Molecular bases of classic galactosemia : searching for new therapeutic strategies

Tese de doutoramento, Farm��cia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farm��cia, 2014

An atypical 7q11.23 deletion in a normal IQ Williams-Beuren syndrome patient.

Williams-Beuren syndrome (WBS; OMIM no. 194050) is a multisystemic neurodevelopmental disorder caused by a hemizygous deletion of 1.55 Mb on chromosome 7q11.23 spanning 28 genes. Haploinsufficiency of the ELN gene was shown to be responsible for supravalvular aortic stenosis and generalized arteriopathy, whereas LIMK1, CLIP2, GTF2IRD1 and GTF2I genes were suggested to be linked to the specific cognitive profile and craniofacial features. These insights for genotype-phenotype correlations came from the molecular and clinical analysis of patients with atypical deletions and mice models. Here we report a patient showing mild WBS physical phenotype and normal IQ, who carries a shorter 1 Mb atypical deletion. This rearrangement does not include the GTF2IRD1 and GTF2I genes and only partially the BAZ1B gene. Our results are consistent with the hypothesis that hemizygosity of the GTF2IRD1 and GTF2I genes might be involved in the facial dysmorphisms and in the specific motor and cognitive deficits observed in WBS patients.

Molecular screening of ADAMTSL2 gene in 33 patients reveals the genetic heterogeneity of geleophysic dysplasia.

Background Geleophysic dysplasia (GD, OMIM 231050) is an autosomal recessive disorder characterised by short stature, small hands and feet, stiff joints, and thick skin. Patients often present with a progressive cardiac valvular disease which can lead to an early death. In a previous study including six GD families, we have mapped the disease gene on chromosome 9q34.2 and identified mutations in the A Disintegrin And Metalloproteinase with Thrombospondin repeats-like 2 gene (ADAMTSL2). Methods Following this study, we have collected the samples of 30 additional GD families, including 33 patients and identified ADAMTSL2 mutations in 14/33 patients, comprising 13 novel mutations. The absence of mutation in 19 patients prompted us to compare the two groups of GD patients, namely group 1, patients with ADAMTSL2 mutations (n=20, also including the 6 patients from our previous study), and group 2, patients without ADAMTSL2 mutations (n=19). Results The main discriminating features were facial dysmorphism and tip-toe walking, which were almost constantly observed in group 1. No differences were found concerning heart involvement, skin thickness, recurrent respiratory and ear infections, bronchopulmonary insufficiency, laryngo-tracheal stenosis, deafness, and radiographic features. Conclusions It is concluded that GD is a genetically heterogeneous condition. Ongoing studies will hopefully lead to the identification of another disease gene.

Subtelomeric deletion of chromosome 10p15.3: clinical findings and molecular cytogenetic characterization.

We describe 19 unrelated individuals with submicroscopic deletions involving 10p15.3 characterized by chromosomal microarray (CMA). Interestingly, to our knowledge, only two individuals with isolated, submicroscopic 10p15.3 deletion have been reported to date; however, only limited clinical information is available for these probands and the deleted region has not been molecularly mapped. Comprehensive clinical history was obtained for 12 of the 19 individuals described in this study. Common features among these 12 individuals include: cognitive/behavioral/developmental differences (11/11), speech delay/language disorder (10/10), motor delay (10/10), craniofacial dysmorphism (9/12), hypotonia (7/11), brain anomalies (4/6) and seizures (3/7). Parental studies were performed for nine of the 19 individuals; the 10p15.3 deletion was de novo in seven of the probands, not maternally inherited in one proband and inherited from an apparently affected mother in one proband. Molecular mapping of the 19 individuals reported in this study has identified two genes, ZMYND11 (OMIM 608668) and DIP2C (OMIM 611380; UCSC Genome Browser), mapping within 10p15.3 which are most commonly deleted. Although no single gene has been identified which is deleted in all 19 individuals studied, the deleted region in all but one individual includes ZMYND11 and the deleted region in all but one other individual includes DIP2C. There is not a clearly identifiable phenotypic difference between these two individuals and the size of the deleted region does not generally predict clinical features. Little is currently known about these genes complicating a direct genotype/phenotype correlation at this time. These data however, suggest that ZMYND11 and/or DIP2C haploinsufficiency contributes to the clinical features associated with 10p15 deletions in probands described in this study.

