Darier disease: molecular study of a two-generation Portuguese family

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2018Background Darier Disease is a rare autosomal dominant disorder, which predominantly affects the skin of individuals regardless their gender or ethnicity. Clinical manifestations usually involve nail abnormalities and focal skin lesions such as greasy, brownish-redish keratotic papules, which are distributed in seborrheic areas of the body. Affected individuals are usually heterozygous for mutation in ATPase sarcoplasmic/endoplasmic reticulum Calcium transporting 2 (ATP2A2) gene, responsible for encoding Sarco/Endoplasmic Reticulum Calcium ATPase 2 (SERCA2). Although ATP2A2 mutations significantly reduce ER Ca2+ stores in both lesional and non lesional keratinocytes, scientists suggest that a threshold in ER Ca2+ depletion is required to disrupt adhesion molecules, as seen in lesional epidermis. Nonetheless, DD hallmarks (acantholysis and dyskeratosis) remain to be fully elucidated. Haploinsufficiency or dominant-negative effect are the postulated pathogenic mechanisms. Objective This study consists in the molecular characterization of a two-generation Portuguese family with DD history, including co-associated phenotypes and intrafamilial phenotypic variability. Methods All exons and intron-exon borders of ATP2A2 were bidirectionally sequenced from DNA and RNA extracted from the five subjects enrolled in this study (four affected individuals and one unaffected individual). Relative levels SERCA2 mRNA and protein were quantified by RT-qPCR and western blotting, respectively. Results A splice-site mutation (c.1287+1G>T or IVS10+1G>T) was identified in all four affected individuals, who carry the mutation in heterozigosity, whereas the unaffected individual was shown to carry the wild-type ATP2A2 sequence in both alleles. This mutation leads to the skipping of full exon 10, which consequently generates a frameshift followed by a premature translation termination codon in exon 11. Results from RT-PCR of a fragment spanning exons 8-13 suggest that a small amount of the mutant transcript escapes NMD and less than 50% expression of the wild-type transcript is detected. In agreement, relative wild-type and mutant SERCA2 mRNA expression levels assessed by qPCR revealed wild-type gene expression alone revealed a 36% expression, while mutant mRNA expression exhibited residual levels of 18%. In contrast, western blot results showed 40 to 50% expression of wild-type SERCA2 (115 kDa) in DD patients when compared to the healthy individual, while no sign of the putative mutant SERCA2 (45 kDa) was detected. These findings suggest that despite the escape of a small portion of abnormal SERCA2 mRNA from NMD, there is still no generation of mutant protein. Therefore, haploinsufficiency appears to be the mechanism underlying DD pathology in these patients. Finally, no genotype-phenotype correlations were found; however, this study contributed to alert physicians to the wide range of defects predicted by ATP2A2 mutations, including neuropsychiatric features, hearing loss, corneal viral infections by HSV, and glomerular nephritis which is, to the best of our knowledge, here firstly reported in association with DD. Conclusion As the first study of Portuguese patients, it adds knowledge to the worldwide mutational and clinical spectrum of DD and, importantly, contributes to genetic counseling since it allowed to discard a possible carrier status of the unaffected member of the family.Introdução A doença de Darier é uma genodermatose rara, de transmissão autossómica dominante, que não distingue sexo ou etnia. As manifestações clínicas caraterísticas desta doença envolvem anomalias nas unhas e lesões cutâneas focais, tais como pápulas queratóticas acastanhadas-avermelhadas distribuídas pelas áreas seborreicas do corpo. Os indivíduos afetados são geralmente heterozigóticos para uma mutação no gene ATP2A2, responsável pela codificação da proteína SERCA2. Embora as mutações no gene ATP2A2 reduzam significativamente as reservas de Ca2+ no retículo endoplasmático em células lesionadas e não lesionadas, os cientistas sugerem a necessidade de um limite na depleção destas reservas para interferir no funcionamento de moléculas de adesão, como observado nos queratinócitos acantolíticos da epiderme lesionada. Haploinsuficiência ou efeito dominante negativo são os mecanismos patogénicos postulados para o fenótipo de Darier. Objetivo Este projeto consiste na caraterização molecular duas gerações de uma família Portuguesa de com histórico de doença de Darier, incluindo co-fenótipos e variabilidade fenotípica intrafamiliar. Métodos Todos os exões e limites exão-intrão do gene ATP2A2 foram sequenciados bidirecionalmente a partir de DNA e RNA extraídos dos cinco indivíduos envolvidos neste estudo (quatro afetados e um não afetado). Os níveis relativos de RNA mensageiro e proteína SERCA2 foram quantificados por PCR em tempo real e Western blotting, respetivamente. Resultados A mutação patogénica (c.1287 + 1G> T ou IVS10 + 1G> T) foi identificada em todos os quatro indivíduos afetados, que exibem a mutação em heterozigotia, enquanto o indivíduo não afetado exibe a sequência normal de ATP2A2 em ambos os alelos. A mutação altera o splicing e leva à eliminação completa do exão 10, que consequentemente altera a leitura da ORF, seguida de um codão de terminação de tradução prematura no exão 11. Os resultados de RT-PCR, correspondente à amplificação de um fragmento abrangendo os exões 8 a 13, sugerem que uma pequena quantidade do transcrito mutado consegue escapar ao NMD e que a expressão de transcrito normal é inferior a 50% relativamente ao indivíduo normal. Em conformidade, os níveis relativos de expressão de RNA mensageiro SERCA2 normal e mutado avaliados por PCR em tempo real revelaram 36% de expressão do gene normal, e aproximadamente 18% de expressão do transcrito mutado. Em contraste, os resultados de western blot mostraram 40 a 50% de expressão de SERCA2 normal (115 kDa) nos indivíduos com a doença, em comparação com o indivíduo saudável, enquanto nenhum sinal da hipotética proteína mutada (45 kDa) foi detetado. Em geral, os resultados obtidos sugerem que, apesar da pequena porção de transcrito mutado escapar ao NMD, não há tradução em proteína mutada, ou esta deve ser imediatamente degradada. Portanto, a haploinsuficiência parece ser o mecanismo responsável pela patologia de Darier nos doentes estudados. Finalmente, não foi possível identificar qualquer correlação genótipo-fenótipo; no entanto, este estudo contribuiu para alertar os médicos dermatologistas sobre a panóplia de alterações provocadas pelas mutações no gene ATP2A2, incluindo sintomas neuropsiquiátricos, perda de audição, infeções virais da córnea causadas por HSV, e nefrite glomerular membranosa que é, pelo que temos conhecimento, relatado neste trabalho pela primeira vez em associação com DD. Conclusão Como primeiro estudo em doentes Portugueses, esta investigação acrescenta conhecimento ao espetro mutacional e clínico mundial da doença de Darier e, mais importante, contribui para o aconselhamento genético, uma vez que permitiu descartar um possível estatuto de portador do membro não afetado da família

