The genetic landscape of mitochondrial diseases in the next-generation sequencing era: a Portuguese cohort study.

Introduction: Rare disorders that are genetically and clinically heterogeneous, such as mitochondrial diseases (MDs), have a challenging diagnosis. Nuclear genes codify most proteins involved in mitochondrial biogenesis, despite all mitochondria having their own DNA. The development of next-generation sequencing (NGS) technologies has revolutionized the understanding of many genes involved in the pathogenesis of MDs. In this new genetic era, using the NGS approach, we aimed to identify the genetic etiology for a suspected MD in a cohort of 450 Portuguese patients. Methods: We examined 450 patients using a combined NGS strategy, starting with the analysis of a targeted mitochondrial panel of 213 nuclear genes, and then proceeding to analyze the whole mitochondrial DNA. Results and Discussion: In this study, we identified disease-related variants in 134 (30%) analyzed patients, 88 with nuclear DNA (nDNA) and 46 with mitochondrial DNA (mtDNA) variants, most of them being pediatric patients (66%), of which 77% were identified in nDNA and 23% in mtDNA. The molecular analysis of this cohort revealed 72 already described pathogenic and 20 novel, probably pathogenic, variants, as well as 62 variants of unknown significance. For this cohort of patients with suspected MDs, the use of a customized gene panel provided a molecular diagnosis in a timely and cost-effective manner. Patients who cannot be diagnosed after this initial approach will be further selected for whole-exome sequencing. Conclusion: As a national laboratory for the study and research of MDs, we demonstrated the power of NGS to achieve a molecular etiology, expanding the mutational spectrum and proposing accurate genetic counseling in this group of heterogeneous diseases without therapeutic options.

Alterations promoted by acid straightening and/or bleaching in hair microstructures.

Human hair is a biopolymer constituted mainly of keratin intermediate filaments, lipids, pigments and water. Cosmetic treatments usually interact with the hair at the molecular level, inducing changes in its components and modifying the physicochemical and mechanical properties of the fibers. Here, the effect of acid straightening on the morphology and ultrastructure of Caucasian hair was investigated by a group of complementary experimental methods: wide-, small- and ultra-small-angle X-ray scattering; high-resolution 3D X-ray microscopy; quasi-elastic neutron scattering and inelastic neutron scattering; thermogravimetry-mass spectrometry; and differential scanning calorimetry (DSC). X-ray diffraction patterns showed that acid straightening associated with a flat iron (∼180°C) changed the cortex of the fiber, shown by denaturation of the intermediate filaments (measured by DSC). The increase in the spacing of the lipid layers and the observation of the dehydration behavior of the fiber provided indications that water may be confined between these layers, while neutron spectroscopy showed alterations in the vibration mode of the CH2 groups of the lipids and an increase of the proton (H+) mobility in the hair structure. The latter may be associated with the extremely low pH of the formulation (pH ≃ 1). Additionally, this investigation showed that bleached hair (one-time bleached) is more damaged by the action of acid straightening than virgin hair, which was shown by a threefold increase in the percentage of total porosity of the tresses. The obtained results demonstrate that the investigation approach proposed here can provide very important thermodynamic and structural information on induced changes of hair structure, and certainly can be applied for the evaluation of the action mode and efficiency of cosmetic treatments.

RFT1-CDG: Absence of Epilepsy and Deafness in Two Patients with Novel Pathogenic Variants.

This report is on two novel patients with RFT1-CDG. Their phenotype is characterized by mild psychomotor disability, behavioral problems, ataxia, and mild dysmorphism. Neither of them shows signs of epilepsy, which was observed in all RFT1-CDG patients reported to date (n = 14). Also, deafness, which is often associated with this condition, was not observed in our patients. Molecular analysis of RFT1 showed biallelic missense variants including three novel ones: c.827G > A (p.G276D), c.73C > T (p.R25W), and c.208T > C (p.C70R).

Nutritional status in patients with phenylketonuria using glycomacropeptide as their major protein source.

