Optimised molecular genetic diagnostics of Fanconi anaemia by whole exome sequencing and functional studies.

PURPOSE: Patients with Fanconi anaemia (FA), a rare DNA repair genetic disease, exhibit chromosome fragility, bone marrow failure, malformations and cancer susceptibility. FA molecular diagnosis is challenging since FA is caused by point mutations and large deletions in 22 genes following three heritability patterns. To optimise FA patients' characterisation, we developed a simplified but effective methodology based on whole exome sequencing (WES) and functional studies. METHODS: 68 patients with FA were analysed by commercial WES services. Copy number variations were evaluated by sequencing data analysis with RStudio. To test FANCA missense variants, wt FANCA cDNA was cloned and variants were introduced by site-directed mutagenesis. Vectors were then tested for their ability to complement DNA repair defects of a FANCA-KO human cell line generated by TALEN technologies. RESULTS: We identified 93.3% of mutated alleles including large deletions. We determined the pathogenicity of three FANCA missense variants and demonstrated that two FANCA variants reported in mutations databases as 'affecting functions' are SNPs. Deep analysis of sequencing data revealed patients' true mutations, highlighting the importance of functional analysis. In one patient, no pathogenic variant could be identified in any of the 22 known FA genes, and in seven patients, only one deleterious variant could be identified (three patients each with FANCA and FANCD2 and one patient with FANCE mutations) CONCLUSION: WES and proper bioinformatics analysis are sufficient to effectively characterise patients with FA regardless of the rarity of their complementation group, type of mutations, mosaic condition and DNA source.

A novel mutation in the SLC40A1 gene associated with reduced iron export in vitro.

Ferroportin disease is an inherited disorder of iron metabolism and is caused by mutations in the ferroportin gene (SLC40A1). We present a patient with hyperferritinemia, iron overload in the liver with reticuloendothelial distribution and also in the spleen, and under treatment with erythropheresis. A molecular study of the genes involved in iron metabolism (HFE, HJV, HAMP, TFR2, SLC40A1) was undertaken. In vitro functional studies of the novel mutation found in the SLC40A1 gene was performed. The patient was heterozygous for a novel mutation, c.386T>C (p.L129P) in the SLC40A1 gene; some of his relatives were also heterozygous for this mutation. In vitro functional studies of the L129P mutation on ferroportin showed it impairs its capacity to export iron from cells but does not alter its sensitivity to hepcidin. These findings and the iron overload phenotype of the patient suggest that the novel mutation c.386T>C (p.L129P) in the SLC40A1 gene has incomplete penetrance and causes the classical form of ferroportin disease.

Iron deficiency is associated with Hypothyroxinemia and Hypotriiodothyroninemia in the Spanish general adult population: Di@bet.es study.

Previous studies have suggested that iron deficiency (ID) may impair thyroid hormone metabolism, however replication in wide samples of the general adult population has not been performed. We studied 3846 individuals free of thyroid disease, participants in a national, cross sectional, population based study representative of the Spanish adult population. Thyroid stimulating hormone (TSH), free thyroxin (FT4) and free triiodothyronine (FT3) were analyzed by electrochemiluminescence (E170, Roche Diagnostics). Serum ferritin was analyzed by immunochemiluminescence (Architect I2000, Abbott Laboratories). As ferritin levels decreased (>100, 30-100, 15-30, <15 µg/L) the adjusted mean concentrations of FT4 (p < 0.001) and FT3 (p < 0.001) descended, whereas TSH levels remained unchanged (p = 0.451). In multivariate logistic regression models adjusted for age, sex, UI, BMI and smoking status, subjects with ferritin levels <30 µg/L were more likely to present hypothyroxinemia (FT4 < 12.0 pmol/L p5): OR 1.5 [1.1-2.2] p = 0.024, and hypotriiodothyroninemia (FT3 < 3.9 pmol/L p5): OR 1.8 [1.3-2.6] p = 0.001 than the reference category with ferritin ≥30 µg/L. There was no significant heterogeneity of the results between men, pre-menopausal and post-menopausal women or according to the iodine nutrition status. Our results confirm an association between ID and hypothyroxinemia and hypotriiodothyroninemia in the general adult population without changes in TSH.

