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- Germline mutations in MAP3K6 are associated with familial gastric cancerPublication . Gaston, D.; Hansford, S.; Oliveira, C.; Nightingale, M.; Pinheiro, H.; Macgillivray, C.; Kaurah, P.; Rideout, A.; Steele, P.; Soares, G.; Huang, W.; Whitehouse, S.; Blowers, S.; LeBlanc, M.; Jiang, H.; Greer, W.; Samuels, M.; Orr, A.; Fernandez, C.; Majewski, J.; Ludman, M.; Dyack, S.; Penney, L.; McMaster, C.; Huntsman, D.; Bedard, K.Gastric cancer is among the leading causes of cancer-related deaths worldwide. While heritable forms of gastric cancer are relatively rare, identifying the genes responsible for such cases can inform diagnosis and treatment for both hereditary and sporadic cases of gastric cancer. Mutations in the E-cadherin gene, CDH1, account for 40% of the most common form of familial gastric cancer (FGC), hereditary diffuse gastric cancer (HDGC). The genes responsible for the remaining forms of FGC are currently unknown. Here we examined a large family from Maritime Canada with FGC without CDH1 mutations, and identified a germline coding variant (p.P946L) in mitogen-activated protein kinase kinase kinase 6 (MAP3K6). Based on conservation, predicted pathogenicity and a known role of the gene in cancer predisposition, MAP3K6 was considered a strong candidate and was investigated further. Screening of an additional 115 unrelated individuals with non-CDH1 FGC identified the p.P946L MAP3K6 variant, as well as four additional coding variants in MAP3K6 (p.F849Sfs*142, p.P958T, p.D200Y and p.V207G). A somatic second-hit variant (p.H506Y) was present in DNA obtained from one of the tumor specimens, and evidence of DNA hypermethylation within the MAP3K6 gene was observed in DNA from the tumor of another affected individual. These findings, together with previous evidence from mouse models that MAP3K6 acts as a tumor suppressor, and studies showing the presence of somatic mutations in MAP3K6 in non-hereditary gastric cancers and gastric cancer cell lines, point towards MAP3K6 variants as a predisposing factor for FGC.
- Assessment of a targeted resequencing assay as a support tool in the diagnosis of lysosomal storage disordersPublication . Fernández-Marmiesse, A.; Morey, M.; Pineda, M.; Eiris, J.; Couce, M.; Castro-Gago, M.; Fraga, J.; Lacerda, L.; Gouveia, S.; Pérez-Poyato, M.; Armstrong, J.; Castiñeiras, D.; Cocho, J.BACKGROUND: With over 50 different disorders and a combined incidence of up to 1/3000 births, lysosomal storage diseases (LSDs) constitute a major public health problem and place an enormous burden on affected individuals and their families. Many factors make LSD diagnosis difficult, including phenotype and penetrance variability, shared signs and symptoms, and problems inherent to biochemical diagnosis. Developing a powerful diagnostic tool could mitigate the protracted diagnostic process for these families, lead to better outcomes for current and proposed therapies, and provide the basis for more appropriate genetic counseling. METHODS: We have designed a targeted resequencing assay for the simultaneous testing of 57 lysosomal genes, using in-solution capture as the enrichment method and two different sequencing platforms. A total of 84 patients with high to moderate-or low suspicion index for LSD were enrolled in different centers in Spain and Portugal, including 18 positive controls. RESULTS: We correctly diagnosed 18 positive blinded controls, provided genetic diagnosis to 25 potential LSD patients, and ended with 18 diagnostic odysseys. CONCLUSION: We report the assessment of a next-generation-sequencing-based approach as an accessory tool in the diagnosis of LSDs, a group of disorders which have overlapping clinical profiles and genetic heterogeneity. We have also identified and quantified the strengths and limitations of next generation sequencing (NGS) technology applied to diagnosis.