Caract��risation clinique et g��n��tique d���une nouvelle forme d���ataxie autosomique r��cessive dans la population qu��b��coise

Les ataxies autosomiques r��cessives sont un groupe de troubles neurologiques h��t��rog��nes caract��ris��s par une incoordination brute des mouvements musculaires impliquant le dysfonctionnement nerveux du cervelet qui coordonne le mouvement. Plusieurs formes h��r��ditaires ont ��t�� d��crites dont la plus connue : l���ataxie de Friedriech. Dans cette th��se nous rapportons l'identification et la caract��risation d���une nouvelle forme dans la population qu��b��coise. L���ataxie r��cessive spastique avec leucoenc��phalopathie (ARSAL; aussi connue comme l���ataxie autosomique r��cessive spastique de type 3 (SPAX3); OMIM 611390) est la deuxi��me ataxie spastique d��crite dans la population canadienne fran��aise. En effet, pr��s de 50 % de nos cas sont originaires de la r��gion de Portneuf. En 2006, nous avons d��crit les caract��ristiques cliniques de cette nouvelle forme d���ataxie. Un premier criblage du g��nome entier, constitu�� de plus de 500 marqueurs microsatellites, a permis la localisation du locus sur le chromosome 2q33-34. Suite au s��quen��age de plus de 37 g��nes candidats et afin de r��tr��cir cet intervalle candidat, nous avons utilis�� une micro-puce d���ADN constitu��e de marqueurs SNP ��single nucleotide polymorphism�� et nous avons identifi�� un deuxi��me intervalle candidat de 0.658Mb au locus 2q33 dans lequel se trouvent moins de 9 g��nes. L���identification et la caract��risation de ces mutations a n��cessit�� l���utilisation de diverses technologies de pointe. Trois mutations (une d��l��tion et deux r��arrangements complexes) dans le g��ne mitochondrial tRNA-synthetase (MARS2) ont ��t�� identifi��es dans notre cohorte. Nous ��mettons l���hypoth��se que la nature des mutations complexes est responsable d���un d��r��glement de la transcription du g��ne, ce qui a un impact n��faste sur la fonction mitochondriale et le tissu neuronal.

��tude clinique et g��n��tique d���une nouvelle forme d���ataxie spinoc��r��belleuse pure associ��e �� l�����rythrok��ratodermie

Nous pr��sentons ici la description clinique et g��n��tique d���un syndrome neurocutan�� unique. Le laboratoire du Dr Cossette a entrepris la caract��risation clinique et g��n��tique d'une famille canadienne-fran��aise qui a ��t�� identifi��e par les Drs Giroux et Barbeau en 1972 et qui comprend plus de 100 personnes sur six g��n��rations. Les membres atteints de cette famille pr��sentent des l��sions typiques d'��rythrok��ratodermie (EK) (OMIM 133190, EKV1 et EKV2), associ��es �� une ataxie spinoc��r��belleuse pure. Dans cette famille, l'ataxie est caract��ris��e par des troubles de la coordination et de la d��marche caus��s par une d��g��n��rescence du cervelet et de la moelle ��pini��re. Cette ataxie est transmise selon un mode autosomique dominant. Une ��tude ant��rieure de cette variante d'EK avec ataxie avait sugg��r�� une liaison sur le chromosome 1p34-p35, soit la m��me r��gion que les formes EKV de type 1 et 2, caus��es respectivement par des mutations dans les g��nes connexin-31 (GJB3; OMIM 603324) et connexin-30.3 (GJB4; OMIM 605425). Cependant, aucune mutation n'a ��t�� retrouv��e dans ces g��nes pour la famille canadienne-fran��aise. Nous avons r��cemment recontact�� la famille et effectu�� des examens d��taill��s, incluant une imagerie par r��sonance magn��tique (IRM) et un ��lectromyogramme (EMG). Les manifestations neurologiques des individus atteints sont compatibles avec une nouvelle forme d���ataxie c��r��belleuse pure �� transmission autosomique dominante (ADCA de type III dans la classification de Harding) que nous avons appel��e SCA34. Une cartographie compl��te du g��nome nous a permis de localiser le g��ne SCA34 sur le chromosome 6p12.3-q16.2. ��galement, en collaboration avec les Drs Alexis Brice (H��pital Piti��-La Salp��tri��re, Paris) et Alfredo Brusco (H��pital San Giovanni Battista di Torino, Italie), nous avons confirm�� que trois autres familles europ��ennes avec SCA inexpliqu��e ��taient ��galement li��es au locus SCA34. Notre laboratoire a r��cemment entrepris la recherche des mutations responsables de SCA34. Les r��sultats de ce criblage de g��nes candidats sont pr��sent��s dans le chapitre 3 de cette th��se.

