Context: Previous studies have shown that double RET mutations may be associated with unusual multiple endocrine neoplasia type 2 (MEN 2) phenotypes. Objective: Our objective was to report the clinical features of patients harboring a previously unreported double mutation of the RET gene and to characterize this mutation in vitro. Patients: Sixteen patients from four unrelated families and harboring the C634Y/Y791F double RET germline mutation were included in the study. Results: Large pheochromocytomas measuring 6.0-14 cm and weighing upto 640 g were identified in the four index cases. Three of the four tumors were bilateral. High penetrance of pheochromocytoma was also seen in the C634Y/Y791F-mutation-positive relatives (seven of nine, 77.7%). Of these, two cases had bilateral tumors, one presented with multifocal tumors, two cases had large tumors (>5 cm), and one case, which was diagnosed with a large (5.5 x 4.5 x 4.0 cm) pheochromocytoma, reported early onset of symptoms of the disease (14 yr old). The overall penetrance of pheochromocytoma was 84.6% (11 of 13). Development of medullary thyroid carcinoma in our patients seemed similar to that observed in patients with codon 634 mutations. Haplotype analysis demonstrated that the mutation did not arise from a common ancestor. In vitro studies showed the double C634Y/Y791F RET receptor was significantly more phosphorylated than either activated wild-type receptor or single C634Y and Y791F RET mutants. Conclusions: Our data suggest that the natural history of the novel C634Y/Y791F double mutation carries a codon 634-like pattern of medullary thyroid carcinoma development...

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide is used to kill phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91-phox, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients. This article lists all mutations identified in CYBB in the X-linked form of CGD. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of future disease-causing mutations. (C) 2010 Elsevier Inc. All rights reserved.; CGD Research Trust, London, UK; CGD Research Trust, London, UK; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[2005/59568]; Slovenian Research Agency[L3-0624]; Slovenian Research Agency, Slovenia; U.S. National Institutes of Health (NIH); National Cancer Institute, National Institutes of Health (NIH)[HHSN261200800001E]

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Aim: The aim of this study was to investigate genetic changes of the TP53 (tumor protein p53) and FHIT (fragile histidine triad) genes in precursor lesion such as chronic esophagitis (CE) and in esophageal squamous cell carcinoma (ESCC). Materials and Methods: PCR-Single Strand Conformation Polymorphism (SSCP) analysis and DNA sequencing in 30 CE and 10 ESCC specimens were performed. Results: DNA sequencing indicated two novel mutations in the TP53 exons 5 (codon 147) and 6 (codon 197) in 219 SSCP positive cases of ESCC, but no mutation was found in the CE. The SIFT (Sorting Intolerant From Tolerant) web-based program showed that missense variant at codon 197 of TP53 could affect the protein function. Additionally, polymorphisms of the TP53 exon 4 (codon 36 and 72) and of the FHIT exon 7 (codon 88) were observed in 4/11(36%) cases of CE and 619 (67%) SSCP positive cases of ESCC after DNA sequencing. Conclusion: TP53 gene mutations are not common events in CE, but are frequent in ESCC, and as polymorphisms of TP53 and FHIT may confer a greater risk for the development of esophageal carcinoma, further studies are required.

O CASR pertence à família C dos receptores que se acoplam à proteína G e é ativado quando interage com o cálcio extracelular, sendo responsável pelo ajuste do ?set point? do cálcio extracelular por meio da regulação da secreção de PTH e excreção de cálcio. Mutações no Receptor Sensor de Cálcio (CASR) estão associadas a FHH (Hipercalcemia Hipocalciúrica Familiar) e NSHTP (Hiperparatireoidismo Neonatal Grave) quando inativadoras do receptor e ADH (Hipoparatireoidismo Autossômico Dominante) quando ativadoras. O Hiperparatireoidismo Neonatal Grave (NSHPT) é uma doença rara caracterizada por calcemias elevadas, próximas às consideradas incompatíveis à vida, associada ao aumento da concentração de PTH, desmineralização óssea grave e sintomas neonatais como hipotonia e baixo ganho ponderal. Trata-se de uma doença familiar, com pais portadores de Hipercalcemia Hipocalciúrica Familiar (FHH), uma doença autossômica dominante, geralmente assintomática, com calcemias elevadas ou no limite superior da normalidade, associada a concentrações de PTH normais, porém não suprimidas e hipocalciúria. ADH, por sua vez, cursam com desregulação no ajuste da concentração de cálcio extracelular, onde baixas concentrações de cálcio ativam o receptor e inibem a secreção de PTH pelas paratireóides e aumentam a excreção de cálcio pelos rins. Indivíduos afetados apresentam hipocalcemia...

