Next-generation sequencing of hereditary hemochromatosis-related genes: Novel likely pathogenic variants found in the Portuguese population.

Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by excessive iron absorption resulting in pathologically increased body iron stores. It is typically associated with common HFE gene mutation (p.Cys282Tyr and p.His63Asp). However, in Southern European populations up to one third of HH patients do not carry the risk genotypes. This study aimed to explore the use of next-generation sequencing (NGS) technology to analyse a panel of iron metabolism-related genes (HFE, TFR2, HJV, HAMP, SLC40A1, and FTL) in 87 non-classic HH Portuguese patients. A total of 1241 genetic alterations were detected corresponding to 53 different variants, 13 of which were not described in the available public databases. Among them, five were predicted to be potentially pathogenic: three novel mutations in TFR2 [two missense (p.Leu750Pro and p.Ala777Val) and one intronic splicing mutation (c.967-1G>C)], one missense mutation in HFE (p.Tyr230Cys), and one mutation in the 5'-UTR of HAMP gene (c.-25G>A). The results reported here illustrate the usefulness of NGS for targeted iron metabolism-related gene panels, as a likely cost-effective approach for molecular genetics diagnosis of non-classic HH patients. Simultaneously, it has contributed to the knowledge of the pathophysiology of those rare iron metabolism-related disorders.

Distribution and quantitation of skin iron in primary haemochromatosis: correlation with total body iron stores in patients undergoing phlebotomy.

Measurement of the concentration of iron in the skin, if correlated with total body iron stores, may enable better informed decisions on when to initiate, change or stop therapy in hereditary heamochromatosis. Naïve haemochromatosis patients with iron overload and with C282Y and/or H63D HFE mutations were evaluated at the following time-points: disease diagnosis, end of the therapy programme, and 6 months after the end of therapy. The distribution and concentration of iron in the skin were assessed by quantitative nuclear microscopy methods, in parallel with serum and plasma iron concentration. Iron content in the liver was determined by nuclear magnetic resonance. Iron accumulated in the epidermis; its concentration increased from outer to inner layers, being maximal in the basal layer (7.33 ± 0.98 µmol/g). At all 3 time-points, most of the iron was associated with the extracellular space. During the phlebotomy programme the iron content of the skin and the liver decreased by a factor of 2. These data suggest that measurements of iron concentration in the epidermis, which is a readily accessible tissue, reflect iron overload in the liver.

The functional significance of E277K and V295A HFE mutations.

Hereditary haemochromatosis (HH) is an autosomal recessive disorder characterized by excessive intestinal iron absorption resulting in increased pathological body iron stores. It is typically associated with homozygosity for the c.845G>A (p.C282Y) mutation in the HFE gene. However, other HFE alterations have been reported in affected individuals but their association with the disease is unclear. This study analysed the functional consequences of two HFE mutations, c.829G>A (p.E277K) and c.884T>C (p.V295A). Firstly, it was shown that c.829G>A affects the HFE splicing by diminishing the full length HFE and ivs4_66bp inclusion transcript levels, while increasing the amount of exon 4 skipping transcript. Immunofluorescent techniques showed that the HFE_E277K protein had a diffuse distribution (similar to HFE_C282Y) while HFE_V295A presented at the cell surface and perinuclear compartments (resembling HFE_wt). Immunoprecipitation assays revealed a decreased association of HFE_E277K and HFE_V295A with both ß2-microglobulin (B2M; 38 ± 7% and 66 ± 8%, respectively) and transferrin receptor (TFRC, also termed TFR1) (58 ± 2% and 49 ± 16%, respectively). Herein, we prove that both mutations partially abrogate HFE association with B2M and TFRC, crucial for its correct processing and cell surface presentation. Although E277K has a more deleterious effect than V295A, we propose that both mutations may play a role in the development of hereditary haemochromatosis.

Severe hypercalcemia without hypercalciuria in a previously healthy infant.

