Solving a case of allelic dropout in the GNPTAB gene: implications in the molecular diagnosis of mucolipidosis type III alpha/beta

While being well known that the diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing, not rarely, however, genetic testing needs much perseverance and cunning strategies to identify the causative mutation(s). Here we present a case of a thorny molecular diagnosis of mucolipidosis type III alpha/beta, which is an autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the α/β-subunits of the GlcNAc-1-phosphotransferase. We used both cDNA and gDNA analyses to characterize a mucolipidosis type III alpha/beta patient whose clinical diagnosis was already confirmed biochemically. In a first stage only one causal mutation was identified in heterozygosity, the already described missense mutation c.1196C>T(p.S399F), both at cDNA and gDNA levels. Only after conducting inhibition of nonsense-mediated mRNA decay (NMD) assays and after the utilization of another pair of primers the second mutation, the c.3503_3504delTC deletion, was identified. Our findings illustrate that allelic dropout due to the presence of polymorphisms and/or of mutations that trigger the NMD pathway can cause difficulties in current molecular diagnosis tests.M.F. Coutinho is grantee from the FCT (SFRH/BPD/101965/2014).info:eu-repo/semantics/publishedVersio

Effects of planting dates on growth and nutrient accumulation of carrots in the brazilian semi-arid

Fertilization plays an important role on carrot’s yield, root quality, storage, plant growth and on the environment. It was aimed to evaluate the plant growth and macronutrients accumulation of carrot cultivars as a function of two planting dates, under high temperatures in the Brazilian semi-arid. The experiments were carried out in randomized blocks design, with ten treatments and four repetitions. Treatments consisted of ten carrot cultivars sowed in two Planting dates. The characteristics that were evaluated were: plant growth (plant height, number of leaves, plant dry matter accumulation, mean fresh mass of the root) and macronutrient accumulation (N, P, K, Ca and Mg) in plant, leaves and root. Plant’s mean height ranged from 42.53 cm (Melinda) to 49.25 cm (Nativa); the highest plant dry matter was obtained by BRS Planalto (12.36 g) and Kuronan (12.18 g); the mean number of leaves was the lowest in Melinda and Nativa: 8.64 and 7.64 leaves plant-1. The root’s fresh weight had a significant decrease among the planting dates for the Brasília, Francine and Suprema cultivars. The nutrient accumulation varied accordingly to the planting date and cultivar

Reflexões acerca das nanotecnologias e as novas densidades técnicas-científicas-informacionais na agricultura

As pesquisas sobre os potenciais usos das nanotecnologias no agronegócio suscitam enormes dúvidas sobre os reais benefícios ou malefícios em relação à adoção desta inovação. Daí a preocupação desta investigação em incluir as ciências humanas nos debates sobre a difusão das nanotecnologias. Pressupõe-se que, dada a efetiva incorporação dos novos produtos e processos nanotecnológicos, irá se configurar um aprofundamento das densidades técnicas-científicas- informacionais no espaço agrário brasileiro

Perfis de Vulnerabilidade Feminina ao HIV/aids em Belo Horizonte e Recife: comparando brancas e negras

