Analysis of the genetic basis of periodic fever with aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome.

PFAPA syndrome is the most common autoinflammatory syndrome in children from Western countries. In spite of its strong familial clustering, its genetic basis and inheritance pattern are still unknown. We performed a comprehensive genetic study on 68 individuals from 14 families. Linkage analysis suggested a susceptibility locus on chromosome 8, but direct molecular sequencing did not support this initial statistical finding. Exome sequencing revealed the absence of any gene that was mutated in all patients. Exhaustive screening of genes involved in other autoinflammatory syndromes or encoding components of the human inflammasome showed no DNA variants that could be linked to PFAPA molecular pathology. Among these, the previously-reported missense mutation V198M in the NLRP3 gene was clearly shown not to co-segregate with PFAPA. Our results on this relatively large cohort indicate that PFAPA syndrome is unlikely to be a monogenic condition. Moreover, none of the several genes known to be involved in inflammation or in autoinflammatory disorders seem to be relevant, alone, to its etiology, suggesting that PFAPA results from oligogenic or complex inheritance of variants in multiple disease genes and/or non-genetic factors.

KARYOTYPE ANALYSIS IN GRAPEVINES

ABSTRACT Seven species of Vitis L., V. champinii Planchon, V. cinerea (Engelm in Gray) Engelm, V. girdiana Munson, V. labrusca L., V. rotundifolia Michaux, V. rupestres Scheels and V. vinifera L. were studied with the purpose of complementing the karyomorphometric information for further comparative analyses. Based on ideograms and on chromosome measures obtained it was possible to see several differences among the species, which were enough to distinguish at least V. champinii and V. girdiana from the others as well as V. labrusca for the lowest measures and V. rotundifolia for the highest mean value of arm ratio. It seems that during the species diversification process the most crucial differences among them did not involve drastic changes in chromosome morphometry.

Impact of a cis-associated gene expression SNP on chromosome 20q11.22 on bipolar disorder susceptibility, hippocampal structure and cognitive performance.

BackgroundBipolar disorder is a highly heritable polygenic disorder. Recent enrichment analyses suggest that there may be true risk variants for bipolar disorder in the expression quantitative trait loci (eQTL) in the brain.AimsWe sought to assess the impact of eQTL variants on bipolar disorder risk by combining data from both bipolar disorder genome-wide association studies (GWAS) and brain eQTL.MethodTo detect single nucleotide polymorphisms (SNPs) that influence expression levels of genes associated with bipolar disorder, we jointly analysed data from a bipolar disorder GWAS (7481 cases and 9250 controls) and a genome-wide brain (cortical) eQTL (193 healthy controls) using a Bayesian statistical method, with independent follow-up replications. The identified risk SNP was then further tested for association with hippocampal volume (n = 5775) and cognitive performance (n = 342) among healthy individuals.ResultsIntegrative analysis revealed a significant association between a brain eQTL rs6088662 on chromosome 20q11.22 and bipolar disorder (log Bayes factor = 5.48; bipolar disorder P = 5.85×10(-5)). Follow-up studies across multiple independent samples confirmed the association of the risk SNP (rs6088662) with gene expression and bipolar disorder susceptibility (P = 3.54×10(-8)). Further exploratory analysis revealed that rs6088662 is also associated with hippocampal volume and cognitive performance in healthy individuals.ConclusionsOur findings suggest that 20q11.22 is likely a risk region for bipolar disorder; they also highlight the informative value of integrating functional annotation of genetic variants for gene expression in advancing our understanding of the biological basis underlying complex disorders, such as bipolar disorder.