Background: The diagnosis of Rett syndrome (RTT) is based on a set of clinical criteria, irrespective of mutation status. The aims of this study were (1) to define the clinical differences existing between patients with Rett syndrome with (Group I) and without a MECP2 mutation (Group II), and (2) to characterize the phenotypes associated with the more common MECP2 mutations. Patients and methods: We analyzed 87 patients fulfilling the clinical criteria for RTT. All were observed and videotaped by the same paediatric neurologist. Seven common mutations were considered separately, and associated clinical features analysed. Results: Comparing Group I and II, we found differences concerning psychomotor development prior to onset, acquisition of propositive manipulation and language, and evolving autistic traits. Based on age at observation, we found differences in eye pointing, microcephaly, growth, number of stereotypies, rigidity, ataxia and ataxic-rigid gait, and severity score. Patients with truncating differed from those with missense mutations regarding acquisition of propositive words and independent gait, before the beginning of the disease, and microcephaly, growth, foot length, dystonia, rigidity and severity score, at the time of observation. Patients with the R168X mutation had a more severe phenotype...

Missense mutations within the central DNA binding region of p53 are the most prevalent mutations found in human cancer. Numerous studies indicate that ‘hot-spot’ p53 mutants (which comprise ∼30% of human p53 gene mutations) are largely devoid of transcriptional activity. However, a growing body of evidence indicates that some non-hot-spot p53 mutants retain some degree of transcriptional activity in vivo, particularly against strong p53 binding sites. We have modified a previously described yeast-based p53 functional assay to readily identify such partial loss of function p53 mutants. We demonstrate the utility of this modified p53 functional assay using a diverse panel of p53 mutants.

The INK4a/ARF tumor suppressor locus is implicated in the senescence-like growth arrest provoked by oncogenic Ras in primary cells. INK4a and ARF are distinct proteins encoded by transcripts in which a shared exon is decoded in alternative reading frames. Here we analyze dermal fibroblasts (designated Q34) from an individual carrying independent missense mutations in each copy of the common exon. Both mutations alter the amino acid sequence of INK4a and functionally impair the protein, although they do so to different degrees. Only one of the mutations affects the sequence of ARF, causing an apparently innocuous change near its carboxy terminus. Unlike normal human fibroblasts, Q34 cells are not permanently arrested by Ras or its downstream effectors Ets1 and Ets2. Moreover, ectopic Ras enables the cells to grow as anchorage-independent colonies, and in relatively young Q34 cells anchorage independence can be achieved without addition of telomerase or perturbation of the p53 pathway. Whereas ARF plays the principal role in Ras-induced arrest of mouse fibroblasts, our data imply that INK4a assumes this role in human fibroblasts.

As the central component of the human endotoxin sensor, Toll-like receptor 4 (TLR4) functions in the early detection and response to Gram-negative infection. We therefore examined a large collection of patients with meningococcal sepsis, comparing the frequency of rare TLR4 coding changes to those in an ethnically matched control population. TLR2 sequences were also acquired and compared. Total nucleotide variation at TLR4 and TLR2 loci was assayed by using a novel computational method. A total of 3.01 megabases of coding sequence was captured at these loci from white subjects with or without meningococcal disease. Authentic mutations were found and high-quality, bidirectional coverage was measured across the coding region by using mutationseeker, a program specifically designed to assay locus-specific genetic load. Using a method that obviates the confounding effect of linkage disequilibrium, we observed that rare heterozygous missense mutations of TLR4 contribute to the development of systemic meningococcal disease among white populations of the southern United Kingdom (P = 0.02; odds ratio 8.2). When results from all white populations were pooled, an overwhelmingly significant excess of such mutations was observed among individuals with disease (P = 2 × 10−6; odds ratio 27.0). The common white TLR4 variant (TLR4B)...