OBJECTIVE: To delineate and compare profiles of white and "Black" (either Black or mixed) women, 18 to 59 years-old, residents of Belo Horizonte and Recife (Brazil), focusing on their knowledge and attitudes about HIV/AIDS, as well as their socio-demographic characteristics. METHODS: Data come from the survey SRSR (Reproductive Health, Sexuality, and Race/Skin Color), conducted by Cedeplar in 2002 and the only one of its kind with representativeness at the municipality (county) level. Grade of Membership (GoM) was used to generate four profiles of women for each county. RESULTS: In Belo Horizonte and Recife, women who are more likely to be White are also more likely to have better education, health insurance, a stable partner in the year before the survey, and more power in their sexual partnership. Regarding the "Black" women in Belo Horizonte, they are more likely to have low education, no health insurance, and less power with their sexual partners. CONCLUSIONS: The comparison among the profiles of White and "Black" women in Belo Horizonte and Recife points to differences regarding their vulnerability to HIV/AIDS. The differences between the two racial groups are more evident in Belo Horizonte.OBJETIVO: Delinear e comparar os perfis das mulheres brancas e negras entre 18 e 59 anos, residentes em Belo Horizonte e Recife, enfocando características sociodemográficas e de conhecimento, além de atitudes em relação ao HIV/aids. MÉTODOS: Os dados são oriundos da pesquisa amostral SRSR - Saúde Reprodutiva, Sexualidade e Raça/Cor, conduzida pelo Cedeplar/UFMG em 2002 e única desta natureza com representatividade municipal. O método utilizado foi o Grade of Membership (GoM), a partir do qual foram gerados quatro perfis extremos para cada município. RESULTADOS: Tanto em Belo Horizonte quanto em Recife, as mulheres com maior probabilidade de serem brancas são também aquelas com maior probabilidade de ter escolaridade mais elevada, possuir plano de saúde, ter tido parceiro estável no ano anterior à pesquisa e ter poder na relação sexual. Quanto às negras, apenas em Belo Horizonte elas têm maior probabilidade de serem de baixa escolaridade, não possuírem plano de saúde, além de se sentirem desempoderadas diante do parceiro sexual. CONCLUSÕES: A comparação dos perfis de brancas e negras em Belo Horizonte e Recife revela diferenças na vulnerabilidade dessas mulheres ao HIV/aids. As diferenças entre os dois grupos são mais evidentes em Belo Horizonte

NPC1 silent variant induces skipping of exon 11 (p.V562V) and unfolded protein response was found in a specific Niemann‐Pick type C patient

Background: Niemann-Pick type C (NPC, MIM #257220) is a neuro-visceral disease, caused predominantly by pathogenic variants in the NPC1 gene. Here we studied patients with clinical diagnosis of NPC but inconclusive results regarding the molecular analysis. Methods: We used a Next-Generation Sequencing (NGS)-panel followed by cDNA analysis. Latter, we used massively parallel single-cell RNA-seq (MARS-Seq) to address gene profiling changes and finally the effect of different variants on the protein and cellular levels. Results: We identified novel variants and cDNA analysis allowed us to establish the functional effect of a silent variant, previously reported as a polymorphism. We demonstrated that this variant induces the skipping of exon 11 leading to a premature stop codon and identified it in NPC patients from two unrelated families. MARS-Seq analysis showed that a number of upregulated genes were related to the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in one specific patient. Also, for all analyzed variants, the NPC1 protein was partially retained in the ER. Conclusion: We showed that the NPC1 silent polymorphism (p.V562V) is a disease-causing variant in NPC and that the UPR is upregulated in an NPC patient.info:eu-repo/semantics/publishedVersio

Novel method for picking up large heterozygous deletions with semiquantitative PCR in patients with mucolipidosis III alpha/beta

Mucolipidosis II (ML II alpha/beta) or I-cell disease is a rare genetic disease in which the activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent. GlcNAc-phosphotransferase is a multimeric enzyme encoded by two genes: GNPTAB and GNPTG. Although a wide spectrum of mutations in GNPTAB has recently been reported to cause ML II alpha/beta, there are some patients that were previously reported as possessing only one heterozygous mutation while the second one was missing. Here we support the idea that some of these cases may be attributable to the methodological problem of direct sequencing since large heterozygous deletions are undetectable through this method. Methods: We developed a semiquantitative approach by multiplex PCR and capillary electrophoresis. In order to do so, we co-amplified two exon fragments in each PCR reaction, including the one corresponding to GNPTAB exons and an internal fragment that would work as a control (primer pairs for LIPA exons, the gene that codes for acid lipase, another lysosomal enzyme). Results: After testing this technique in the two Portuguese patients that present heterozygous missense mutations but with the second mutation missing, we found evidences that support the idea that, in both patient, the last exons (20 and 21) of the GNPTAB gene are missing in one of the alleles. Conclusion: Here we present the first molecular evidence of the existence of large deletions in the GNPTAB, underlying Mucolipidosis type III as well as a novel method for detecting these types of alterations when present in heterozygous patients. This semiquantitative PCR approach is a valuable tool that allows the screening of large deletions in the GNPTAB gene. In conclusion, ML III cases with only one heterozygous mutation already detected trhpugh direct sequencing methods should be further screened for the possible presence of a large heterozygous deletion mutation