Molecular analysis of NOTCH2 in patients with primary open-angle glaucoma

Le glaucome est la seconde cause de cécité dans le monde après la cataracte et est caractérisé par la perte progressive de cellules ganglionnaires de la rétine allant vers la dégénérescence du nerf optique. On distingue deux formes de glaucome; le glaucome à angle fermé et le glaucome à angle ouvert. L'hérédité du glaucome est souvent sporadique, parfois autosomique dominante. Une pression intraoculaire de plus de 21 mmHg représente un facteur de risque important pour son développement. Actuellement, la mutation la plus fréquente, observée dans 5% des cas de glaucome héréditaire, est retrouvée dans le gène MYOC (trabecular meshwork inducible glucocorticoide response). À ce jour, les causes et mécanismes moléculaires sous-jacent ne sont que partiellement compris. Récemment, il a été démontré qu'une souris transgénique exprimant le gène Notch2 dans luvée, développait un glaucome. Pour cette raison, nous avons analysé le gène NOTCH2 chez l'homme afin de déterminer s'il était impliqué. NOTCH2 est composé de 34 exons sur le chromosome 1 et code une protéine transmembranaire essentielle à la prolifération, l'apoptose, la différenciation cellulaire et le destin cellulaires. L'expression du gène est localisée dans le segment antérieur de l'oeil, le segment externe du corps ciliaire et le trabéculum. Les fonctions principales de ces deux tissus sont la production et le drainage de l'humeur aqueuse. Pour mémoire, une perturbation du flux peut générer une augmentation de la pression intraoculaire. Le but de cette étude était de rechercher d'éventuelles mutations du gène NOTCH2 chez des patients souffrant de glaucome. 130 patients ont été vu à l'hôpital ophtalmique Jules- Gonin et un échantillon d'ADN a été récolté afin d'identifier l'origine moléculaire de leur pathologie. L'analyse moléculaire s'est fait étape par étape. Premièrement, j'ai séquencé l'exon 3 du gène MYOC. Deuxièment, la Chromatographie en phase liquide à haute performance a été utilisée pour l'analyse des 34 exons du gène NOTCH2. Troisièment, tous les exons présentant une courbe suspecte au chromatogramme ont été séquencés selon la méthode de Sanger. Dans la première partie de l'étude, j'ai analysé l'exon 3 du gène MYOC afin de déterminer les éventuels porteurs d'une mutation dominante. Aucune mutation pathogénique n'a été mis en évidence mais 4 patients sur les 130 étaient porteurs d'un variant connu et fréquent. Dans la deuxième partie de mon étude, j'ai analysé les 34 exons du gène NOTCH2, qui n'ont révélé aucune mutation. Bien que les méthodes utilisées dans cette étude montrent quelques limitations, il est peu probable que des mutations dans les régions codantes de NOTCH2 soient un facteur de risque important dans le glaucome primaire à angle ouvert.

TP53 mutations and loss of heterozygosity of chromosome 17 in colorectal tumors

The incidence of TP53 point mutations and loss of heterozygosity (LOH) of chromosome 17 in colorectal tumors was determined in a group of Brazilian patients. We screened DNA samples from tumors and distal normal mucosa of 39 patients with colorectal cancer, for TP53 mutations by PCR-SSCP (single-strand conformation polymorphism) analysis. Chromosome 17 LOH was investigated using six PCR-based polymorphic markers and one VNTR probe. TP53 mutations were demonstrated in 15/39 of the cases. Mutations were distributed among all exons examined (five to eight), the majority of them being G/C to A/T transitions. LOH of chromosome 17p and 17q was detected in 70 and 46% of the tumors, respectively. There was a significant association between TP53 mutations and LOH in chromosome 17p (P = 0.0035) and 17q (P = 0.03). Although no correlation was observed between TP53 genetic alterations and clinical/ pathological characteristics, the association of TP53 mutations with loss of both chromosome 17 arms may indicate that TP53 inactivation provokes an unstable phenotype in tumor cells in colorectal tumors.

Analysis of HLA-A antigens and C282Y and H63D mutations of the HFE gene in Brazilian patients with hemochromatosis

The hemochromatosis gene, HFE, is located on chromosome 6 in close proximity to the HLA-A locus. Most Caucasian patients with hereditary hemochromatosis (HH) are homozygous for HLA-A3 and for the C282Y mutation of the HFE gene, while a minority are compound heterozygotes for C282Y and H63D. The prevalence of these mutations in non-Caucasian patients with HH is lower than expected. The objective of the present study was to evaluate the frequencies of HLA-A antigens and the C282Y and H63D mutations of the HFE gene in Brazilian patients with HH and to compare clinical and laboratory profiles of C282Y-positive and -negative patients with HH. The frequencies of HLA-A and C282Y and H63D mutations were determined by PCR-based methods in 15 male patients (median age 44 (20-72) years) with HH. Eight patients (53%) were homozygous and one (7%) was heterozygous for the C282Y mutation. None had compound heterozygosity for C282Y and H63D mutations. All but three C282Y homozygotes were positive for HLA-A3 and three other patients without C282Y were shown to be either heterozygous (N = 2) or homozygous (N = 1) for HLA-A3. Patients homozygous for the C282Y mutation had higher ferritin levels and lower age at onset, but the difference was not significant. The presence of C282Y homozygosity in roughly half of the Brazilian patients with HH, together with the findings of HLA-A homozygosity in C282Y-negative subjects, suggest that other mutations in the HFE gene or in other genes involved in iron homeostasis might also be linked to HH in Brazil.

