Multiple endocrine neoplasia type 1 in Brazil: MEN1 founding mutation, clinical features, and bone mineral density profile

Objective: Only few large families with multiple endocrine neoplasia type 1 (MEN1) have been documented. Here, we aimed to investigate the clinical features of a seven-generation Brazilian pedigree. which included 715 at-risk family members. Design: Genealogical and geographic analysis was used to identify the MEN1 pedigree. Clinical and genetic approach was applied to characterize the phenotypic and genotypic features of the family members. Results: Our genetic data indicated that a founding mutation in the MEN1 gene has occurred in this extended Brazilian family. Fifty family members were diagnosed with MEN1. Very high frequencies of functioning and non-functioning MEN1-related tumors were documented and the prevalence of prolactinoma (29.6%) was similar to that previously described in prolactinoma-variant Burin (32%). In addition, bone mineral density analysis revealed severe osteoporosis (T,-2.87 +/- 0.32) of compact bone (distal radius) in hyperparathyroidism (HPT)/MEN1 patients. while marked bone mineral loss in the lumbar spine (T,-1.95 +/- 0.39). with most cancellous bone, and femoral neck (mixed composition: T,-1.48 +/- 0.27) were also present. Conclusions: In this study, we described clinically and genetically the fifth largest MEN1 family in the literature. Our data confirm previous findings suggesting that prevalence of MEN1-related tumors in large families may differ from reports combining cumulative data of small families. Furthermore. we were able to evaluate the bone status in HPT/MEN1 cases, a subject that has been incompletely approached in the literature. We discussed the bone loss pattern found in our MEN1 patients comparing with that of patients with sporadic primary HPT.

Cytogenetic and Molecular Evaluation and 20-Year Follow-Up of a Patient With Ring Chromosome 14

We present a 20-year follow-up on a patient with a ring chromosome 14. The ring chromosome was studied by fluorescence in-situ hybridization (FISH), multiplex-ligation probe amplification (MLPA), and genome wide SNP array, and no deletions of chromosome 14 were detected, although the telomeric repeat sequence was absent from the ring chromosome. The patient had skeletal abnormalities, and susceptibility to infections, as well as seizures and retinal pigmentation, which are commonly found in individuals with a ring 14. Our patient corroborates the idea that even when no genes are lost during ring formation, a complete ring chromosome can produce phenotypic alterations, which presumably result from ring instability or gene silencing due to the new chromosomal architecture. (C) 2010 Wiley-Liss, Inc.

Further delineation of the 15q13 microdeletion and duplication syndromes: a clinical spectrum varying from non-pathogenic to a severe outcome

Background: Recurrent 15q13.3 microdeletions were recently identified with identical proximal (BP4) and distal (BP5) breakpoints and associated with mild to moderate mental retardation and epilepsy. Methods: To assess further the clinical implications of this novel 15q13.3 microdeletion syndrome, 18 new probands with a deletion were molecularly and clinically characterised. In addition, we evaluated the characteristics of a family with a more proximal deletion between BP3 and BP4. Finally, four patients with a duplication in the BP3-BP4-BP5 region were included in this study to ascertain the clinical significance of duplications in this region. Results: The 15q13.3 microdeletion in our series was associated with a highly variable intra-and inter-familial phenotype. At least 11 of the 18 deletions identified were inherited. Moreover, 7 of 10 siblings from four different families also had this deletion: one had a mild developmental delay, four had only learning problems during childhood, but functioned well in daily life as adults, whereas the other two had no learning problems at all. In contrast to previous findings, seizures were not a common feature in our series (only 2 of 17 living probands). Three patients with deletions had cardiac defects and deletion of the KLF13 gene, located in the critical region, may contribute to these abnormalities. The limited data from the single family with the more proximal BP3-BP4 deletion suggest this deletion may have little clinical significance. Patients with duplications of the BP3-BP4-BP5 region did not share a recognisable phenotype, but psychiatric disease was noted in 2 of 4 patients. Conclusions: Overall, our findings broaden the phenotypic spectrum associated with 15q13.3 deletions and suggest that, in some individuals, deletion of 15q13.3 is not sufficient to cause disease. The existence of microdeletion syndromes, associated with an unpredictable and variable phenotypic outcome, will pose the clinician with diagnostic difficulties and challenge the commonly used paradigm in the diagnostic setting that aberrations inherited from a phenotypically normal parent are usually without clinical consequences.