- Prevalence of use of preimplantation genetic diagnosis in Unidade Clínica de Paramiloidose from Centro Hospitalar do PortoPublication . Valdrez, K.; Alves, E.; Coelho, T.; Silva, S.Introduction: The Familial Amyloid Polyneuropathy, with the world’s largest focus in Portugal, is recognized by the National Board of Assisted Reproductive Technologies as a serious disease eligible for Preimplantation Genetic Diagnosis. This study aims to determine the prevalence of the use of Preimplantation Genetic Diagnosis in FAP carriers followed in Unidade Clínica de Paramiloidose, Centro Hospitalar do Porto, and to identify the associated factors. Material and Methods: Between January and May 2013, a representative sample of Portuguese Familial Amyloid Polyneuropathy carriers, aged between 18 and 55 years, was systematically recruited. The analysis is based on 111 carriers with previous familial diagnosis, who reported having ever tried to get pregnant after 2001. Data on sociodemographic characteristics and use of Preimplan¬tation Genetic Diagnosis were collected through a self-administered questionnaire. Proportions were compared using the chi-square test. Crude and adjusted odds ratios (OR) and the respective confidence intervals of 95% (95% CI) were estimated using multivariate logistic regression. Results: The prevalence of use of Preimplantation Genetic Diagnosis was 20.7% (95% CI: 13.6-29.5). After adjustment, a household income above 1000 €/month (OR = 11.87; 95% CI 2.87-49.15) was directly associated with the use of Preimplantation Genetic Diag¬nosis, while carriers with an individual diagnosis (OR = 0.15; 95% CI 0.04-0.57) and children born after 2001 (OR = 0.07; 95% CI 0.02- 0.32) revealed a prevalence of use significantly lower than those with a individual diagnosis and children born before 2001. Discussion: The low prevalence of use of Preimplantation Genetic Diagnosis, as well as the less frequent use of the technique by those with a lower household income, shows the importance of improving access to Preimplantation Genetic Diagnosis in the case of Familial Amyloid Polyneuropathy. Conclusion: This work contributes to increase the sensitivity of health professionals around the use and accessibility to Preimplanta¬tion Genetic Diagnosis among Familial Amyloid Polyneuropathy carriers.
- MAN1B1 Deficiency: An Unexpected CDG-IIPublication . Rymen, D.; Peanne, R.; Millón, M.; Race, V.; Sturiale, L.; Garozzo, D.; Mills, P.; Clayton, P.; Asteggiano, C.; Quelhas, D.; Cansu, A.; Martins, E.; Nassogne, M.; Gonc¸alves-Rocha, M.; Topaloglu, H.; Jaeken, J.; Foulquier, F.; Matthijs, G.Congenital disorders of glycosylation (CDG) are a group of rare metabolic diseases, due to impaired protein and lipid glycosylation. In the present study, exome sequencing was used to identify MAN1B1 as the culprit gene in an unsolved CDG-II patient. Subsequently, 6 additional cases with MAN1B1-CDG were found. All individuals presented slight facial dysmorphism, psychomotor retardation and truncal obesity. Generally, MAN1B1 is believed to be an ER resident alpha-1,2-mannosidase acting as a key factor in glycoprotein quality control by targeting misfolded proteins for ER-associated degradation (ERAD). However, recent studies indicated a Golgi localization of the endogenous MAN1B1, suggesting a more complex role for MAN1B1 in quality control. We were able to confirm that MAN1B1 is indeed localized to the Golgi complex instead of the ER. Furthermore, we observed an altered Golgi morphology in all patients' cells, with marked dilatation and fragmentation. We hypothesize that part of the phenotype is associated to this Golgi disruption. In conclusion, we linked mutations in MAN1B1 to a Golgi glycosylation disorder. Additionally, our results support the recent findings on MAN1B1 localization. However, more work is needed to pinpoint the exact function of MAN1B1 in glycoprotein quality control, and to understand the pathophysiology of its deficiency.