NEOTROPICAL XENARTHRANS: a data set of occurrence of xenarthran species in the Neotropics

Xenarthrans—anteaters, sloths, and armadillos—have essential functions for ecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosystem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts with domestic dogs, these species have been threatened locally, regionally, or even across their full distribution ranges. The Neotropics harbor 21 species of armadillos, 10 anteaters, and 6 sloths. Our data set includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae (3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data on Dasypus pilosus (Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized, but new genetic studies have revealed that the group is represented by seven species. In this data paper, we compiled a total of 42,528 records of 31 species, represented by occurrence and quantitative data, totaling 24,847 unique georeferenced records. The geographic range is from the southern United States, Mexico, and Caribbean countries at the northern portion of the Neotropics, to the austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regarding anteaters, Myrmecophaga tridactyla has the most records (n = 5,941), and Cyclopes sp. have the fewest (n = 240). The armadillo species with the most data is Dasypus novemcinctus (n = 11,588), and the fewest data are recorded for Calyptophractus retusus (n = 33). With regard to sloth species, Bradypus variegatus has the most records (n = 962), and Bradypus pygmaeus has the fewest (n = 12). Our main objective with Neotropical Xenarthrans is to make occurrence and quantitative data available to facilitate more ecological research, particularly if we integrate the xenarthran data with other data sets of Neotropical Series that will become available very soon (i.e., Neotropical Carnivores, Neotropical Invasive Mammals, and Neotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure, habitat loss, fragmentation effects, species invasion, and climate change effects will be possible with the Neotropical Xenarthrans data set. Please cite this data paper when using its data in publications. We also request that researchers and teachers inform us of how they are using these data

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field