CYLD mutations in familial skin appendage tumours

Background: Germ-line mutations in CYLD are found in patients with familial skin appendage tumours. The protein product functions as a deubiquitinase enzyme, which negatively regulates NF-kappa B and c-Jun N-terminal kinase signalling. Brooke-Spiegler syndrome (BSS) is characterised by cylindromas, trichoepitheliomas and spiradenomas, whereas in familial cylindromatosis (FC) patients present with cylindromas and in multiple familial trichoepitheliomas (MFT) with trichoepitheliomas as the only skin tumour type. Although described as distinct entities, recent studies suggest that they are within the spectrum of a single entity. Objective: To investigate the mutation spectrum of CYLD and possible genotype-phenotype correlations. Methods: 25 families including 13 BSS, 3 FC, and 9 MFT families were examined and evaluated for mutations in the CYLD gene. Results: In total, 18 mutations in CYLD, including 6 novel mutations, were identified in 25 probands (72%). The mutation frequencies among distinct phenotypes were 85% for BSS, 100% for FC, and 44% for MFT. The majority of the mutations were insertions, deletions or nonsense mutations leading to formation of truncated proteins. All mutations were located between exons 9 to 20, encoding the NEMO binding site and the catalytic domain. Genotype-phenotype analysis failed to reveal a correlation between the types of mutations and their location within the gene and the patients` phenotypes and disease severity. Conclusions: This study provides further evidence on the role of CYLD in the pathogenesis of skin appendage tumours characterised by cylindromas, trichoepitheliomas and/or spiradenomas, but the molecular mechanisms of CYLD in skin tumorigenesis and the reasons for phenotypic variability remain to be explored.

Endocrine signatures underlying plasticity in postembryonic development of a lower termite, Cryptotermes secundus (Kalotermitidae)

Wood-dwelling termites are characterized by an extremely high and unique developmental flexibility that allows workers, which are immatures, to explore all caste options. The endocrine signatures underlying this flexibility are only vaguely understood. We determined juvenile hormone (JH) and ecdysteroid hemolymph titers during postembryonic development and in terminal instars of the drywood termite Cryptotermes secundus using field and laboratory colonies. Postembryonic development is characterized by a drop in JH titers at the transition from larval (individuals without wing buds) to nymphal (individuals with wing buds) instars. JH titers were low in winged sexuals and reproducing primary reproductives (< 200 pg/mu l) but were by an order of magnitude higher in neotenic replacement reproductives. The unique regressive molts of termites seem to be characterized by elevated JH titers, compared with progressive or stationary molts. Ecdysteroid titers were generally low in nymphal instars and in primary reproductives (< 50 pg/mu l). It was only during the third and fourth nymphal instars and in winged sexuals where some individuals showed elevated ecdysteroid titers. These results are the most comprehensive endocrinological data set available for any lower termite, with the potential to serve as baseline for understanding the extreme developmental flexibility underlying the evolution of social life in termites.

Turbulent blood flow plays an essential localizing role in the development of atherosclerotic lesions in experimentally induced hypercholesterolaemia in rats

Taking into account that atherosclerosis is a focal disease and high levels of plasma cholesterol are closely correlated with its pathogenesis, it is a challenge to explain how equal concentrations of cholesterol bathing the endothelium can produce local, rather than global, effects on arteries. The focal distribution of atherosclerotic lesions has been considered to be dependent, at least in part, on hydrodynamic factors. The present study was carried out to further test the hypothesis that these forces are an important localizing factor in rats feeding a hypercholesterolaemic diet and submitted to infra-diaphragmatic aortic constriction. These animals develop a normotensive prestenotic region with laminar blood flow that serves as control for a normotensive poststenotic region with turbulent blood flow. Our findings clearly demonstrated that the combination of turbulent blood flow and low wall shear stress (WSS) in the presence of hypercholesterolaemia and oxidative stress creates conditions to the formation of focally distributed incipient atherosclerotic lesions observed in the poststenotic segment. In contrast, only diffuse fatty streaks could be observed in the normotensive prestenotic segment with laminar blood flow and normal WSS in the presence of hypercholesterolaemia and oxidative stress. Although haemodynamic forces are not by themselves responsible for the pathogenesis of atherosclerosis, they prime the local vascular wall in which the lesion develop. Further studies are required to establish how haemodynamic forces are detected and transduced into chemical signalling by the cells of the artery wall and then converted into pathophysiologically relevant phenotypic changes.