Polimorfismos del gen Butirilcolinesterasa responsables de reacciones adversas en pacientes consumidores de ���coca��na���

La butirilcolinesterasa humana (BChE; EC 3.1.1.8) es una enzima polim��rfica sintetizada en el h��gado y en el tejido adiposo, ampliamente distribuida en el organismo y encargada de hidrolizar algunos ��steres de colina como la proca��na, ��steres alif��ticos como el ��cido acetilsalic��lico, f��rmacos como la metilprednisolona, el mivacurium y la succinilcolina y drogas de uso y/o abuso como la hero��na y la coca��na. Es codificada por el gen BCHE (OMIM 177400), habi��ndose identificado m��s de 100 variantes, algunas no estudiadas plenamente, adem��s de la forma m��s frecuente, llamada usual o silvestre. Diferentes polimorfismos del gen BCHE se han relacionado con la s��ntesis de enzimas con niveles variados de actividad catal��tica. Las bases moleculares de algunas de esas variantes gen��ticas han sido reportadas, entre las que se encuentra las variantes At��pica (A), fluoruro-resistente del tipo 1 y 2 (F-1 y F-2), silente (S), Kalow (K), James (J) y Hammersmith (H). En este estudio, en un grupo de pacientes se aplic�� el instrumento validado Lifetime Severity Index for Cocaine Use Disorder (LSI-C) para evaluar la gravedad del consumo de ���coca��na��� a lo largo de la vida. Adem��s, se determinaron Polimorfismos de Nucle��tido Simple (SNPs) en el gen BCHE conocidos como responsables de reacciones adversas en pacientes consumidores de ���coca��na��� mediante secuenciaci��n del gen y se predijo el efecto delos SNPs sobre la funci��n y la estructura de la prote��na, mediante el uso de herramientas bio-inform��ticas. El instrumento LSI-C ofreci�� resultados en cuatro dimensiones: consumo a lo largo de la vida, consumo reciente, dependencia psicol��gica e intento de abandono del consumo. Los estudios de an��lisis molecular permitieron observar dos SNPs codificantes (cSNPs) no sin��nimos en el 27.3% de la muestra, c.293A>G (p.Asp98Gly) y c.1699G>A (p.Ala567Thr), localizados en los exones 2 y 4, que corresponden, desde el punto de vista funcional, a la variante At��pica (A) [dbSNP: rs1799807] y a la variante Kalow (K) [dbSNP: rs1803274] de la enzima BChE, respectivamente. Los estudios de predicci��n In silico establecieron para el SNP p.Asp98Gly un car��cter patog��nico, mientras que para el SNP p.Ala567Thr, mostraron un comportamiento neutro. El an��lisis de los resultados permite proponer la existencia de una relaci��n entre polimorfismos o variantes gen��ticas responsables de una baja actividad catal��tica y/o baja concentraci��n plasm��tica de la enzima BChE y algunas de las reacciones adversas ocurridas en pacientes consumidores de coca��na.