BACKGROUND/OBJECTIVES: Low phenylalanine (PHE), glycomacropeptide-based protein substitute (GMP) is an alternative to traditional L-amino acid supplements (AA) used in the dietary management of phenylketonuria (PKU). In a retrospective, longitudinal study, we report the nutritional status of PKU patients taking AA and GMP. SUBJECTS/METHODS: Eleven PKU patients aged 27±10 years (1 HPA, 4 mild and 6 classical PKU) on dietary treatment were evaluated (anthropometry, body composition, blood pressure measurements, biochemical markers including vitamin, mineral, lipids, carbohydrates and protein status/metabolism, and nutritional intake assessment) at two different annual reviews. The mean time taking AA was 13±5 months and GMP 13±7 months. Blood phenylalanine (PHE) and tyrosine (TYR) were analysed before and after GMP introduction. RESULTS: Both GMP and AA protein substitutes provided similar protein equivalent intake (0.85 vs 0.75 g/kg/day, P=0.182). In the GMP group, it contributed 57% (27-100%) of the protein substitute intake (with AA delivering the rest of protein substitute intake), providing an additional 34±12 mg/day PHE. Nutritional intake, anthropometry and body composition measurements were similar in both the groups. Median blood PHE did not change (P=0.594), although values within target range improved (36 vs 46%), but this was not statistically significant. Mean blood TYR increased (52.0±19.2 vs 63.2±25.6 µmol/l, P=0.033), and all biochemical markers remained stable, except for a lower A1C haemoglobin (P=0.011). CONCLUSIONS: Partial GMP contribution to total protein substitute intake did not affect nutritional status in patients with PKU. Blood PHE control was not adversely affected. The increased blood TYR after GMP introduction necessitates further study.

Living with inborn errors of cholesterol biosynthesis: lessons from adult patients.

In the last decades, nine inherited errors of the distal part of cholesterol biosynthesis have been recognized. Affected patients present complex malformation syndromes involving different organs and systems with variable degrees of severity. We report on the phenotype evolution of three patients with enzymatic defects at three distinct steps of such pathway: Smith-Lemli-Opitz syndrome, X-linked dominant chondrodysplasia punctata type 2 and congenital hemidysplasia with ichthyosiform erythroderma and limb defects syndrome. The patients' natural history, from childhood to adulthood, is thoroughly described in order to contribute for a better knowledge of these diseases. Our ultimate goals are to contribute for a better characterization of the long-term course of these metabolic disorders and for the recognition of such diseases in older patients.

Morphometric and quantitative analysis of theafferent renal artery variation

The kidney is a retroperitoneal organ that weight from 125 to 170 g in the adult men and 115to 155 g in adult women. Irrigation kidney is characterized by the presence of large anatomical variability thatmay be influenced by ethnic and to a lesser extent by gender. Among the variations, there may be the presenceof an accessory renal artery that is projected into the upper or lower end of the kidney. This research aimsto observe the incidence of anatomical variations of the afferent renal artery and quantify both right and leftkidney weight.Materials and Methods:We analyzed kidney weights and accessory renal artery variations in48 adult kidneys of both genders obtained from Institute of Anatomy of the University of Severino Sombra.Subsequently, we compared the mean weights of kidneys in order to ascertain whether there was significantdiscrepancy between the left and the right kidney. For this, we performed the Student t test considering aP < 0.05.Results:The mean weight of the right kidney was 140.4 ± 22.6 g and the left was 148.8 ± 20.5 g.In 40% of right kidneys was observed anatomic variation with the presence of accessory renal artery. To the leftkidney was observed a variation of 35%.Conclusion:We found that the accessory renal artery, when present,was more closely related to the end of the kidney especially in the right kidney.

Methionine Adenosyltransferase I/III Deficiency in Portugal: High Frequency of a Dominantly Inherited Form in a Small Area of Douro High Lands.