Two genes (rpsL and rrs) with mutations associated with streptomycin resistance in Mycobacterium tuberculosis were characterized in 78 streptomycin-resistant and 61 streptomycin-susceptible isolates recovered from patients living in the United States, South America, Europe, Africa, and Asia. Fifty-four percent of the 78 resistant organisms had missense mutations in codon 43 of rpsL resulting in a K-43-->R substitution. Mutations in codon 88 of rpsL were also identified in four Asian isolates.

The Escherichia coli grpE gene (along with dnaK, dnaJ, groEL, and groES) was originally identified as one of the host factors required for phage lambda growth. The classical grpE280 mutation was the only grpE mutation that resulted from the initial screen and shown to specifically block the initiation of lambda DNA replication. Here we report the isolation of several new grpE missense mutations, again using phage lambda resistance as a selection. All mutants fall into two groups based on their temperature-dependent phenotype for lambda growth. Members of the first group (I), including grpE17 and grpE280, which was obtained again, are resistant to lambda growth at both 30 and 42 degrees C. Members of the second group (II), including grpE25, grpE66, grpE103, grpE13a, grpE57b, and grpE61, are sensitive to lambda growth at 30 degrees C but resistant at 42 degrees C. All mutations are recessive, since an E. coli grpE null mutant strain carrying these mutant alleles on low-copy-number plasmids are sensitive to infection by the lambda grpE+ transducing phage. Both group I and group II mutants are temperature sensitive for E. coli growth above 42 degrees C. The nucleotide changes were identified by sequencing analyses and shown to be dispersed throughout the latter 75% of the grpE coding region. Most of the amino acid changes occur at conserved residues...

X-linked chondrodysplasia punctata (CDPX) is a congenital disorder characterized by abnormalities in cartilage and bone development. Mutations leading to amino acid substitutions were identified recently in CDPX patients, in the coding region of the arylsulfatase E (ARSE) gene, a novel member of the sulfatase gene family. Transfection of the ARSE full-length cDNA, in Cos7 cells, allowed us to establish that its protein product is a 60-kD precursor, which is subject to N-glycosylation, to give a mature 68-kD form that, unique among sulfatases, is localized to the Golgi apparatus. Five missense mutations found in CDPX patients were introduced into wild-type ARSE cDNA by site-directed mutagenesis. These mutants were transfected into Cos7 cells, and the arylsulfatase activity and biochemical properties were determined, to study the effect of these substitutions on the ARSE protein. One of the mutants behaves as the wild-type protein. All four of the other mutations resulted in a complete lack of arylsulfatase activity, although the substitutions do not appear to affect the stability and subcellular localization of the protein. The loss of activity due to these mutations confirms their involvement in the clinical phenotype and points to the importance of these residues in the correct folding of a catalytically active ARSE enzyme.

Mutations in the Bruton agammaglobulinemia tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA). Unfolded or misfolded proteins can trigger stress pathways in the endoplasmic reticulum (ER), known as unfolded protein response (UPR). The aim was to clarify the involvement of UPR in XLA pathophysiology. By reverse transcription-quantitative PCR, we evaluated the expression of BTK and 12 UPR-related genes in eight patients. Moreover, we assessed the BTK protein expression and pattern in the patients' monocytes by flow cytometry and fluorescence immunocytochemistry. We found a reduced BTK expression in patients with stop codon mutations (P < 0.02). However, missense mutations did not affect BTK expression. Flow cytometry showed a reduction of BTK in patients which was corroborated by an absent or nonfunctional protein synthesis revealed by immunocytochemistry. In contrast with the other UPR-related genes, X-box binding protein 1 (XBP1) was markedly upregulated in the patients (P < 0.01), suggesting Toll-like receptor (TLR) activation since BTK directly interacts with TLRs as a negative regulator and XBP1 can be activated in direct response to TLR ligation. Different BTK mutations can be identified by the BTK expression. Inasmuch as UPR-related genes were downregulated or unaltered in patients...

