction monitoring mode using mass-to-charge transitions of 547.0 → 467.9/396.1 and 456.0 → 395.8/324.1, respectively. The limit of quantification was 0.58 mg/L and linearity was observed in the range 0.58-160 mg/L. Coefficients of variation and absolute relative biases were <9.8 and 8.4%. The mean recovery for ceftazidime was 74.4 ± 8.1%. Evaluation of the matrix effect showed ion enhancement, and no carry-over was observed. The validated method could be applied to daily clinical laboratory practice to measure the concentration of ceftazidime in plasma.

Measurement of total and free docetaxel concentration in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry.

Docetaxel is a semi-synthetic taxane with cytotoxic anti-neoplastic activity and, currently used as anticancer agent in several types of cancer. Docetaxel is highly bound to plasma proteins, and this significantly determines its clearance and activity. Therefore, measurement of free docetaxel in plasma is pharmacologically important when pharmacokinetics is investigated. We developed and validated chromatographic methods by ultra-performance liquid chromatography-tandem mass spectrometry to measure total and free docetaxel concentration in human plasma. The final validated methods involved liquid-liquid extraction followed by dryness under nitrogen evaporation. To measure free docetaxel concentration, sample preparation was preceded by ultrafiltration. Chromatographic separation was achieved using an Acquity(®) UPLC(®) BEH™ (2.1×100 mm id, 1.7 µm) reverse-phase C18 column at a flow rate of 0.4 mL/min, using isocratic elution mode containing ammonium acetate/formic acid in water/methanol (30:70 v/v) as mobile phase. Docetaxel and its internal standard (paclitaxel) were detected by electrospray ionization mass spectrometry in positive ion multiple reaction monitoring mode using mass-to-charge (m/z) transitions of 808.3→527.0 (quantifier) and 808.3→509.0 (qualifier); and 854.3→569.0 (quantifier) and 854,3→509,0 (qualifier), respectively. The run time per sample was 3.5 min. The limits of quantification were 1,95 and 0.42 µg/L and linearity was observed between 1.95 and 1000 and 0.42-100 µg/L for total and free docetaxel, respectively. Coefficients of variation and absolute relative biases were less than 13.8% and 10.0%. Recovery values were greater than 79.4%. Evaluation of the matrix effect showed ion suppression and no carry-over was observed. The validated methods could be useful for both therapeutic drug monitoring and pharmacokinetic studies. They could be applied to daily clinical laboratory practice to measure the concentration of total and free docetaxel in plasma.

Measurement of meropenem concentration in different human biological fluids by ultra-performance liquid chromatography-tandem mass spectrometry.

Meropenem is a broad-spectrum antibiotic, often used for the empirical treatment of infections in critically ill patients with acute kidney injury. Meropenem has clinically insignificant protein binding and, as a carbapenem antibiotic, shows time-dependent bacterial killing, meaning that the unbound or free antibiotic concentration in blood should be maintained above the minimal inhibitory concentration of the pathogen for at least 40 % of the dosing interval. We developed and validated simple chromatographic methods by ultra-performance liquid chromatography-tandem mass spectrometry to measure plasma, filtrate-dialysate, and urine concentrations of meropenem. Chromatographic separation was achieved using an Acquity(®) UPLC(®) BEH(TM) (2.1 × 100 mm id, 1.7 µm) reverse-phase C(18) column, with a water/acetonitrile linear gradient containing 0.1 % formic acid at a 0.4-mL/min flow rate. Meropenem and its internal standard (ertapenem) were detected by electrospray ionization mass spectrometry in positive ion multiple reaction monitoring mode. The limits of quantification were 0.27, 0.24, and 1.22 mg/L, and linearity was observed between 0.27-150, 0.24-150, and 1.22-2,000 mg/L for plasma, filtrate-dialysate, and urine samples, respectively. Coefficients of variation and relative biases were less than 13.5 and 8.0 % for all biological fluids. Recovery values were greater than 68.3 %. Evaluation of the matrix effect showed ion suppression for meropenem and ertapenem. No carry-over was observed. The validated methods are useful for both therapeutic drug monitoring and pharmacokinetic studies. It could be applied to daily clinical laboratory practice to measure the concentration of meropenem in plasma, filtrate-dialysate, and urine.

Apolipoprotein alleles and the response to interferon-ß-1b in multiple sclerosis.