TAC3/TACR3 Mutations Reveal Preferential Activation of Gonadotropin-Releasing Hormone Release by Neurokinin B in Neonatal Life Followed by Reversal in Adulthood

Context: Mutations in TAC3 and TACR3 (encoding neurokinin B and its receptor) have been identified in Turkish patients with idiopathic hypogonadotropic hypogonadism (IHH), but broader populations have not yet been tested and genotype-phenotype correlations have not been established. Objective: A broad cohort of normosmic IHH probands was screened for mutations in TAC3/TACR3 to evaluate the prevalence of such mutations and define the genotype/phenotype relationships. Design and Setting: The study consisted of sequencing of TAC3/TACR3, in vitro functional assays, and neuroendocrine phenotyping conducted in tertiary care centers worldwide. Patients or Other Participants: 345 probands, 18 family members, and 292 controls were studied. Intervention: Reproductive phenotypes throughout reproductive life and before and after therapy were examined. Main Outcome Measure: Rare sequence variants in TAC3/TACR3 were detected. Results: In TACR3, 19 probands harbored 13 distinct coding sequence rare nucleotide variants [three nonsense mutations, six nonsynonymous, four synonymous (one predicted to affect splicing)]. In TAC3, one homozygous single base pair deletion was identified, resulting in complete loss of the neurokinin B decapeptide. Phenotypic information was available on 16 males and seven females with coding sequence variants in TACR3/TAC3. Of the 16 males, 15 had microphallus; none of the females had spontaneous thelarche. Seven of the 16 males and five of the seven females were assessed after discontinuation of therapy; six of the seven males and four of the five females demonstrated evidence for reversibility of their hypogonadotropism. Conclusions: Mutations in the neurokinin B pathway are relatively common as causes of hypogonadism. Although the neurokinin B pathway appears essential during early sexual development, its importance in sustaining the integrity of the hypothalamic-pituitary-gonadal axis appears attenuated over time. (J Clin Endocrinol Metab 95: 2857-2867, 2010)

Kallmann syndrome and mirror movements: White matter quantitative evaluation with magnetic resonance imaging

Kallmann syndrome (KS), characterized by the association of hypogonadotropic hypogonadism and anosmia, may present many other phenotypic abnormalities, including neurologic features as involuntary movements, called mirror movements (MM). MM etiology probably involves a complex mechanism comprising corticospinal tract abnormal development associated with deficient contralateral motor cortex inhibitory system. In this study, in order to address previous hypotheses concerning MM etiology, we identified and quantified white matter (WM) alterations in 21 KS patients, comparing subjects with and without MM and 16 control subjects, using magnetization transfer ratio (MTR) and T2 relaxometry (R2). Magnetization transfer and 12 double-echo images were acquired in a 1.5 T system. MTR and R2 were calculated pixel by pixel to initially create individual maps, and then, group average maps, co-registered with MNI305 stereotaxic coordinate system. After analysis of selected regions of interest, we demonstrated areas with higher 12 relaxation time and lower MTR values in KS patients, with and without MM, differently involving corticospinal tract projection, frontal lobes and corpus callosum. Higher MTR was observed only in pyramidal decussation when compared in both groups of patients with controls. In conclusion, we demonstrated that patients with KS have altered WM areas, presenting in a different manner in patients with and without MM. These data suggest axonal loss or disorganization involving abnormal pyramidal tracts and other associative/connective areas, relating to the presence or absence of MM. We also found a different pattern of alteration in pyramidal decussation, which can represent the primary area of neuronal disarrangement. (C) 2010 Elsevier B.V. All rights reserved.

Hypoxia modulates phenotype, inflammatory response, and leishmanial infection of human dendritic cells

Development of hypoxic areas occurs during infectious and inflammatory processes and dendritic cells (DCs) are involved in both innate and adaptive immunity in diseased tissues. Our group previously reported that macrophages exposed to hypoxia were infected with the intracellular parasite Leishmania amazonensis, but showed reduced susceptibility to the parasite. This study shows that although hypoxia did not alter human DC viability, it significantly altered phenotypic and functional characteristics. The expression of CD1a, CD80, and CD86 was significantly reduced in DCs exposed to hypoxia, whereas CD11c, CD14, CD123, CD49 and HLA-DR expression remained unaltered in DCs cultured in hypoxia or normoxia. DC secretion of IL-12p70, the bioactive interleukin-12 (IL-12), a cytokine produced in response to inflammatory mediators, was enhanced under hypoxia. In addition, phagocytic activity (Leishmania uptake) was not impaired under hypoxia, although this microenviroment induced infected DCs to reduce parasite survival, consequently controlling the infection rate. All these data support the notion that a hypoxic microenvironment promotes selective pressure on DCs to assume a phenotype characterized by pro-inflammatory and microbial activities in injured or inflamed tissues and contribute to the innate immune response.