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease. A recent study showed that male KLF10-encoded TGFβ Inducible Early Gene-1 knock-out mice (TIEG-/-) develop HCM with 13-fold up-regulation of PTTG1-encoded pituitary tumor-transforming gene 1. We hypothesized TIEG1 could be a novel candidate gene in the pathogenesis of genotype negative HCM in humans, possibly through a loss of its repression on PTTG1 expression. A cohort of 923 unrelated patients from two independent HCM centers was analyzed for mutations in TIEG's 4 translated exons using DHPLC and direct DNA-sequencing. Site directed mutagenesis was performed to clone novel variants. The effect of TIEG1 mutations on SMAD7 and PTTG1 promoters was studied using transient transfection and luciferase-assays. Altered expression of PTTG1 in cardiac tissue was studied by immunohistochemistry (IHC) to determine levels of PTTG1 protein in hypertrophic diseases. Six novel TIEG1 missense mutations were discovered in 6 patients (2 males/4 females, mean age at diagnosis 56.2 ± 23 years, MLVWT 20.8 ± 4 mm). Compared to WT TIEG1, 5 TIEG1 mutants significantly increased PTTG1 promoter function similar to TIEG1-/--mice. By IHC, PTTG1-protein expression was significantly increased in multiple models of hypertrophic cardiac disease...

Gene mutations that encode retinoschisin (RS1) cause X-linked retinoschisis (XLRS), a form of juvenile macular and retinal degeneration that affects males. RS1 is an adhesive protein which is proposed to preserve the structural and functional integrity of the retina, but there is very little evidence of the mechanism by which protein changes are related to XLRS disease. Here, we report molecular modeling of the RS1 protein and consider perturbations caused by mutations found in human XLRS subjects. In 60 XLRS patients who share 27 missense mutations, we then evaluated possible correlations of the molecular modeling with retinal function as determined by the electroretinogram (ERG) a- and b-waves. The b/a-wave ratio reflects visual-signal transfer in retina. We sorted the ERG b/a-ratios by patient age and by the mutation impact on protein structure. The majority of RS1 mutations caused minimal structure perturbation and targeted the protein surface. These patients' b/a-ratios were similar across younger and older subjects. Maximum structural perturbations from either the removal or insertion of cysteine residues or changes in the hydrophobic core were associated with greater difference in the b/a-ratio with age, with a significantly smaller ratio at younger ages...

Motivation: The folding free energy is an important characteristic of proteins stability and is directly related to protein's wild-type function. The changes of protein's stability due to naturally occurring mutations, missense mutations, are typically causing diseases. Single point mutations made in vitro are frequently used to assess the contribution of given amino acid to the stability of the protein. In both cases, it is desirable to predict the change of the folding free energy upon single point mutations in order to either provide insights of the molecular mechanism of the change or to design new experimental studies.

Cleidocranial dysplasia (CCD) is an autosomal dominant human skeletal disorder comprising hypoplastic clavicles, wide cranial sutures, supernumerary teeth, short stature, and other skeletal abnormalities. It is known that mutations in the human RUNX2 gene mapped at 6p21 are responsible for CCD. We analyzed the mutation patterns of the RUNX2 gene by direct sequencing in six Taiwanese index cases with typical CCD. One of the patients was a familial case and the others were sporadic cases. Sequencing identified four mutations. Three were caused by single nucleotide substitutions, which created a nonsense (p.R391X), two were missense mutations (p.R190W, p.R225Q), and the forth was a novel mutation (c.1119delC), a one-base deletion. Real time quantitative PCR adapted to determine copy numbers of the promoter, all exons and the 3'UTR region of the RUNX2 gene detected the deletion of a single allele in a sporadic case. The results extend the spectrum of RUNX2 mutations in CCD patients and indicate that complete deletions of the RUNX2 gene should be considered in those CCD patients lacking a point mutation detected by direct sequencing.