Consumo de sustancias y noxas prenatales en madres de pacientes con s��ndrome de M��bius y M��bius Like

Introducci��n: El s��ndrome de M��bius y M��bius Like es una entidad poco frecuente caracterizada principalmente por par��lisis cong��nita del VI y VII par craneal. Su etiolog��a es poco conocida aunque se ha asociado a inductores del aborto. El objetivo de este estudio es describir factores an��malos, t��xicos o nocivos que hayan estado presentes en el embarazo de las madres de estos pacientes. Metodolog��a: se realiz�� una encuesta auto-diligenciable a 15 madres de pacientes con el diagn��stico, indagando sobre condiciones an��malas y/o exposicionales del embarazo, el padre y el ambiente. Resultados: Las madres se encontraban entre los 16 y 34 a��os al momento de quedar embarazadas, en su mayor��a eran solteras, estudiantes y sin planes de embarazo. Once en total usaron alg��n medicamento y/o sustancias durante la gestaci��n; seis de ellas Misoprostol (40%). Las otras sustancias utilizadas incluyeron: alternativas, cigarrillo, alcohol, ibuprofeno, anticonceptivos, otros. Como anomal��as del periodo prenatal se reportaron sangrado activo y/o amenaza de aborto, infecci��n, exposici��n a qu��micos ambientales y enfermedad materna activa. Las condiciones paternas descritas fueron alcoholismo y/o drogadicci��n, enfermedad y edad ��� 40 a��os en bajo porcentaje. Conclusi��n: El s��ndrome de M��bius y M��bius Like es una patolog��a poco frecuente de la cual a��n se debe seguir investigando sobre su etiolog��a, para plantear posibles medidas de prevenci��n.

Eficacia del diagn��stico prenatal no invasivo por c��lulas fetales libres en sangre materna 1990-2014. Revisi��n sistem��tica

ANTECEDENTES:��El aislamiento de c��lulas fetales libres o ADN fetal en sangre materna abre una ventana de posibilidades diagn��sticas no invasivas para patolog��as monog��nicas y cromos��micas, adem��s de permitir la identificaci��n del sexo y del RH fetal. Actualmente existen m��ltiples estudios que eval��an la eficacia de estos m��todos, mostrando resultados costo-efectivos y de menor riesgo que el est��ndar de oro. Este trabajo describe la evidencia encontrada acerca del diagn��stico prenatal no invasivo luego de realizar una revisi��n��sistem��tica de la literatura. OBJETIVOS:��El objetivo de este estudio fue��reunir la evidencia que��cumpla��con los criterios de b��squeda, en el tema del diagn��stico fetal no invasivo por c��lulas fetales libres en sangre materna para determinar su utilidad diagn��stica.�� M��TODOS:��Se realiz�� una revisi��n��sistem��tica de la literatura con el fin de determinar si��el diagn��stico prenatal no invasivo por c��lulas fetales libres en sangre materna es efectivo como m��todo de diagn��stico.�� RESULTADOS:��Se encontraron 5,893 art��culos que cumpl��an con los criterios de b��squeda;��67 cumplieron los criterios de inclusi��n: 49.3% (33/67) correspondieron a estudios de corte transversal, 38,8% (26/67) a estudios de cohortes y el 11.9% (8/67) a estudios casos y controles.��Se obtuvieron resultados de sensibilidad, especificidad y tipo de prueba. CONCLUSI��N:��En la presente revisi��n sistem��tica, se evidencia como el diagn��stico prenatal no invasivo es una t��cnica feasible, reproducible y sensible para el diagn��stico fetal, evitando el riesgo de un diagn��stico invasivo.

Oculoauriculovertebral spectrum: report of nine familial cases with evidence of autosomal dominant inheritance and review of the literature

Oculoauriculovertebral spectrum (OAVS; OMIM 164210) is a complex condition characterized by defects of aural, oral, mandibular and vertebral development. The aetiology of this condition is likely to be heterogeneous; most cases are sporadic, however, familial cases suggesting autosomal recessive end autosomal dominant inheritance have been reported. In this study, we describe the clinical aspects of nine familial cases with evidence of autosomal dominant inheritance and compare them with reports in the literature. Interfamilial and intrafamilial clinical variabilities were observed in this study (reinforcing the necessity of careful examination of familial members). We suggest that oculoauriculovertebral spectrum with autosomal dominant inheritance is characterized mainly by bilateral auricular involvement and rarely presents extracranial anomalies. Clin Dysmorphol 18:67-77 (C) 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes

Interferon regulatory factor 6 (IRF6) belongs to a family of nine transcription factors that share a highly conserved helix-turn-helix DNA-binding domain and a less conserved protein-binding domain. Most IRFs regulate the expression of interferon-alpha and -beta after viral infection(1), but the function of IRF6 is unknown. The gene encoding IRF6 is located in the critical region for the Van der Woude syndrome (VWS; OMIM 119300) locus at chromosome 1q32-q41 (refs 2,3). The disorder is an autosomal dominant form of cleft lip and palate with lip pits(4), and is the most common syndromic form of cleft lip or palate. Popliteal pterygium syndrome (PPS; OMIM 119500) is a disorder with a similar orofacial phenotype that also includes skin and genital anomalies(5). Phenotypic overlap(6) and linkage data(7) suggest that these two disorders are allelic. We found a nonsense mutation in IRF6 in the affected twin of a pair of monozygotic twins who were discordant for VWS. Subsequently, we identified mutations in IRF6 in 45 additional unrelated families affected with VWS and distinct mutations in 13 families affected with PPS. Expression analyses showed high levels of Irf6 mRNA along the medial edge of the fusing palate, tooth buds, hair follicles, genitalia and skin. Our observations demonstrate that haploinsufficiency of IRF6 disrupts orofacial development and are consistent with dominant-negative mutations disturbing development of the skin and genitalia.

Frequ��ncia da neoplasia end��crina m��ltipla tipo 1 em grupos de pacientes com adenoma hipofis��rio: aspectos cl��nicos e estudo gen��tico familiar

Coordena����o de Aperfei��oamento de Pessoal de N��vel Superior (CAPES)

Copy number variation in Williams-Beuren syndrome: suitable diagnostic strategy for developing countries

Abstract Background Williams-Beuren syndrome (WBS; OMIM 194050) is caused by a hemizygous contiguous gene microdeletion at 7q11.23. Supravalvular aortic stenosis (SVAS), mental retardation, and overfriendliness comprise typical symptoms of WBS. Although fluorescence in situ hybridization (FISH) is considered the gold standard technique, the microsatellite DNA markers and multiplex ligation-dependent probe amplification (MLPA) could be used for to confirm the diagnosis of WBS. Results We have evaluated a total cohort of 88 patients with a suspicion clinical diagnosis of WBS using a collection of five markers (D7S1870, D7S489, D7S613, D7S2476, and D7S489_A) and a commercial MLPA kit (P029). The microdeletion was present in 64 (72.7%) patients and absent in 24 (27.3%) patients. The parental origin of deletion was maternal in 36 of 64 patients (56.3%) paternal in 28 of 64 patients (43.7%). The deletion size was 1.55 Mb in 57 of 64 patients (89.1%) and 1.84 Mb in 7 of 64 patients (10.9%). The results were concordant using both techniques, except for four patients whose microsatellite markers were uninformative. There were no clinical differences in relation to either the size or parental origin of the deletion. Conclusion MLPA was considered a faster and more economical method in a single assay, whereas the microsatellite markers could determine both the size and parental origin of the deletion in WBS. The microsatellite marker and MLPA techniques are effective in deletion detection in WBS, and both methods provide a useful diagnostic strategy mainly for developing countries.