- Phenotypic and functional consequences of haploinsufficiency of genes from exocyst and retinoic acid pathway due to a recurrent microdeletion of 2p13.2Publication . Wen, J.; Lopes, F.; Soares, G.; Farrell, S.; Nelson, C.; Qiao, Y.; Martell, S.; Badukke, C.; Bessa, C.; Ylstra, B.; Lewis, S.; Isoherranen, N.; Maciel, P.; Rajcan-Separovic, E.Background Rare, recurrent genomic imbalances facilitate the association of genotype with abnormalities at the “whole body” level. However, at the cellular level, the functional consequences of recurrent genomic abnormalities and how they can be linked to the phenotype are much less investigated. Method and results We report an example of a functional analysis of two genes from a new, overlapping microdeletion of 2p13.2 region (from 72,140,702-72,924,626). The subjects shared intellectual disability (ID), language delay, hyperactivity, facial asymmetry, ear malformations, and vertebral and/or craniofacial abnormalities. The overlapping region included two genes, EXOC6B and CYP26B1, which are involved in exocytosis/Notch signaling and retinoic acid (RA) metabolism, respectively, and are of critical importance for early morphogenesis, symmetry as well as craniofacial, skeleton and brain development. The abnormal function of EXOC6B was documented in patient lymphoblasts by its reduced expression and with perturbed expression of Notch signaling pathway genes HES1 and RBPJ, previously noted to be the consequence of EXOC6B dysfunction in animal and cell line models. Similarly, the function of CYP26B1 was affected by the deletion since the retinoic acid induced expression of this gene in patient lymphoblasts was significantly lower compared to controls (8% of controls). Conclusion Haploinsufficiency of CYP26B1 and EXOC6B genes involved in retinoic acid and exocyst/Notch signaling pathways, respectively, has not been reported previously in humans. The developmental anomalies and phenotypic features of our subjects are in keeping with the dysfunction of these genes, considering their known role. Documenting their dysfunction at the cellular level in patient cells enhanced our understanding of biological processes which contribute to the clinical phenotype.
- Newborn screening for medium-chain acyl-CoA dehydrogenase deficiency: regional experience and high incidence of carnitine deficiencyPublication . Couce, M.; Sánchez-Pintos, P.; Diogo, L.; Leão-Teles, E.; Martins, E.; Santos, H.; Amor Bueno, M.; Delgado-Pecellín, C.; Castiñeiras, D.; Cocho, J.; García-Villoria, J.; Ribes, A.; Fraga, J.; Rocha, HBackground Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common inherited defect in the mitochondrial fatty acid oxidation pathway, resulting in significant morbidity and mortality in undiagnosed patients. Newborn screening (NBS) has considerably improved MCADD outcome, but the risk of complication remains in some patients. The aim of this study was to evaluate the relationship between genotype, biochemical parameters and clinical data at diagnosis and during follow-up, in order to optimize monitoring of these patients. Methods We carried out a multicenter study in southwest Europe, of MCADD patients detected by NBS. Evaluated NBS data included free carnitine (C0) and the acylcarnitines C8, C10, C10:1 together with C8/C2 and C8/C10 ratios, clinical presentation parameters and genotype, in 45 patients. Follow-up data included C0 levels, duration of carnitine supplementation and occurrence of metabolic crises. Results C8/C2 ratio and C8 were the most accurate biomarkers of MCADD in NBS. We found a high number of patients homozygous for the prevalent c.985A > G mutation (75%). Moreover, in these patients C8, C8/C10 and C8/C2 were higher than in patients with other genotypes, while median value of C0 was significantly lower (23 μmol/L vs 36 μmol/L). The average follow-up period was 43 months. To keep carnitine levels within the normal range, carnitine supplementation was required in 82% of patients, and for a longer period in patients homozygotes for the c.985A>G mutation than in patients with other genotypes (average 31 vs 18 months). Even with treatment, median C0 levels remained lower in homozygous patients than in those with other genotypes (14 μmol/L vs 22 μmol/L). Two patients died and another three suffered a metabolic crisis, all of whom were homozygous for the c.985 A>G mutation. Conclusions Our data show a direct association between homozygosity for c.985A>G and lower carnitine values at diagnosis, and a higher dose of carnitine supplementation for maintenance within the normal range. This study contributes to a better understanding of the relationship between genotype and phenotype in newborn patients with MCADD detected through screening which could be useful in improving follow-up strategies and clinical outcome.