Publicación ISI; Context: T-3 action in neurons is essential for brain development. Recent evidence indicates that monocarboxylate transporter 8 (MCT8) is important for neuronal T-3 uptake. Hemizygous mutations have been identified in the X-linked MCT8 gene in boys with severe psychomotor retardation and elevated serum T-3 levels. Objective: The objective of this study was to determine the functional consequences of MCT8 mutations regarding transport of T-3. Design: MCT8 function was studied in wild-type or mutant MCT8-transfected JEG3 cells by analyzing: 1) T-3 uptake, 2) T-3 metabolism in cells cotransfected with human type 3 deiodinase, 3) immunoblotting, and 4) immunocytochemistry. Results: The mutations identified in MCT8 comprise four deletions (24.5 kb, 2.4 kb, 14 bp, and 3 bp), three missense mutations (Ala224Val, Arg271His, and Leu471Pro), a nonsense mutation (Arg245stop), and a splice site mutation (94 amino acid deletion). All tested mutants were inactive in uptake and metabolism assays, except MCT8 Arg271His, which showed approximately 20% activity vs. wild-type MCT8. Conclusion: These findings support the hypothesis that the severe psychomotor retardation and elevated serum T-3 levels in these patients are caused by inactivation of the MCT8 transporter...

The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses and its loss causes fatal autoimmunity in mice. We investigated a large autosomal-dominant family with five individuals presenting with a complex immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with novel splice site and missense mutations in CTLA4. While clinical penetrance was incomplete (eight adults of a total of 19 CTLA4 mutation carriers were considered unaffected), CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in patients and carriers with CTLA4 mutations. Whilst Treg cells were generally present at elevated numbers, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers and antibody levels. Taken together, mutations in CTLA-4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding results in a complex syndrome with features of both autoimmunity and immunodeficiency.

OBJECTIVE: The aim of this study was to evaluate the prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation (AF). METHODS: A cohort of 136 unrelated patients with idiopathic atrial fibrillation and 200 unrelated, ethnically matched healthy controls were enrolled. The coding exons and splice junctions of the Nkx2.5 gene were sequenced in 136 atrial fibrillation patients, and the available relatives of mutation carriers and 200 controls were subsequently genotyped for the identified mutations. The functional characteristics of the mutated Nkx2.5 gene were analyzed using a dual-luciferase reporter assay system. RESULTS: Two novel heterozygous Nkx2.5 mutations (p.N19D and p.F186S) were identified in 2 of the 136 unrelated atrial fibrillation cases, with a mutational prevalence of approximately 1.47%. These missense mutations co-segregated with atrial fibrillation in the families and were absent in the 400 control chromosomes. Notably, 2 mutation carriers also had congenital atrial septal defects and atrioventricular block. Multiple alignments of the Nkx2.5 protein sequences across various species revealed that the altered amino acids were completely conserved evolutionarily. Functional analysis demonstrated that the mutant Nkx2.5 proteins were associated with significantly reduced transcriptional activity compared to their wild-type counterpart. CONCLUSION: These findings associate the Nkx2.5 loss-of-function mutation with atrial fibrillation and atrioventricular block and provide novel insights into the molecular mechanism involved in the pathogenesis of atrial fibrillation. These results also have potential implications for early prophylaxis and allele-specific therapy of this common arrhythmia.

Background: Five children from two consanguineous families presented with epilepsy beginning in infancy and severe ataxia, moderate sensorineural deafness, and a renal salt-losing tubulopathy with normotensive hypokalemic metabolic alkalosis. We investigated the genetic basis of this autosomal recessive disease, which we call the EAST syndrome (the presence of epilepsy, ataxia, sensorineural deafness, and tubulopathy). Methods: Whole-genome linkage analysis was performed in the four affected children in one of the families. Newly identified mutations in a potassium-channel gene were evaluated with the use of a heterologous expression system. Protein expression and function were further investigated in genetically modified mice. Results: Linkage analysis identified a single significant locus on chromosome 1q23.2 with a lod score of 4.98. This region contained the KCNJ10 gene, which encodes a potassium channel expressed in the brain, inner ear, and kidney. Sequencing of this candidate gene revealed homozygous missense mutations in affected persons in both families. These mutations, when expressed heterologously in xenopus oocytes, caused significant and specific decreases in potassium currents. Mice with Kcnj10 deletions became dehydrated...