[Vitamin D deficiency and morbimortality in critically ill paediatric patients]. / Déficit de vitamina D y morbimortalidad en pacientes críticos pediátricos.

OBJECTIVES: To determine the prevalence and risks factors of vitamin D deficiency, as well as its relationship with morbidity and mortality in a PICU. MATERIAL AND METHODS: An observational prospective study in a tertiary children's University Hospital PICU conducted in two phases: i: cohorts study, and ii: prevalence study. The study included 340 critically ill children with ages comprising 6 months to 16 years old. EXCLUSION CRITERIA: Chronic kidney disease, known parathyroid disorders, and vitamin D supplementation. Total 25-hydroxyvitamin D [25(OH)D] was measured in the first 48hours of admission to a PICU. Parathormone, calcium, phosphate, blood gases, blood count, C-reactive protein, and procalcitonin were also analysed. A record was also made of demographic features, characteristics of the episode, and complications during the PICU stay. RESULTS: The overall prevalence rate of vitamin D deficiency was 43.8%, with a mean of 22.28 (95% CI 21.15-23.41) ng/ml. Patients with vitamin D deficiency were older (61 vs 47 months, P=.039), had parents with a higher level of academic studies (36.5% vs 20%, P=.016), were admitted more often in winter and spring, had a higher PRISM-III (6.8 vs 5.1, P=.037), a longer PICU stay (3 vs 2 days, P=.001), and higher morbidity (61.1% vs 30.4%, P<001) than the patients with sufficient levels of 25(OH)D. Patients who died had lower levels of 25(OH)D (14±8.81ng/ml versus 22.53±10.53ng/ml, P=.012). Adjusted OR for morbidity was 5.44 (95%CI; 2.5-11.6). CONCLUSIONS: Vitamin D deficiency is frequent in critically ill children, and it is related to both morbidity and mortality, although it remains unclear whether it is a causal relationship or it is simply a marker of severity in different clinical situations.

Reference values for TSH may be inadequate to define hypothyroidism in persons with morbid obesity: Di@bet.es study.

OBJECTIVE: To analyze the reference range of thyroid-stimulating hormone (TSH) in different BMI categories and its impact on the classification of hypothyroidism. METHODS: The study included 3,928 individuals free of thyroid disease (without previous thyroid disease, no interfering medications, TSH <10 µUI/mL and thyroid peroxidase antibodies [TPO Abs] <50 IU/mL) who participated in a national, cross-sectional, population-based study and were representative of the adult population of Spain. Data gathered included clinical and demographic characteristics, physical examination, and blood and urine sampling. TSH, free thyroxine, free triiodothyronine, and TPO Ab were analyzed by electrochemiluminescence (E170, Roche Diagnostics, Basel, Switzerland). RESULTS: The reference range (p2.5-97.5) for TSH was estimated as 0.6 to 4.8 µUI/mL in the underweight category (BMI<20 kg/m2 ), 0.6 to 5.5 µUI/mL in the normal-weight category (BMI 20-24.9 kg/m2 ), 0.6 to 5.5 µUI/mL in the overweight category (BMI 25-29.9 kg/m2 ), 0.5 to 5.9 µUI/mL in the obesity category (BMI 30-39.9 kg/m2 ), and 0.7 to 7.5 µUI/mL in the morbid obesity category (BMI ≥40). By using the reference criteria for the normal-weight population, the prevalence of high TSH levels increased threefold in the morbid obesity category (P < 0.01). CONCLUSIONS: Persons with morbid obesity might be inappropriately classified if the standard ranges of normality of TSH for the normal-weight population are applied to them.

Use of newer prognostic indices for patients with myelodysplastic syndromes in the low and intermediate-1 risk categories: a population-based study.