Williams-Beuren syndrome (WBS) is a multisystem disorder that has a broad range of clinical findings including characteristic facial appearance, supravalvular aortic stenosis, dental and developmental abnormalities, and endocrinologic disorders including but not limited to the development of hypercalcemia. We present the case of a 10-month-old girl, with a history of intrauterine growth restriction, who presented with symptoms of weight loss and poor feeding. She was found to have severe elevation of her serum calcium to 20 mg/dL. She was subsequently diagnosed with WBS by fluorescent in situ hybridization analysis. The exact etiology of hypercalcemia in patients with WBS is unknown, but there are several hypotheses. Treatment of hypercalcemia in WBS is achieved with intravenous (IV) fluids, loop diuretics, and a low calcium diet; bisphosphonate therapy is required if adequate decreases in the serum calcium level are not achieved with traditional therapy.

Does pelvic exam in the emergency department add useful information?

OBJECTIVE: Physicians are taught that the pelvic exam is a key part of the evaluation of a woman presenting with abdominal pain or vaginal bleeding. However, the exam is time consuming and invasive, and its use in the emergency department (ED) has not been prospectively evaluated. We evaluated how often the findings of the pelvic exam changed management in a cohort of consecutive female patients presenting with acute abdominal pain or vaginal bleeding. METHODS: We enrolled women who required a pelvic exam together with the providers caring for them in an academic ED from September 2004 to August 2005. We collected the results of the general history and physical exam. The provider was asked to predict the findings of the pelvic exam, and these were compared with the actual findings of the exam. RESULTS: One hundred eighty-three patients were prospectively entered into the study. When compared with predicted findings, the pelvic exam was as expected in 131 patients (72%). In a further 40 patients (22%), the findings of the pelvic exam were not as predicted, but resulted in no change in the clinical plan. In 12 cases (6%) the exam revealed a finding that was both unexpected and changed the clinical plan. Only one of these patients was admitted. Of the 24 patients who were admitted, four had a pelvic exam that revealed unexpected results, but only one of these cases caused the physician to change the care planned for the patient. CONCLUSION: In 94% of women with acute abdominal pain or vaginal bleeding, the results of the pelvic exam were either predictable or had no effect on the clinical plan. This suggests that there may be a subset of women with abdominal pain or vaginal bleeding in whom a pelvic exam may safely be deferred.

Non-classical hereditary hemochromatosis in Portugal: novel mutations identified in iron metabolism-related genes.

The most frequent genotype associated with Hereditary hemochromatosis is the homozygosity for C282Y, a common HFE mutation. However, other mutations in HFE, transferrin receptor 2 (TFR2), hemojuvelin (HJV) and hepcidin (HAMP) genes, have also been reported in association with this pathology. A mutational analysis of these genes was carried out in 215 Portuguese iron-overloaded individuals previously characterized as non-C282Y or non-H63D homozygous and non-compound heterozygous. The aim was to determine the influence of these genes in the development of iron overload phenotypes in our population. Regarding HFE, some known mutations were found, as S65C and E277K. In addition, three novel missense mutations (L46W, D129N and Y230F) and one nonsense mutation (Y138X) were identified. In TFR2, besides the I238M polymorphism and the rare IVS5 -9T-->A mutation, a novel missense mutation was detected (F280L). Concerning HAMP, the deleterious mutation 5'UTR -25G-->A was found once, associated with Juvenile Hemochromatosis. In HJV, the A310G polymorphism, the novel E275E silent alteration and the novel putative splicing mutation (IVS2 +395C-->G) were identified. In conclusion, only a few number of mutations which can be linked to iron overload was found, revealing their modest contribution for the development of this phenotype in our population, and suggesting that their screening in routine diagnosis is not cost-effective.

[The maximum surgical blood order schedule]. / Utilização de sangue em cirurgias programadas.

For elective surgeries over ordering of blood is a common practice. The goal of the Maximum Surgical Blood Order Schedule (MSBOS) is to promote efficient use of blood. The purpose of this Portuguese study is to analyse the crossmatched blood units for elective surgery and the real amount of transfused blood, the crossmatch:transfusion rate (C:T) which must be less than 2.5. As in other Blood Banks, the differences are more evident in the situations in which the need for blood is very small, the so called Type and Screen. MSBOS should be re-evaluated regularly because of operative environment such as surgeons, operative methods, haemostatic drugs and preoperative conditions of patients can change.