Una base dati per il knowledge discovery in genetica medica

L'innovazione delle tecnologie di sequenziamento negli ultimi anni ha reso possibile la catalogazione delle varianti genetiche nei campioni umani, portando nuove scoperte e comprensioni nella ricerca medica, farmaceutica, dell'evoluzione e negli studi sulla popolazione. La quantit�� di sequenze prodotta �� molto cospicua, e per giungere all'identificazione delle varianti sono necessari diversi stadi di elaborazione delle informazioni genetiche in cui, ad ogni passo, vengono generate ulteriori informazioni. Insieme a questa immensa accumulazione di dati, �� nata la necessit�� da parte della comunit�� scientifica di organizzare i dati in repository, dapprima solo per condividere i risultati delle ricerche, poi per permettere studi statistici direttamente sui dati genetici. Gli studi su larga scala coinvolgono quantit�� di dati nell'ordine dei petabyte, il cui mantenimento continua a rappresentare una sfida per le infrastrutture. Per la variet�� e la quantit�� di dati prodotti, i database giocano un ruolo di primaria importanza in questa sfida. Modelli e organizzazione dei dati in questo campo possono fare la differenza non soltanto per la scalabilit��, ma anche e soprattutto per la predisposizione al data mining. Infatti, la memorizzazione di questi dati in file con formati quasi-standard, la dimensione di questi file, e i requisiti computazionali richiesti, rendono difficile la scrittura di software di analisi efficienti e scoraggiano studi su larga scala e su dati eterogenei. Prima di progettare il database si �� perci�� studiata l���evoluzione, negli ultimi vent���anni, dei formati quasi-standard per i flat file biologici, contenenti metadati eterogenei e sequenze nucleotidiche vere e proprie, con record privi di relazioni strutturali. Recentemente questa evoluzione �� culminata nell���utilizzo dello standard XML, ma i flat file delimitati continuano a essere gli standard pi�� supportati da tools e piattaforme online. �� seguita poi un���analisi dell���organizzazione interna dei dati per i database biologici pubblici. Queste basi di dati contengono geni, varianti genetiche, strutture proteiche, ontologie fenotipiche, relazioni tra malattie e geni, relazioni tra farmaci e geni. Tra i database pubblici studiati rientrano OMIM, Entrez, KEGG, UniProt, GO. L'obiettivo principale nello studio e nella modellazione del database genetico �� stato quello di strutturare i dati in modo da integrare insieme i dati eterogenei prodotti e rendere computazionalmente possibili i processi di data mining. La scelta di tecnologia Hadoop/MapReduce risulta in questo caso particolarmente incisiva, per la scalabilit�� garantita e per l���efficienza nelle analisi statistiche pi�� complesse e parallele, come quelle riguardanti le varianti alleliche multi-locus.

Examination of FGFRL1 as a candidate gene for diaphragmatic defects at chromosome 4p16.3 shows that Fgfrl1 null mice have reduced expression of Tpm3, sarcomere genes and Lrtm1 in the diaphragm

Fgfrl1 (also known as Fgfr5; OMIM 605830) homozygous null mice have thin, amuscular diaphragms and die at birth because of diaphragm hypoplasia. FGFRL1 is located at 4p16.3, and this chromosome region can be deleted in patients with congenital diaphragmatic hernia (CDH). We examined FGFRL1 as a candidate gene for the diaphragmatic defects associated with 4p16.3 deletions and re-sequenced this gene in 54 patients with CDH. We confirmed six known coding single nucleotide polymorphisms (SNPs): c.209G > A (p.Pro20Pro), c.977G > A (p.Pro276Pro), c.1040T > C (p.Asp297Asp), c.1234C > A (p.Pro362Gln), c.1420G > T (p.Arg424Leu), and c.1540C > T (p.Pro464Leu), but we did not identify any gene mutations. We genotyped additional CDH patients for four of these six SNPs, including the three non-synonymous SNPs, to make a total of 200 chromosomes, and found that the allele frequency for the four SNPs, did not differ significantly between patients and normal controls (p > or = 0.05). We then used Affymetrix Genechip Mouse Gene 1.0 ST arrays and found eight genes with significantly reduced expression levels in the diaphragms of Fgfrl1 homozygous null mice when compared with wildtype mice-Tpm3, Fgfrl1 (p = 0.004), Myl2, Lrtm1, Myh4, Myl3, Myh7 and Hephl1. Lrtm1 is closely related to Slit3, a protein associated with herniation of the central tendon of the diaphragm in mice. The Slit proteins are known to regulate axon branching and cell migration, and inhibition of Slit3 reduces cell motility and decreases the expression of Rac and Cdc42, two genes that are essential for myoblast fusion. Further studies to determine if Lrtm1 has a similar function to Slit3 and if reduced Fgfrl1 expression can cause diaphragm hypoplasia through a mechanism involving decreased myoblast motility and/or myoblast fusion, seem indicated.