BACKGROUND: Results for the e4/e2 alleles of the ApoE gene as markers of susceptibility, clinical and radiological progression, and cognitive deterioration in patients with multiple sclerosis (MS) are contradictory. AIM: The usefulness of these markers in predicting the response to interferon-ß-1b (IFNß-1b) was evaluated. MATERIAL AND METHODS: 95 patients with relapsing-remitting MS treated with IFNß-1b (mean follow-up 7.44 years) were studied. We correlated the e4 and e2 alleles with the time to the first relapse or to a 1-point worsening on the Expanded Disability Status Scale, time to moderate disability, progression index, and treatment discontinuation due to inefficacy. RESULTS: We found no association between the e4 allele and any of the variables. The e2 allele was associated with increased time to moderate disability. CONCLUSION: The e4 allele of ApoE has no prognostic value for the response to IFNß-1b. The e2 allele delayed the progression of disability in our MS patient cohort.

Interaction between APOA5 -1131T>C and APOE polymorphisms and their association with severe hypertriglyceridemia.

BACKGROUND: Apolipoprotein A5 gene (APOA5) has been shown to modulate plasma triglyceride concentrations. The apolipoprotein E gene (APOE) has been implicated in cholesterol and triglyceride homeostasis in humans and plays an important role in atherogenesis. The aim of this study was to determine the genotypic distribution of the APOA5 -1131T>C and APOE polymorphisms and to identify the combined association of these variants between patients with and without severe hypertriglyceridemia (HTG). METHODS: We genotyped 96 individuals who had reached plasma TG concentrations of more than 10 mmol/L and 225 ischemic patients without severe HTG. RESULTS: Minor allele carriers were significantly more frequent in HTG group for all three polymorphisms (APOA5, APOE2 and APOE4). Adjusted individual risks for severe HTG were: APOA5 -1131C, OR=4.1 (95%CI:2.02-8.24); APOE2, OR=1.6 (95%CI:0.73-3.58); APOE4, OR=3.0 (95%CI:1.68-5.86). Adjusted risks for APOA5-APOE combinations were: APOA5 -1131C/APOE2, OR=45.2 (95%CI:4.92-415.5); APOA5 -1131C/APOE4, OR=6.4 (95%CI:2.28-18.01). CONCLUSIONS: These data provide evidence that APOA5 -1131T>C polymorphism is associated with risk for severe HTG. Furthermore, this effect is strongly increased when -1131C variant is combined with APOE variants.

[Apolipoprotein A5 gene: association with triglyceride metabolism and cardiovascular disease]. / Gen de la apolipoproteína A5: asociación con el metabolismo de los triglicéridos y las enfermedades cardiovasculares.

Hypertriglyceridemia constitutes an independent risk factor for coronary disease. The gene apolipoprotein A5 (ApoA5) is a newly discovered member in the ApoA1/C3/A4/A5 cluster, and its product, apolipoprotein A5, influences on triglycerides through an unknown mechanism. Recently, there have been described the clinical consequences and the functional effects over more than 10 variants of the ApoA5 gene, associated with atherosclerosis. In different studies carried out in different ethnic groups, it has been observed a great variation in the distribution of the ApoA5 genotypes for the respective polymorphisms. Different authors have described associations among the ApoA5 gene, high triglyceride concentrations and an increase in the cardiovascular risk. In this context, the ApoA5 gene is considered as a probable biochemical and genetic marker of increased triglyceride concentrations and also a risk factor of coronary disease in some populations.

[Lack of association between the APOE genotype and the response to statin treatment in patients with acute ischemic episodes]. / Ausencia de asociación entre el genotipo de la apolipoproteína E y la respuesta al tratamiento con estatinas en los pacientes con episodios isquémicos agudos.

BACKGROUND AND OBJECTIVE: APOE genotype has been shown to have an influence on lipid concentrations. However, its relation with response to lipid-lowering treatment is not well established. The aim of our work was to analyze whether this genotype is associated with changes in the lipid profile in response to statins treatment. PATIENTS AND METHOD: A total of 222 consecutive patients with acute ischemic episodes and subjected to treatment with statins were included in a retrospective study. The patients' lipid profile was determined at the first visit to the Lipids Unit and after one year on a statin regime. APOE genotypes were determined by PCR-RFLP, and separated in three groups: E2 (E2 carriers), E4 (E4 carriers) and E3 (E3/E3). E2/E4 patients were not included in the study. RESULTS: Relative frequencies of alleles epsilon2, epsilon3 and epsilon4 were 10.5%, 70.9% and 18.6% respectively. Significant differences among groups (p = 0.039) were observed for c-LDL concentrations. E2 group had lower c-LDL than E3 group (p = 0.017) and E4 group (p = 0.01). No significant differences in c-LDL, c-HDL and c-HDL/CT were observed among the three groups with regard to variation after statin treatment. CONCLUSION: APOE genotype does not significantly affect the lipid response in patients with acute ischemic episodes after statin treatment.