Mineralocorticoid Receptor Mutations Differentially Affect Individual Gene Expression Profiles in Pseudohypoaldosteronism Type 1

Context: Type 1 pseudohypoaldosteronism (PHA1), a primary form of mineralocorticoid resistance, isdueto inactivating mutations of the NR3C2 gene, coding for the mineralocorticoid receptor (MR). Objective: The objective of the study was to assess whether different NR3C2 mutations have distinct effects on the pattern of MR-dependent transcriptional regulation of aldosterone-regulated genes. Design and Methods: Four MR mutations affecting residues in the ligand binding domain, identified in families with PHA1, were tested. MR proteins generated by site-directed mutagenesis were analyzed for their binding to aldosterone and were transiently transfected into renal cells to explore the functional effects on the transcriptional activity of the receptors by cis-trans-cotrans-activation assays and by measuring the induction of endogenous gene transcription. Results: Binding assays showed very low or absent aldosterone binding for mutants MR(877Pro), MR(848Pro), and MR(947stop) and decreased affinity for aldosterone of MR(843Pro). Compared with wildtype MR, the mutations p.Leu843Pro and p.Leu877Pro displayed half-maximal aldosterone-dependent transactivation of reporter genes driven by mouse mammary tumor virus or glucocorticoid response element-2 dependent promoters, whereas MR(848Pro) and MR(947stop) nearly or completely lost transcriptional activity. Although MR(848Pro) and MR(947stop) were also incapable of inducing aldosterone-dependent gene expression ofendogenoussgk1, GILZ, NDRG2, and SCNN1A, MR(843Pro) retained complete transcriptional activity on sgk1 and GILZ gene expression, and MR(877Pro) negatively affected the expression of sgk1, NDRG2, and SCNN1A. Conclusions: Our data demonstrate that MR mutations differentially affect individual gene expression in a promoter-dependent manner. Investigation of differential gene expression profiles in PHA1 may allow a better understanding of the molecular substrate of phenotypic variability and to elucidate pathogenic mechanisms underlying the disease. (J Clin Endocrinol Metab 96: E519-E527, 2011)

INCREASED SARCOLEMMAL PERMEABILITY AS AN EARLY EVENT IN EXPERIMENTAL SEPTIC CARDIOMYOPATHY: A POTENTIAL ROLE FOR OXIDATIVE DAMAGE TO LIPIDS AND PROTEINS

This study describes increased sarcolemmal permeability and myofilamentar damage that occur together with lipid peroxidation and protein nitration in the myocardium in severe sepsis induced by cecal ligation and puncture. Male C57BL/6 mice were submitted to moderate and severe septic injury and sham operation. Using light and laser confocal microscopy, diffuse foci of myocytolysis associated with focal disruption of the actin/myosin contractile apparatus could be seen in hearts with severe septic injury. The myocardial expressions of the sarcomeric proteins myosin and actin were downregulated by both severe and moderate injuries. The detection of albumin staining in the cytoplasm of myocytes to evaluate sarcolemmal permeability provided evidence of severe and mild injury of the plasma membrane in hearts with severe and moderate septic injury, respectively. The administration of a superoxide scavenger caused marked reduction of sarcolemmal permeability, indicating the involvement of free radicals in its genesis. On electron microscopy, these changes were seen to correspond to spread blocks of a few myocytes with fragmentation and dissolution of myofibrils, intracellular edema, and, occasionally, rupture of the sarcolemma. In addition, oxidative damage to lipids, using anti-4-hydroxynonenal, an indicator of oxidative stress and disruption of plasma membrane lipids, and to proteins, using antinitrotyrosine, a stable biomarker of peroxynitrite-mediated protein nitration, was demonstrated. These findings make plausible the hypothesis that increased sarcolemmal permeability might be a primary event in myocardial injury in severe sepsis possibly due to oxidative damage to lipids and proteins that could precede phenotypic changes that characterize a septic cardiomyopathy.

Systemic lupus erythematosus: Association with KIR and SLC11A1 polymorphisms, ethnic predisposition and influence in clinical manifestations at onset revealed by ancestry genetic markers in an urban Brazilian population