Methionine adenosyltransferase deficiency (MAT I/III deficiency) is an inborn error of metabolism resulting in isolated hypermethioninemia, and usually inherited as an autosomal recessive trait, although a dominant form has been reported in several families.During the last 6 years, approximately 520,000 newborns were screened in the Portuguese Newborn Screening Laboratory by MS/MS, and 21 cases of persistent hypermethioninemia were found. One case was confirmed to be a deficiency of cystathionine ß-synthase and 20 cases were confirmed by MAT1A gene analysis to have an elevation of methionine due to MAT I/III deficiency, which indicates an incidence for this condition of 1/26,000. Twelve of the MAT I/III deficient newborns, belonging to 11 families, were identified in the northern region of Portugal and sent to the same treatment center, where they are under follow-up. Clinical, biochemical, and genetic characteristics of individuals from these 11 families are presented. Plasma methionine and homocysteine concentrations were found to be moderately increased in all newborns, and molecular analysis revealed that they all were heterozygous for R264H mutation. Normal growth, development, and neurological examination were observed in all cases, and cerebral MRI performed in six cases revealed myelination abnormalities in one case. Plasma methionine concentration for all 12 cases was always below 300 µM, and they are all on a normal diet for their age.

Abundance and stratification of soil macroarthropods in a Caatinga Forest in Northeast Brazil.

In arid and semiarid environments, seasonality usually exerts a strong influence on the composition and dynamics of the soil community. The soil macroarthropods were studied in a Caatinga forest located in the Reserva Particular do Patrimônio Natural (RPPN) Fazenda Almas, São José dos Cordeiros, Paraíba, Brazil. Samples were collected during the dry and rainy seasons following the method proposed by the Tropical Soil Biology and Fertility Program (TSBF), with minor modifications. At each station, 15 soil blocks (20 × 20 × 30 cm: 12 L) were extracted and divided into three layers: A (0-10 cm), B (10-20 cm), and C (20-30 cm). In the rainy and dry seasons 1,306 ± 543(se) and 458 ± 212 ind.m-2 macroarthropods were found, respectively, with 35 and 18 respective taxa recorded. The abundance of individuals and taxa were significantly higher in the rainy season. Isoptera (57.8%) was the most abundant taxon, followed by Hymenoptera: Formicidae (17.2%), Coleoptera larvae (7.3%), and Araneae (3.5%). In the rainy season, abundance in layer A (576 ± 138 ind.m-2) was significantly higher than that of layer C (117 ± 64 ind.m-2), but was not different from layer B (613 ± 480 ind.m-2). There was also no difference between the layer B and C abundances. In the dry season, abundance in layer B (232 ± 120 ind.m-2) was not significantly different compared to layer A (182 ± 129 ind.m-2), but was significantly higher than abundance in layer C (44 ± 35 ind.m-2). During the rainy season, layer A (34 taxa) was significantly richer in taxa than layers B (19 taxa) and C (11 taxa). On the other hand, during the dry season the richness of layers A (12 taxa) and B (12 taxa) was equal, but significantly higher than that of layer C (6 taxa). Richness of taxa and abundance were positively correlated with soil organic matter and negatively correlated with soil temperature. The community of soil macroarthropods in the area of Caatinga studied has taxonomic and functional structures that are relatively complex and is therefore likely to exert an influence on ecosystem productivity due to its physical effects on soil profile and necromass fragmentation, as occurs in other arid and semiarid ecosystems throughout the world.

[Pseudohypoparathyroidism type Ia - a novel mutation]. / Seudohipoparatiroidismo tipo Ia. Una mutación original.

Pseudohypoparathyroidism Ia (PHP-Ia) results from a specific deficiency of the alpha subunit of stimulatory G protein, manifested by resistance to parathormone and a characteristic phenotype, referred to as Albright hereditary osteodystrophy (AHO). Several mutations were identified in the GNAS1 gene in individuals with PHP-Ia and pseudopseudohypoparathyroidism (PPHP). A single GNAS1 mutation may be responsible for both PHP-Ia e PPHP in the same family, when inherited from the maternal and the paternal allele, respectively. The authors present the case of a teenage boy with PHP- Ia. The study revealed the GNAS1 mutation c.899A >T (p.Lys300Ile) in exon 11. After the genetic study of his parents, we have identified the same mutation in the mother, who had only somatic alterations (AHO), not associated with hormone resistance (PPHP). This is an original mutation, not yet described in the literature.

Solitaire stent for endovascular treatment of intracranial aneurysms: immediate and mid-term results in 15 patients with 17 aneurysms.