Prevalence of Y chromosome deletions in a Brazilian population of nonobstructive azoospermic and severely oligozoospermic men

We determined the prevalence of Y chromosome deletions in a population of 60 Brazilian nonobstructive azoospermic and severely oligozoospermic men. PCR-based screening of microdeletions was performed on lymphocyte DNA for the presence of 14 sequence-tagged sites (STS) located in the azoospermic factor (AZF) on the Yq chromosome. All STS were amplified efficiently in samples from 12 fertile men tested, but failed to be amplified in samples from fertile women, indicating the specificity of PCR conditions for Yq screening. Overall, 4 of the 60 infertile patients tested (6.7%) exhibited deletion of the Y chromosome, 2 of them being severely oligozoospermic patients (P10 and P32) and 2 azoospermic men (patients P47 and P57). Patients P47 and P57 presented larger deletions in the AZFa, AZFb and AZFc subregions, with apparent loss of Yq material evidenced by karyotype analysis. Patients P10 and P32 presented deletions confined to the AZFc region, involving the DAZ locus. Male relatives of patients P10 and P32 had no Y chromosome deletions and presented a normal karyotype, suggesting a de novo status of the deletions found. Our data add to the growing literature showing that microdeletions of the Y chromosome can be the cause of male idiopathic infertility.

Hereditary motor and autonomic neuronopathy 1 maps to chromosome 20q13.2-13.3

The spinal muscular atrophies (SMA) or hereditary motor neuronopathies result from the continuous degeneration and death of spinal cord lower motor neurons, leading to progressive muscular weakness and atrophy. We describe a large Brazilian family exhibiting an extremely rare, late-onset, dominant, proximal, and progressive SMA accompanied by very unusual manifestations, such as an abnormal sweating pattern, and gastrointestinal and sexual dysfunctions, suggesting concomitant involvement of the autonomic nervous system. We propose a new disease category for this disorder, `hereditary motor and autonomic neuronopathy', and attribute the term, `survival of motor and autonomic neurons 1' (SMAN1) to the respective locus that was mapped to a 14.5 cM region on chromosome 20q13.2-13.3 by genetic linkage analysis and haplotype studies using microsatellite polymorphic markers. This locus lies between markers D20S120 and D20S173 showing a maximum LOD score of 4.6 at D20S171, defining a region with 33 known genes, including several potential candidates. Identifying the SMAN1 gene should not only improve our understanding of the molecular mechanisms underlying lower motor neuron diseases but also help to clarify the relationship between motor and autonomic neurons.

Genomic analysis of Brazilian patients with Fabry disease

Fabry disease is an X-linked lysosomal disorder due to a-galactosidase A deficiency that causes storage of globotriaosylceramide. The gene coding for this lysosomal enzyme is located on the long arm of the X chromosome, in region Xq21.33-Xq22. Disease progression leads to vascular disease secondary to involvement of kidney, heart and the central nervous system. Detection of female carriers based solely on enzyme assays is often inconclusive. Therefore, mutation analysis is a valuable tool for diagnosis and genetic counseling. Many mutations of the a-galactosidase A gene have been reported with high genetic heterogeneity, being most mutations private found in only one family. The disease is panethnic, and estimates of incidence range from about 1 in 40,000 to 60,000 males. Our objective was to describe the analysis of 6 male and 7 female individuals belonging to 4 different Fabry disease families by automated sequencing of the seven exons of the a-galactosidase gene. Sequencing was performed using PCR fragments for each exon amplified from DNA extracted from peripheral blood. Three known mutations and one previously described in another Brazilian family were detected. Of 7 female relatives studied, 4 were carriers. Although the present study confirms the heterogeneity of mutations in Fabry disease, the finding of the same mutation previously detected in another Fabry family from our region raises the possibility of some founder effect, or genetic drift. Finally, the present study highlights the importance of molecular analysis for carrier detection and genetic counseling.

Bioinformatics analysis of biomarkers and transcriptional factor motifs in Down syndrome