Systemic lupus erythematosus (SLE) is an autoimmune disorder of the connective tissue with a wide and heterogeneous spectrum of manifestations, with renal and neurological involvement usually related to worse prognosis. SLE more frequently affects females of reproductive age, and a high prevalence and renal manifestation seem to be associated with non-European ethnicity. The present study aims to investigate candidate loci to SLE predisposition and evaluate the influence of ethnic ancestry in the disease risk and clinical phenotypic heterogeneity of lupus at onset. Samples represented by 111 patients and 345 controls, originated from the city of Belem, located in the Northern Region of Brazil, were investigated for polymorphisms in HLA-G, HLA-C, SLC11A1, MTHFR, CASP8 and 15 KIR genes, in addition to 89 Amerindian samples genotyped for SLC11A1. We also investigated 48 insertion/deletion ancestry markers to characterize individual African, European and Amerindian ancestry proportions in the samples. Predisposition to SLE was associated with GTGT deletion at the SLC11A1 3`UTR, presence of KIR2DS2 +/KIR2DS5 +/KIR3DS1 + profile, increased number of stimulatory KIR genes, and European and Amerindian ancestries. The ancestry analysis ruled out ethnic differences between controls and patients as the source of the observed associations. Moreover, the African ancestry was associated with renal manifestations. Lupus (2011) 20, 265-273.

Cancer stem cell immunophenotypes in oral squamous cell carcinoma

Background: The presence of cancer stem cell (CSC) antigens can be evidenced in some human tumors by phenotypic analysis through immunostaining. This study aims to identify a putative CSC immunophenotype in oral squamous cell carcinoma (OSCC) and determine its influence on prognosis. Methods: The following data were retrieved from 157 patents: age, gender, primary anatomic site, smoking and alcohol intake, recurrence, metastases, histologic classification, treatment, disease-free survival (DFS), and overall survival (OS). An immunohistochemical study for CD44 and CD24 was performed in a tissue microarray of 157 paraffin blocks of OSCCs. Results: In univariate analysis, the immunostaining pattern showed significant influences in relation to OS for alcohol intake and treatment, as well as for the CD44+ and CD44-/CD24- immunophenotypes. The multivariate test confirmed these associations. Conclusions: Based on our results, the CD44 immunostaining and the absence of immunoexpression of these two investigated markers can be used in combination with other clinicopathologic information to improve the assessment of prognosis in OSCC.

Tissue diffusion and retention of metalloproteinases in ascending aortic aneurysms and dissections

Histopathological alterations in human aneurysms and dissections of the thoracic ascending aorta include areas of mucoid degeneration within the medial layer, colocalized with areas of cell disappearance and disruption of extracellular matrix elastic and collagen fibers. We studied the presence of matrix metalloproteinases in relation to their capacity to diffuse through the tissue or to be retained in areas of mucoid degeneration in aneurysms and dissections of the ascending aorta. Ascending aortas from 9 controls, 33 patients with aneurysms, and 14 with acute dissections, all collected at surgery, were analyzed. The morphological aspect was similar whatever the etiology or phenotypic expression of the pathological aortas, involving areas of extracellular matrix breakdown and cell rarefaction associated with mucoid degeneration. Release of proMMP-2, constitutively expressed by smooth muscle cells, was not different between controls and aneurysmal aortas, whereas the aneurysmal aortas released more of the active form. Release of pro and active MMP-9 was also similar between controls and aneurysmal aortas. Immunohistochemical staining of MMP-2 and MMP-9 was weak in both control and pathological aortas. In contrast, released MMP-7 (matrilysin) and MMP-3 (stromelysin-1) could not be detected in conditioned media but were present in tissue extracts with no detectable quantitative difference between controls and pathological aortas. Immunohistochemical staining of MMP-7 and MMP-3 revealed their retention in areas of mucoid degeneration, and semiquantitative evaluation of immunostaining showed more MMP-7 in pathological aortas than in controls. In conclusion, areas of mucoid degeneration, the hallmark of aneurysms, and dissections of thoracic ascending aortas, whatever their etiology, are not inert and can retain specific proteases. (c) 2009 Elsevier Inc. All rights reserved.

Physical therapy assessment tools to evaluate disease progression and phenotype variability in Golden Retriever muscular dystrophy

Dogs suffering from Golden Retriever muscular dystrophy (GRMD) present symptoms that are similar to human patients with Duchenne muscular dystrophy (DMD). Phenotypic variability is common in both cases and correlates with disease progression and response to therapy. Physical therapy assessment tools were used to study disease progression and assess phenotypic variability in dogs with GRMD. At 5 (TO), 9 (T1), 13 (T2) and 17 (T3) months of age, the physical features, joint ranges of motion (ROM), limb and thorax circumferences, weight and creatine kinase (CK) levels were assessed in 11 dogs with GRMD. Alterations of physical features were higher at 13 months, and different disease progression rates were observed. Passive ROM decreased until 1 year old, which was followed by a decline of elbow and tarsal ROM. Limb and thorax circumferences, which were corrected for body weight, decreased significantly between TO and T3. These measurements can be used to evaluate disease progression in dogs with GRMD and to help discover new therapies for DMD patients. (C) 2011 Elsevier Ltd. All rights reserved.