INTRODUCTION: The Solitaire stent is the first fully retractable stent for endovascular treatment (EVT) of intracranial aneurysms. The aim of this study was to evaluate its use in a prospective series with mid-term follow-up. METHODS: A retrospective review of our prospectively maintained database identified all patients treated with a Solitaire stent. Clinical charts, procedural data, angiographic results were reviewed. RESULTS: Between June 2008 and September 2009, 15 patients with 17 wide-necked or fusiform aneurysms (16 unruptured/one ruptured) were identified. EVT was successfully performed in all but one patient in whom the stent was removed because it induced flow reduction in the 1.8-mm parent artery. Among 14 treated patients, 13 had an excellent outcome and one had a good outcome. In this latter patient, the first stent could not be delivered and was changed for another one that was successfully deployed. The patient experienced a thrombo-embolic complication 6 hours after EVT and kept a slight hand paresis. In all cases but one, the stent was thus easily navigated and positioned despite a relative poor visibility. Angiographic results included eight complete occlusions, two neck remnants, and six incomplete occlusions. Six-month control in 14 aneurysms showed 13 complete occlusions and one incomplete occlusion. CONCLUSION: The Solitaire stent is useful for EVT of complex intracranial aneurysms because it is fully retractable, easy to navigate and to precisely place. However, it should be used with caution in arteries less than 2mm in diameter.

Endovascular treatment of anterior choroidal artery aneurysms.

INTRODUCTION: Endovascular treatment (EVT) of anterior choroidal artery aneurysms (AChAA) may be challenging because of the close relationship with the parent artery. The aim of this study was to report our experience with EVT of AChAA. METHODS: A retrospective review of our prospectively maintained database identified all AChAA treated by embolization. The clinical charts, procedural data and angiographic results were reviewed. RESULTS: From April 2004-August 2008, 11 patients were identified. Five patients presented with a subarachnoid hemorrhage (SAH) and six patients were asymptomatic. Aneurysms size varied from two to 13 mm (mean size=3.6 mm) and nine had an unfavourable neck/sac ratio (>or=0.7). The anterior choroidal artery was arising from the sac (n=6) or from the neck (n=5). Endovascular treatment consisted of balloon-assisted coiling (n=8), coiling (n=2) and stent-assisted coiling (n=1). No procedural complication occurred and all patients had an excellent outcome except one patient who died because of severe vasospasm 8 days after an uneventful EVT. Immediate angiographic control showed six complete occlusions, one neck remnant and four incomplete occlusions. Follow-up controls (mean=18 months) were obtained in eight patients and showed five stable occlusions and three further thrombosis achieving complete occlusion. CONCLUSION: EVT of AChAA is associated with very good clinical and long-term anatomical results. Because of their small size, unfavourable neck/sac ratio and close relationship with the parent artery, EVT frequently requires the use of adjunctive techniques such as balloon or stent-assisted coiling.

Unilateral hypopharyngitis, cellulitis, and a multinodular goiter: a triad of findings suggestive of acute suppurative thyroiditis.

Acute suppurative thyroiditis is an uncommon disorder, which has been associated with pre-existing thyroid disease, especially a multinodular goiter. We describe a case of a woman who presented clinically with an acute inflammatory condition of the neck. On CT examination, she had a triad of a multinodular goiter, a unilateral hypopharyngitis, and a surrounding cellulitis. We suggest that the constellation of these imaging findings should alert the radiologist to the diagnosis of acute thyroiditis.

Prevalence of pulmonary hypertension in patients with schistosomal liver fibrosis.

Between the April and July of 2007, patients undergoing treatment for schistosomal liver fibrosis, at a university hospital in north-eastern Brazil, were examined by transthoracic Doppler echocardiography (TTE). The main aim was to determine the prevalence of pulmonary hypertension in the patients. The thorax of each patient who had such hypertension, as indicated by an estimated pulmonary arterial systolic pressure (PASP) in excess of 35 mmHg, was then investigated by contrast-enhanced multidetector-row computed tomography (MDCT). The 84 patients (53 women and 31 men) enrolled in the study had a mean (S.D.) age of 50.06 (12.25) years. Nine (10.7%, with a 95% confidence interval of 5.0%-19.4%) of the patients were found to have pulmonary hypertension, with PASP ranging from 40-126 mmHg, and a median (S.D.) PASP of 58.78 (28.01) mmHg. The contrast-enhanced thoracic MDCT indicated that most of the patients with pulmonary hypertension had a pulmonary artery trunk that was unusually wide (67%) and more than 1.1-fold wider than the ascending aorta (56%), dilatation of the main pulmonary arteries (100%), a segmental artery that, in diameter, was more than 1.1-fold larger than the adjacent bronchi (89%), tapering of the peripheral pulmonary arteries (78%), and cardiac enlargement (78%). No patient suffered pulmonary embolism as a result of the investigations. The prevalence of pulmonary hypertension in the patients with schistosomal liver fibrosis (10.7%) justifies the screening of such patients by TTE.