BACKGROUND: We aimed to compare the ability of recently developed prognostic indices for myelodysplastic syndromes to identify patients with poor prognoses within the lower-risk (low and intermediate-1) categories defined by the International Prognosis Scoring System (IPSS). METHODS: We included patients with de-novo myelodysplastic syndromes diagnosed between Nov 29, 1972, and Dec 15, 2011, who had low or intermediate-1 IPSS scores and were in the Spanish Registry of Myelodysplastic Syndromes. We reclassified these patients with the new prognostic indices (revised IPSS [IPSS-R], revised WHO-based Prognostic Scoring System [WPSS-R], Lower Risk Scoring System [LRSS], and the Grupo Español de Síndromes Mielodisplásicos [Spanish Group of Myelodysplastic Syndromes; GESMD]) and calculated the overall survival of the different risk groups within each prognostic index to identify the groups of patients with overall poor prognoses (defined as an expected overall survival <30 months). We calculated overall survival with the Kaplan-Meier method. FINDINGS: We identified 2373 patients. None of the prognostic indices could be used to identify a population with poor prognoses (median overall survival <30 months) for the patients with low IPSS scores (1290 individuals). In the group with intermediate-1 scores (1083 individuals), between 17% and 47% of patients were identified as having poor prognoses with the new prognostic indices. The LRSS had the best model fit with the lowest value in the Akaike information criteria test, whereas the IPSS-R identified the largest proportion of patients with poor prognoses (47%). Patients with intermediate-1 scores who were classified as having poor prognoses by one or more prognostic index (646 [60%] individuals) had worse median overall survival (33·1 months, 95% CI 28·4-37·9) than did patients who were classified as having low risk by all prognostic indices (63·7 months, 49·5-78·0], HR 1·9, 95% CI 1·6-2·3, p<0·0001) INTERPRETATION: Recently proposed prognostic indices for myelodysplastic syndromes can be used to improve identification of patients with poor prognoses in the group of patients with intermediate-1 IPSS scores, who could potentially benefit from a high-risk treatment approach. FUNDING: None.

Transfusion dependence development and disease evolution in patients with MDS and del(5q) and without transfusion needs at diagnosis.

Patients with isolated del(5q) and MDS are considered to have good prognosis as compared to other MDS subtypes. Most patients suffered of anemia and 50% of them required transfusions at diagnosis. It is known that for patients with MDS and del(5q) in transfusion dependence(TD), Lenalidomide is the first choice treatment. However, there are no data regarding natural evolution of anemia in patients diagnosed in MDS and del(5q) without TD, factors that may impact on the development of TD or disease outcome. In the present study we have performed a retrospective multicenter analysis on 83 patients with low-int 1 MDS and del(5q) without TD. During the study 61 patients became TD at a median of 1.7 years and only the Hb level 9 g/dL was associated with poorer TFS (p = 0.007) in the multivariate analysis. Among these 61 TD patients, 49 received treatment (19 Lenalidomide). Median follow up was 48 months, estimated OS at 2 and 5 year was 92% and 50% respectively. In the multivariate analysis for OS, platelets <100,000 mm(-3) and Lenalidomide treatment retained the statistical significant impact. LFS at 2 and 5 years was 86% and 73% respectively, and median time to sAML was 8.16 years (CI 95%: 6.05-10.27). In the multivariate analysis only thrombocytopenia retained statistical significance. In summary, this retrospective study show that level of Hb is an important parameter in order to determine the time until TD, it should be also stressed the importance of an early treatment in order to prevent TD development and shorter survival.

Sentinel node biopsy in early breast cancer at the Hospital Comarcal La Linea (Spain).