- Expanding the MTM1 mutational spectrum: novel variants including the first multi-exonic duplication and development of a locus-specific databasePublication . Oliveira, J.; Oliveira, M.; Kress, W.; Taipa, R.; Melo-Pires, M.; Hilbert, P.; Baxter, P.; Santos, M.; Buermans, H.; den Dunnen, J.; Santos, R.Myotubular myopathy (MIM#310400), the X-linked form of Centronuclear myopathy (CNM) is mainly characterized by neonatal hypotonia and inability to maintain unassisted respiration. The MTM1 gene, responsible for this disease, encodes myotubularin - a lipidic phosphatase involved in vesicle trafficking regulation and maturation. Recently, it was shown that myotubularin interacts with desmin, being a major regulator of intermediate filaments. We report the development of a locus-specific database for MTM1 using the Leiden Open Variation database software (http://www.lovd.nl/MTM1), with data collated for 474 mutations identified in 472 patients (by June 2012). Among the entries are a total of 25 new mutations, including a large deletion encompassing introns 2-15. During database implementation it was noticed that no large duplications had been reported. We tested a group of eight uncharacterized CNM patients for this specific type of mutation, by multiple ligation-dependent probe amplification (MLPA) analysis. A large duplication spanning exons 1-5 was identified in a boy with a mild phenotype, with results pointing toward possible somatic mosaicism. Further characterization revealed that this duplication causes an in-frame deletion at the mRNA level (r.343_444del). Results obtained with a next generation sequencing approach suggested that the duplication extends into the neighboring MAMLD1 gene and subsequent cDNA analysis detected the presence of a MTM1/MAMLD1 fusion transcript. A complex rearrangement involving the duplication of exon 10 has since been reported, with detection also enabled by MLPA analysis. It is thus conceivable that large duplications in MTM1 may account for a number of CNM cases that have remained genetically unresolved.
- Can Power Laws Help Us Understand Gene and Proteome Information?Publication . Tenreiro-Machado, J.; Costa, A.; Quelhas, M.Proteins are biochemical entities consisting of one or more blocks typically folded in a 3D pattern. Each block (a polypeptide) is a single linear sequence of amino acids that are biochemically bonded together. The amino acid sequence in a protein is defined by the sequence of a gene or several genes encoded in the DNA-based genetic code. This genetic code typically uses twenty amino acids, but in certain organisms the genetic code can also include two other amino acids. After linking the amino acids during protein synthesis, each amino acid becomes a residue in a protein, which is then chemically modified, ultimately changing and defining the protein function. In this study, the authors analyze the amino acid sequence using alignment-free methods, aiming to identify structural patterns in sets of proteins and in the proteome, without any other previous assumptions. The paper starts by analyzing amino acid sequence data by means of histograms using fixed length amino acid words (tuples). After creating the initial relative frequency histograms, they are transformed and processed in order to generate quantitative results for information extraction and graphical visualization. Selected samples from two reference datasets are used, and results reveal that the proposed method is able to generate relevant outputs in accordance with current scientific knowledge in domains like protein sequence/proteome analysis.