In this study, biomarkers and transcriptional factor motifs were identified in order to investigate the etiology and phenotypic severity of Down syndrome. GSE 1281, GSE 1611, and GSE 5390 were downloaded from the gene expression ominibus (GEO). A robust multiarray analysis (RMA) algorithm was applied to detect differentially expressed genes (DEGs). In order to screen for biological pathways and to interrogate the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, the database for annotation, visualization, and integrated discovery (DAVID) was used to carry out a gene ontology (GO) function enrichment for DEGs. Finally, a transcriptional regulatory network was constructed, and a hypergeometric distribution test was applied to select for significantly enriched transcriptional factor motifs. CBR1, DYRK1A, HMGN1, ITSN1, RCAN1, SON, TMEM50B, and TTC3 were each up-regulated two-fold in Down syndrome samples compared to normal samples; of these, SON and TTC3 were newly reported. CBR1, DYRK1A, HMGN1, ITSN1, RCAN1, SON, TMEM50B, and TTC3 were located on human chromosome 21 (mouse chromosome 16). The DEGs were significantly enriched in macromolecular complex subunit organization and focal adhesion pathways. Eleven significantly enriched transcription factor motifs (PAX5, EGR1, XBP1, SREBP1, OLF1, MZF1, NFY, NFKAPPAB, MYCMAX, NFE2, and RP58) were identified. The DEGs and transcription factor motifs identified in our study provide biomarkers for the understanding of Down syndrome pathogenesis and progression.

Analysis of a temperature sensitive mutation affecting aldehyde oxidase activity in Drosophila melanogaster

A strain of Drosophila melanogaster (mid america stock culture no. hl16) has been reported to be deficient in aldehyde oxidase activity (Hickey and Singh 1982). This strain was characterized during the course of this study and compared to other mutant strains known to be deficient in aldehyde oxidase activity. During the course of this investigation, the hl16 strain was found to be temperature sensitive in its viability. It was found that the two phenotypes, the enzyme deficiency, and the temperature sensitive lethality were the result of two different mutations, both mapping to the X-chromosome. These two mutations were found to be separable by recombination. The enzyme deficiency was found to map to the same locus as the cinnamon mutation, another mutation which affects aldehyde oxidase production. The developmental profile of aldehyde oxidase in the hl16 strain was compared to the developmental profile in the Canton S wild type strain. The aldehyde oxidase activity in adult hl16 individuals was also compared to that of various other strains. It was also found that the aldehyde oxidase activity was temperature sensitive in the adult flies. The temperature sensitive lethality mutation was mapped to position 1-0.1.

Cartographie génétique d’une forme familiale autosomale dominante d’anévrysmes du septum inter-ventriculaire et de communications inter-ventriculaires au chromosome 10p15

Les malformations cardiaques congénitales (CHM) représentent 28 % de toutes les malformations congénitales majeures et touchent 8 pour 1000 naissances à terme. Elles sont la cause de mortalité et de morbidité non infectieuse la plus fréquente chez les enfants de moins d’une année de vie. Les communications interventriculaires (VSD) forment le sous-type de CHM le plus fréquent et l’aggrégation familiale est extrêmement rare. Le but de cette étude était d’identifier les facteurs génétiques et les régions chromosomiques contribuant aux VSD. Une grande famille ségréguant diverses formes de pathologies septales, incluant des VSD, des anévrysmes du septum interventriculaire (VSA) et des communications interauriculaires (ASD), a été examinées et caractérisées cliniquement et génétiquement. Dix-huit membres de la famille, sur trois générations, ont pu être étudiés. (10 affectés : 4 VSD, 3 VSA, 2 ASD et une tétralogie de Fallot). L’analyse de liaison multipoint paramétrique démontre un logarithme des probabilités maximal (LOD) de 3.29 liant significativement le chromosome 10p15.3-10p15.2 aux traits observés dans cette famille. Le pointage LOD oriente vers une région pauvre en gènes qui a déjà été associée aux malformations du septum interventriculaire, mais qui est distincte de la région du syndrome de DiGeorge de type 2 sur le chromosome 10p. De plus, plusieurs scénarios d’analyse de liaison suggèrent que la tétralogie de Fallot est une phénocopie et qu’elle est donc génétiquement différente des autres pathologies du septum observées dans cette famille. En bref, cette étude associe une forme rare de VSD/VSA au chromosome 10p15 et permet d’étendre le spectre de l’hétérogénéité des pathologies septales. Mots-clés : Malformations cardiaques congénitales, malformations du septum, tétralogie de Fallot, analyse de liaison, chromosome 10p15, génétique moléculaire

Functional genomics of the Drosophila melanogaster X chromosome and the role of DWnt5 during development