Utility of alpha subunit determination after thyrotropin-releasing hormone stimulation as an indicator of gonadotropinoma persistence and/or recurrence.

OBJECTIVE: Gonadotropinomas are adenomas of the gonadotropic cells of the anterior pituitary. These cells produce and secrete gonadotropins (follicle-stimulating hormone and luteinizing hormone). Most of these tumors show altered production of gonadotropins and their subunits (the p-FSH, a and, less frequently, p-LH subunits). The thyrotropin-releasing hormone (TRH) stimulation test could differentiate these tumors from nonfunctioning tumors. Equally, this test could be able to distinguish between postsurgical changes and tumoral remnants after surgery. SUBJECTS AND METHOD: We studied 24 patients with pituitary macroadenoma, 14 of who had a histological diagnosis of gonadotroph adenoma. The TRH stimulation test was performed before and after surgery. RESULTS: Both before and after surgery, a positive result to the TRH test was obtained in 50% of gonadotropinomas. Magnetic resonance imaging (MRI) performed after surgery revealed that 83% of the patients with gonadotropinoma had signs of tumoral persistence or recurrence and/or postsurgical changes. Of these patients, 83% (41.6% of the total) showed positive a subunit stimulation after the TRH test. In the group of non-gonadotropinoma macroadenomas, only 33% had a positive result before surgery and another 33% had a positive result after surgery. In the MRI performed after surgery, all showed tumoral persistence/recurrence or postsurgical inflammatory changes. CONCLUSIONS: This test could be useful in the differential diagnosis of gonadotropinomas as well as in the follow-upand postsurgical evaluation of these tumors.

Postresection parathyroid hormone and parathyroid hormone decline accurately predict hypocalcemia after thyroidectomy.

Hypocalcemia is the most frequent complication after total thyroidectomy. Parathyroid hormone (PTH) measurement has been proposed as an early predictor of this condition. Total thyroidectomy was performed in 39 patients. Hypocalcemia was present in 15 cases (38%). Patients undergoing hemithyroidectomy (n = 13) were considered control subjects not developing hypocalcemia. PTH was measured before surgery and 10 minutes after resection of the gland using a rapid (15 minutes) chemiluminescent immunometric assay. Patients developing hypocalcemia had lower calcium and postresection PTH levels and higher PTH decline than patients not developing hypocalcemia (P < .0001). PTH decline (cutoff value, 62.5%) had the better sensitivity (93.3%) for predicting hypocalcemia, allowing for a fairly safe early discharge. However, the best overall results corresponded to the combination of postresection PTH level (< or = 18 pg/mL [< or = 1.9 pmol/L]) and PTH decline (>62.5%), with a sensitivity of 90% and a specificity of 97.9%. Perioperative PTH measures can accurately predict hypocalcemia after thyroidectomy, granting the laboratory a key role in the immediate decision about calcium supplementation for patients at risk.

Differential expression of MHC class II molecules in highly metastatic breast cancer cells is mediated by the regulation of the CIITA transcription Implication of CIITA in tumor and metastasis development.

We analyzed the differential gene expression between variants of MDA-MB-435 human breast cancer cell line that share an identical genetic background but have different metastatic ability. The major histocompatibility complex class II was found down-regulated in highly metastatic cells and correlated with MHC transactivator (CIITA) expression. Constitutive CIITA expression observed in poorly metastatic is driven by promoters III and IV of CIITA gene. Conversely, both promoters were ineffective in highly metastatic cells. The MHC class II and CIITA expression was restored in these cells upon stimulation with IFNgamma or by the treatment with a hypomethylating agent. Both treatments induced USF-1 and IRF binding complexes to promoter IV but only IFNgamma induced the binding of 435-Lung2 nuclear proteins to an ARE-1 site at the promoter III. Neither Southern blot nor bisulfite sequencing of promoter IV demonstrated strong hypermethylation of this promoter at the IFNgamma-responsive elements such as GAS, E-box or IRF-1. We suggest that partial or hemimethylation of promoter IV is sufficient to silence the CIITA expression in highly metastatic cells and that this epigenetic mechanism is responsible for the lack of MHC-II expression. Forced CIITA expression restored the MHC-II antigen expression in 435-Lung2 cells and abrogates spontaneous lung metastasis in both SCID and nude mice but also affected the tumorigenicity in nude mice. The increase in NK cell infiltration in nude mice bearing CIITA-tumors correlated with sign of tumor cell apoptosis and the increase in the number of NK cells in the spleens, suggesting that NK cells might be responsible for the observed antitumor activity.