Treatment of vasospasm secondary to subarachnoid hemorrhage using intra-arterial nimodipine in low dosage.

SUMMARY: There are currently no consensus criteria to define the dosage of intra-arterial Nimodipine to be used to obtain permanent remission of vasospasm secondary to subarachnoid haemorrhage. The purpose of this paper is to establish a mathematical relation between the number of vessels affected by this kind of vasospasm and the Nimodipine dose that must be used for intra- arterial angioplasty to obtain permanent remission of the vasospasm.

On the ontogeny and physiology of regulatory T cells.

Lymphocytes can interfere with the activity of other lymphocytes in a thousand and one ways. A particular subset of so-called regulatory CD4+ T cells is capable of controlling the activity of other lymphocytes in yet another way. Their function is primarily defined by the ability to protect the integrity of tissues and organs in vivo. This was demonstrated in experimental models of natural tolerance to peripheral tissues, transplantation tolerance and the regulation of immune responses promoted by exogenous antigens at the level of the intestinal mucosa. Moreover, regulatory T cells also play a major role in the systemic homeostatic mechanisms that control total lymphocyte numbers. There is good evidence to support the contention that a significant fraction of the naturally occurring regulatory T cells is generated in the thymus following selection mediated by high avidity T-cell receptor/ligand interactions. Symbolically, self-reactive regulatory T cells do represent the breakthrough of concepts challenging the long-lasting Burnetian dogma that all autoreactive cells should be eliminated or inactivated. Although clonal deletion of self-reactive cells is a fundamental process in T-cell development, controlled autoreactivity is part of the physiology of the immune system. Thus, autoreactive regulatory T cells also protect immunologists from the desperate hunting for the evil of horror autotoxicus.

Embryonic thymic epithelium naturally devoid of APCs is acutely rejected in the absence of indirect recognition.

Transplants of tissues depleted of passenger leukocytes are upon in vitro culture usually accepted in allogeneic recipients. Accordingly, fully allogeneic embryonic thymic epithelium was suggested to be poorly immunogenic. However, this tissue is capable of inducing donor-specific tolerance to peripheral tissues, when restoring T cell development in nude mice, through the production of regulatory cells. In the present work, adult immunocompetent allogeneic recipients were grafted with embryonic tissues isolated at stages before hemopoietic colonization or even before the establishment of circulation. Allogeneic thymic epithelium of day 10 embryos and heart primordium of day 8 embryonic donors were always rejected. Acute rejection of the thymic anlagen takes place in less than 12 days, with maximal CD4(+) and CD8(+) T cell infiltrates at 10 days post-transplant. In addition, a significant infiltrate of NK1.1(+) cells is observed, although without any essential role in this process. Furthermore, recipients lacking the indirect pathway of Ag presentation to CD4(+) T cells do not reveal any significant delay in rejection, even when CD8(+) T cells are also eliminated. Thus, our experimental approach reveals acute allograft rejection in the absence of all known pathways of naive T cell activation and therefore unveils a novel graft rejection mechanism that should be mediated by direct recognition of parenchymal cells. Given the importance of dendritic cells in naive T cell activation, it is likely that cross-reactive memory T cells may also drive rejection.

Major histocompatibility complex class II-positive cortical epithelium mediates the selection of CD4(+)25(+) immunoregulatory T cells.