OBJECTIVE: Our objective was to determine the identification and the percentage of false negatives in sentinel node biopsies in patients with early breast cancer at the Hospital La Línea (Spain), during the period between November 2007 and September 2010. METHODS: We collected 50 patients with early breast cancer, without clinical and ultrasonographic involvement of axillary nodes, from November 2007 to September 2010. We used the vital dye in the first 20 patients and the combined technique of vital dye and albumin labelled with technetium 99 in the other 30 patients. The site of injection for patients using blue dye was subdermal for palpable tumours and periareolar for non-palpable tumours. The technique of injection with the radioisotope for patients for palpable and most non-palpable tumours was the periareolar technique. We used albumin labelled with technetium 99. In seven patients with non-palpable tumours, we used the sentinel node occult lesion localisation (SNOLL) technique. The sentinel node biopsy was examined during surgery, with the frozen section examination and imprint as follows: the sentinel node was cut in three transversal sections along the axis and five frozen sections of each portion were done at a distance of 60 µm each; in total, 15-20 frozen sections and three imprints were done for each sentinel node. The axillary dissection was completed in the first 17 patients, and we performed total axillary dissection on the remaining patients if the sentinel node was positive for metastasis. RESULTS: The sentinel nodes were identified in 49 of 50 patients (98%). The patient in whom we did not identify the sentinel node was a patient in the combined technique. The number of nodes identified in the patients with vital dye was one sentinel node, and with the combined technique, it was two sentinel nodes. The false-negative rate was 8% (four patients); the micrometastasis was the principal factor of the false-negative rate (p < 0.05). The cases of false negatives were present at the beginning of the study with the use of the blue dyes; this factor was statistically significant (p < 0.05). The tumour size, the vascular invasion, and the periganglionar adipose tissue invasion were statistically significant for the presentation of axillary metastasis (p < 0.05). CONCLUSION: This study shows that the micrometastasis and the use of vital dye were the principal factors for the presentation of the false-negative rate. The size of the tumour, the vascular invasion, and the periganglionar adipose tissue invasion were statistically significant for the appearance of the axillary metastasis.

Giant fibrovascular polyp of the esophagus: a novel technical approach.

Fibrovascular polyps (FVPs) of the esophagus are rare, and their course is usually indolent until reaching enormous proportions. It is a dramatic entity owing to its tendency to cause bizarre complications. We describe a 49-year-old female patient with sudden dyspnoea that required digital maneuvers to clear the airway. After diagnosing, an FVP of the esophagus, a video-assisted endocavitary surgery was made. Histopathological examination revealed a fibrovascular polyp. Endoscopic controls after excision show no mass or symptoms recurrence.

[Paroxysmal nocturnal hemoglobinuria therapy with eculizumab: Spanish experience]. / Tratamiento de la hemoglobinuria paroxística nocturna con eculizumab: experiencia en España.

BACKGROUND AND OBJECTIVES: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal disease characterized by complement-mediated hemolysis, bone marrow failure and thrombosis. Eculizumab is a humanized monoclonal antibody that blocks the cytolytic component of the complement system by binding to complement C5. MATERIAL AND METHODS: We report the results of eculizumab treatment in 25 PNH patients from different centers in Spain. Statistical analysis was perfomed with a SPSS v15.0 software. RESULTS: Fifty-eight per cent of the patients achieved transfusional independence after a median of 14 months. Transfusion requirements were reduced in 60% of the remaining cases. Fatigue resolved in 96% of the patients and smooth muscle dystony-related symptoms in all cases. A single case of treatment-related infection was observed. CONCLUSIONS: Eculizumab controls effectively hemolysis and greatly improves clinical symptoms. The drug is safe and well tolerated, without significant adverse effects except meningococcal infection. Patients with suboptimal response to treatment must be assessed for bone marrow insufficiency and extravascular haemolysis.

Evaluation of different solvent systems for the extraction and fractionation of oleoresins from guajillo peppers / Evaluación de diferentes solventes para la extracción y fraccionamiento de oleorresinas a partir de chile guajillo

Dried guajillo peppers were first extracte with four different solvents: ethanol, acetone, ethyl acetate and hexane with the aim of obtaining oleoresins which were futher fractionated into red and paprika extracts. Results showed that as the polarity of the solvent increased the amount of pigment extracted also increased. Acetone had good affinity for pungent (capsaicin) compounds. Utilization of these solvents alone did not produce red and paprika oleoresins that meet commercial specifications. Fractionation of acetone extracted oleoresins with ethanol: water (90:10) yielded a precipitate and a solution. The precipitate and solution produced red and paprika extracts that meet pungency and color specifications. It was possible to obtain red and paprika oleoresins from mild guajillo peppers