The collection of X chromosome insertions (PX) lethal lines, which was isolated from a screen for essential genes on the X chromosome, was characterized by means of cloning the insertion sites, mapping the sites within genomic DNA and determination of the associated reporter gene expresssion patterns. The established STS flanking the P element insertion sites were submitted to EMBL nucleotide databases and their in situ data together with the enhancer trap expression patterns have been deposited in the FlyView database. The characterized lines are now available to be used by the scientific community for a detailed analysis of the newly established lethal gene functions. One of the isolated genes on the X chromosome was the Drosophila gene Wnt5 (DWnt5). From two independent screens, one lethal and three homozygous viable alleles were recovered, allowing the identification of two distinct functions for DWnt5 in the fly. Observations on the developing nervous system of mutant embryos suggest that DWnt5 activity affects axon projection pattern. Elevated levels of DWNT5 activity in the midline cells of the central nervous system causes improper establishment and maintenance of the axonal pathways. Our analysis of the expression and mutant phenotype indicates that DWnt5 function in a process needed for proper organization of the nervous system. A second and novel function of DWnt5 is the control of the body size by regulation of the cell number rather than affecting the size of cells. Moreover, experimentally increased DWnt5 levels in a post-mitotic region of the eye imaginal disc causes abnormal cell cycle progression, resulting in additional ommatidia in the adult eye when compared to wild type. The increased cell number and the effects on the cell cycle after exposure to high DWNT5 levels is the result of a failure to downregulate cyclin B and therefore the unsuccessful establishment of a G1 arrest.

HLA-A Typing in formalin-fixed paraffin embedded tissue samples : towards potential retrospective analysis

El Antígeno Leucocitario Humano (HLA en inglés) ha sido descrito en muchos casos como factor de pronóstico para cáncer. La característica principal de los genes de HLA, localizados en el cromosoma 6 (6p21.3), son sus numerosos polimorfismos. Los análisis de secuencia de nucleótidos muestran que la variación está restringida predominantemente a los exones que codifican los dominios de unión a péptidos de la proteína. Por lo tanto, el polimorfismo del HLA define el repertorio de péptidos que se unen a los alotipos de HLA y este hecho define la habilidad de un individuo para responder a la exposición a muchos agentes infecciosos durante su vida. La tipificación de HLA se ha convertido en un análisis importante en clínica. Muestras de tejido embebidas en parafina y fijadas con formalina (FFPE en inglés) son recolectadas rutinariamente en oncología. Este procedimiento podría ser utilizado como una buena fuente de ADN, dado que en estudios en el pasado los ensayos de recolección de ADN no eran normalmente llevados a cabo de casi ningún tejido o muestra en procedimientos clínicos regulares. Teniendo en cuenta que el problema más importante con el ADN de muestras FFPE es la fragmentación, nosotros propusimos un nuevo método para la tipificación del alelo HLA-A desde muestras FFPE basado en las secuencias del exón 2, 3 y 4. Nosotros diseñamos un juego de 12 cebadores: cuatro para el exón 2 de HLA-A, tres para el exón 3 de HLA-A y cinco para el exón 4 de HLA-A, cada uno de acuerdo las secuencias flanqueantes de su respectivo exón y la variación en la secuencia entre diferentes alelos. 17 muestran FFPE colectadas en el Hospital Universitario de Karolinska en Estocolmo Suecia fueron sometidas a PCR y los productos fueron secuenciados. Finalmente todas las secuencias obtenidas fueron analizadas y comparadas con la base de datos del IMGT-HLA. Las muestras FFPE habían sido previamente tipificadas para HLA y los resultados fueron comparados con los de este método. De acuerdo con nuestros resultados, las muestras pudieron ser correctamente secuenciadas. Con este procedimiento, podemos concluir que nuestro estudio es el primer método de tipificación basado en secuencia que permite analizar muestras viejas de ADN de las cuales no se tiene otra fuente. Este estudio abre la posibilidad de desarrollar análisis para establecer nuevas relaciones entre HLA y diferentes enfermedades como el cáncer también.

"Refined mapping of a quantitative trait locus on chromosome 1 responsible for mouse embryonic death"

Recurrent spontaneous abortion (RSA) is defined as the loss of three or more consecutive pregnancies during the first trimester of embryonic intrauterine development. This kind of human infertility is frequent among the general population since it affects 1 to 5% of women. In half of the cases the etiology remains unelucidated. In the present study, we used interspecific recombinant congenic mouse strains (IRCS) in the aim to identify genes responsible for embryonic lethality. Applying a cartographic approach using a genotype/phenotype association, we identified a minimal QTL region, of about 6 Mb on chromosome 1, responsible for a high rate of embryonic death (,30%). Genetic analysis suggests that the observed phenotype is linked to uterine dysfunction. Transcriptomic analysis of the uterine tissue revealed a preferential deregulation of genes of this region compared to the rest of the genome. Some genes from the QTL region are associated with VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathways. This work may contribute to elucidate the molecular basis of a multifactorial and complex human disorder as RSA.