CD4(+)25(+) T cells are a unique population of immunoregulatory T cells which are critical for the prevention of autoimmunity. To address the thymic selection of these cells we have used two models of attenuated thymic deletion. In K14-A(beta)(b) mice, major histocompatibility complex (MHC) class II I-A(b) expression is limited to thymic cortical epithelium and deletion by hematopoietic antigen-presenting cells does not occur. In H2-DMalpha-deficient mice, MHC class II molecules contain a limited array of self-peptides resulting in inefficient clonal deletion. We find that CD4(+)25(+) T cells are present in the thymus and periphery of K14-A(beta)(b) and H2-DMalpha-deficient mice and, like their wild-type counterparts, suppress the proliferation of cocultured CD4(+)25(-) effector T cells. In contrast, CD4(+)25(+) T cells from MHC class II-deficient mice do not suppress responder CD4(+) T cells in vitro or in vivo. Thus, development of regulatory CD4(+)25(+) T cells is dependent on MHC class II-positive thymic cortical epithelium. Furthermore, analysis of the specificities of CD4(+)25(+) T cells in K14-A(beta)(b) and H2-DMalpha-deficient mice suggests that a subset of CD4(+)25(+) T cells is subject to negative selection on hematopoietic antigen-presenting cells.

CD25+ CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10.

The mechanisms by which the immune system achieves constant T cell numbers throughout life, thereby controlling autoaggressive cell expansions, are to date not completely understood. Here, we show that the CD25(+) subpopulation of naturally activated (CD45RB(low)) CD4 T cells, but not CD25(-) CD45RB(low) CD4 T cells, inhibits the accumulation of cotransferred CD45RB(high) CD4 T cells in lymphocyte-deficient mice. However, both CD25(+) and CD25(-) CD45RB(low) CD4 T cell subpopulations contain regulatory cells, since they can prevent naive CD4 T cell-induced wasting disease. In the absence of a correlation between disease and the number of recovered CD4(+) cells, we conclude that expansion control and disease prevention are largely independent processes. CD25(+) CD45RB(low) CD4 T cells from IL-10-deficient mice do not protect from disease. They accumulate to a higher cell number and cannot prevent the expansion of CD45RB(high) CD4 T cells upon transfer compared with their wild-type counterparts. Although CD25(+) CD45RB(low) CD4 T cells are capable of expanding when transferred in vivo, they reach a homeostatic equilibrium at lower cell numbers than CD25(-) CD45RB(low) or CD45RB(high) CD4 T cells. We conclude that CD25(+) CD45RB(low) CD4 T cells from nonmanipulated mice control the number of peripheral CD4 T cells through a mechanism involving the production of IL-10 by regulatory T cells.

High prevalence of giardiasis and stronglyloidiasis among HIV-infected patients in Bahia, Brazil.

Diarrhea due to intestinal microbial infections is a frequent manifestation among HIV-infected patients. It has been postulated that HIV-infected patients may have special types of intestinal infections, and that immune activation from such parasites may affect the progression of HIV disease. To evaluate these associations, the frequency of infections was examined in HIV-infected patients in Bahia, Brazil. To determine the potential impact of the presence of intestinal parasitic infections on HIV disease progression, a retrospective study approach was used. The medical charts of 365 HIV-infected patients who had been treated at the AIDS Clinic of the Federal University of Bahia Hospital were reviewed, and the prevalence of parasites was compared with 5,243 HIV-negative patients who had attended the hospital during the same period of time. Among HIV-infected subjects, CD(4) count, RNA plasma viral load (VL), and number of eosinophils were compared according to their stool examination results. The overall prevalence of each parasite was similar for HIV-positive and HIV-negative patients. However, the prevalence of S. stercoralis (p<10(-7)) and G. lamblia (p=0.005) was greater for HIV-infected subjects. The mean CD(4) count and viral load of HIV patients in our clinic who had stool examinations was 350 cells +/- 340 and 4.4 +/- 1.4 log RNA viral load, respectively. In this patient group there was no clear association between the level of the absolute CD(4) count or the viral load and a specific parasitic infection. The presence of an intestinal parasitic infection was not associated with faster progression of the HIV disease among HIV-infected patients. We conclude that strongyloidiasis and giardiasis are more frequent in HIV-infected patients in Bahia, Brazil. If this association is due to immune dysregulation, as has been proposed elsewhere, it must occur in patients after only minor shifts in CD(4) count from normal levels, or as a result of immune dysfunction not represented by CD(4) count. These infections do not appear to alter the progression of HIV disease.