- Doença de Pompe Juvenil: Estudo Retrospetivo de Casuística ClínicaPublication . Loureiro-Neves, F.; Garcia, P.; Madeira, N.; Araújo, H.; Rodrigues, F.; Estevão, M.; Lacerda, L.; Diogo-Matos, L.ntrodução: A doença de Pompe ou glicogenose tipo II é uma doença autossómica recessiva por deficiência de maltase ácida. É uma entidade rara, com prevalência de 1/40.000 nas populações holandesa e afro-americana e 1/46000 na população australiana. Embora se distingam três formas de apresentação (infantil, juvenil e do adulto), observa-se um amplo espectro clínico. Em Portugal está disponível terapêutica enzimática de substituição desde 2006.Material e Métodos: Fez-se o estudo retrospetivo de quatro doentes (duas das quais irmãs), baseado na revisão dos processos clínicos.Resultados: Em todas, a doença manifestou-se no segundo ano de vida. O tempo até ao diagnóstico variou entre dois e onze anos. Aquando do diagnóstico, todas apresentavam miopatia com atraso de aquisições motoras e em duas havia hipertrofia miocárdica. A suspeita clínica surgiu por insuficiência respiratória em contexto infeccioso em duas doentes. Em todas havia elevação da creatina quinase e das aminotransferases. Todas evoluíram com insuficiência respiratória crónica por síndrome restritiva. O diagnóstico foi baseado na diminuição da atividade da maltase ácida em fibroblastos (0 a 1,5% do limite inferior do normal). Na biópsia muscular, realizada em três doentes, demonstrou-se acumulação lisossómica de glicogénio. Todas apresentavam a mutação c.1064T > C no exão 6 do gene GAA (glucosidase-alpha-acid), em homozigotia numa delas, associada às mutações c.1666A > G no exão 12 e c.2065G > A no exão 15 nas duas irmãs e à mutação c.380G > T no exão 2 na doente mais nova. Todas iniciaram terapia enzimática de substituiçãologo que disponível, com boa tolerância. A doente mais jovem faleceu pouco depois. As outras mantêm medidas de suporteventilatório e fisioterapia, deslocando-se a mais velha, em cadeira de rodas, mantendo a irmã marcha independente e necessitando a mais nova de andarilho.Conclusão: Os nossos casos incluem-se clinicamente na forma juvenil da doença de Pompe. A hipótese de doença de Pompe deve ser considerada em lactentes com miocardiopatia e nas miopatias progressivas, especialmente as das cinturas e dos músculos respiratórios em qualquer idade. A elevação da creatina quinase é um dado sensível, embora inespecífico. Dada a grande variabilidade dos achados genéticos, a demonstração da redução da atividade da maltase ácida continua a ser o pilar do diagnóstico.
- Development and validation of a multiplex-PCR assay for X-linked intellectual disabilityPublication . Jorge, P.; Oliveira, B.; Marques, I.; Santos, R.BACKGROUND: X-linked intellectual disability is a common cause of inherited cognitive deficit affecting mostly males. There are several genetic causes implicated in this condition, which has hampered the establishment of an accurate diagnosis. We developed a multiplex-PCR assay for the mutational hotspot regions of the FMR1, AFF2 and ARX genes. METHODS: The multiplex-PCR was validated in a cohort of 100 males selected to include known alleles for the FMR1 repetitive region: five full mutations (250-650 CGGs), ten premutations (70-165 CGGs) and eighty-five in the normal range (19-42 CGGs). Sequencing or Southern blotting was used to confirm the results, depending on the allele class. In this cohort, with the exception of one sample showing an AFF2 intermediate-sized allele, all other samples were normal (8-34 CCGs). No ARX variant was found besides the c.429_452dup. The validated assay was applied to 5000 samples (64.4% males and 35.6% females). RESULTS: The normal-allelic range of both FMR1 and AFF2 genes as well as the nature of ARX variants identified was similar in both genders. The rate of homozygosity observed in female samples, 27.5% for FMR1 and 17.8% for AFF2 alleles, is comparable to that published by others. Two FMR1 premutations were identified, in a male (58 CGGs) and a female case [(CGG)(47)/(CGG)(61)], as well as several FMR1 or AFF2 intermediate-sized alleles. One AFF2 premutation (68 CCGs) and two putative full expansions were picked up in male subjects, which seems relevant considering the rarity of reported AFF2 mutations found in the absence of a family history. CONCLUSIONS: We developed a robust multiplex-PCR that can be used to screen the mutational hotspot regions of FMR1, AFF2 and ARX genes. Moreover, this strategy led to the identification of variants in all three genes, representing not only an improvement in allele-sizing but also in achieving a differential diagnosis. Although the distinction between females who are truly homozygous and those with a second pre- or full mutation sized allele, as well as a definitive diagnosis, requires a specific downstream technique, the use of this multiplex-PCR for initial screening is a cost-effective approach which widens the scope of detection.