The Role of a Novel TRMT1 Gene Mutation and Rare GRM1 Gene Defect in Intellectual Disability in Two Azeri Families

Cognitive impairment or intellectual disability (ID) is a widespread neurodevelopmental disorder characterized by low IQ (below 70). ID is genetically heterogeneous and is estimated to affect 1–3% of the world’s population. In affected children from consanguineous families, autosomal recessive inheritance is common, and identifying the underlying genetic cause is an important issue in clinical genetics. In the framework of a larger project, aimed at identifying candidate genes for autosomal recessive intellectual disorder (ARID), we recently carried out single nucleotide polymorphism-based genome-wide linkage analysis in several families from Ardabil province in Iran. The identification of homozygosity-by-descent loci in these families, in combination with whole exome sequencing, led us to identify possible causative homozygous changes in two families. In the first family, a missense variant was found in GRM1 gene, while in the second family, a frameshift alteration was identified in TRMT1, both of which were found to co-segregate with the disease. GRM1, a known causal gene for autosomal recessive spinocerebellar ataxia (SCAR13, MIM#614831), encodes the metabotropic glutamate receptor1 (mGluR1). This gene plays an important role in synaptic plasticity and cerebellar development. Conversely, the TRMT1 gene encodes a tRNA methyltransferase that dimethylates a single guanine residue at position 26 of most tRNAs using S-adenosyl methionine as the methyl group donor. We recently presented TRMT1 as a candidate gene for ARID in a consanguineous Iranian family (Najmabadi et al., 2011). We believe that this second Iranian family with a biallelic loss-of-function mutation in TRMT1 gene supports the idea that this gene likely has function in development of the disorder.     

Cerebrospinal Fluid from Patients with Subarachnoid Haemorrhage and Vasospasm Enhances Endothelin Contraction in Rat Cerebral Arteries

Introduction

Previous studies have suggested that cerebrospinal fluid from patients with subarachnoid hemorrhage (SAH) leads to pronounced vasoconstriction in isolated arteries. We hypothesized that only cerebrospinal fluid from SAH patients with vasospasm would produce an enhanced contractile response to endothelin-1 in rat cerebral arteries, involving both endothelin ET_A and ET_B receptors.

Methods

Intact rat basilar arteries were incubated for 24 hours with cerebrospinal fluid from 1) SAH patients with vasospasm, 2) SAH patients without vasospasm, and 3) control patients. Arterial segments with and without endothelium were mounted in myographs and concentration-response curves for endothelin-1 were constructed in the absence and presence of selective and combined ET_A and ET_B receptor antagonists. Endothelin concentrations in culture medium and receptor expression were measured.

Results

Compared to the other groups, the following was observed in arteries exposed to cerebrospinal fluid from patients with vasospasm: 1) larger contractions at lower endothelin concentrations (p<0.05); 2) the increased endothelin contraction was absent in arteries without endothelium; 3) higher levels of endothelin secretion in the culture medium (p<0.05); 4) there was expression of ET_A receptors and new expression of ET_B receptors was apparent; 5) reduction in the enhanced response to endothelin after ET_B blockade in the low range and after ET_A blockade in the high range of endothelin concentrations; 6) after combined ET_A and ET_B blockade a complete inhibition of endothelin contraction was observed.

Conclusions

Our experimental findings showed that in intact rat basilar arteries exposed to cerebrospinal fluid from patients with vasospasm endothelin contraction was enhanced in an endothelium-dependent manner and was blocked by combined ET_A and ET_B receptor antagonism. Therefore we suggest that combined blockade of both receptors may play a role in counteracting vasospasm in patients with SAH.     

Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.     

Pediatric Chronic Dialysis in Brazil: Epidemiology and Regional Inequalities

Introduction

There are few reports in the literature estimating the epidemiologic characteristics of pediatric chronic dialysis. These patients have impaired physical growth, high number of comorbidities and great need for continuous attention of specialized services with high demand for complex and costly procedures.

Objective

The aim of this study was to estimate the incidence and prevalence rates and describe the characteristics of children and adolescents undergoing chronic dialysis treatment in a Brazilian demographic health survey.

Materials and Methods

A cross-sectional study was performed in a representative sample of dialysis centers (n^c = 239) that was established from the 2011 Brazilian Nephrology Society Census (N^c = 708). We collected data encompassing the five Brazilian macro-regions. We analyzed the data from all patients under 19 years of age. The sample population consisted of 643 children and adolescents who were on chronic dialysis program anytime in 2012. Data collection was carried out in the dialysis services by means of patients'' records reviews and personal interviews with the centers’ leaders.

Results

We estimated that there were a total of 1,283 pediatric patients on chronic dialysis treatment in Brazil, resulting in a prevalence of 20.0 cases per million age-related population (pmarp) (95% CI: 14.8–25.3) and an incidence of 6.6 cases pmarp in 2012 (95% CI: 4.8–8.4). The South region had the highest prevalence and incidence rates of patients under dialysis therapy, 27.7 (95% CI: 7.3–48.1) and 11.0 (95% CI: 2.8–19.3) cases pmarp, respectively; the lowest prevalence and incidence rates were found in the North-Midwest region, 13.8 (95% CI: 6.2–21.4), and in the Northeast region, 3.8 (95% CI: 1.4–6.3) cases pmarp, respectively.

Conclusion

Brazil has an overall low prevalence of children on chronic dialysis treatment, figuring near the rates from others countries with same socioeconomic profile. There are substantial differences among regions related to pediatric chronic dialysis treatment. Joint strategies aiming to reduce inequities and improving access to treatment and adequacy of services across the Brazilian regions are necessary to provide an appropriate care setting for this population group.     

Factors Associated with Mortality and Graft Failure in Liver Transplants: A Hierarchical Approach

Background

Liver transplantation has received increased attention in the medical field since the 1980s following the introduction of new immunosuppressants and improved surgical techniques. Currently, transplantation is the treatment of choice for patients with end-stage liver disease, and it has been expanded for other indications. Liver transplantation outcomes depend on donor factors, operating conditions, and the disease stage of the recipient. A retrospective cohort was studied to identify mortality and graft failure rates and their associated factors. All adult liver transplants performed in the state of São Paulo, Brazil, between 2006 and 2012 were studied.

Methods and Findings

A hierarchical Poisson multiple regression model was used to analyze factors related to mortality and graft failure in liver transplants. A total of 2,666 patients, 18 years or older, (1,482 males; 1,184 females) were investigated. Outcome variables included mortality and graft failure rates, which were grouped into a single binary variable called negative outcome rate. Additionally, donor clinical, laboratory, intensive care, and organ characteristics and recipient clinical data were analyzed. The mortality rate was 16.2 per 100 person-years (py) (95% CI: 15.1–17.3), and the graft failure rate was 1.8 per 100 py (95% CI: 1.5–2.2). Thus, the negative outcome rate was 18.0 per 100 py (95% CI: 16.9–19.2). The best risk model demonstrated that recipient creatinine ≥ 2.11 mg/dl [RR = 1.80 (95% CI: 1.56–2.08)], total bilirubin ≥ 2.11 mg/dl [RR = 1.48 (95% CI: 1.27–1.72)], Na⁺ ≥ 141.01 mg/dl [RR = 1.70 (95% CI: 1.47–1.97)], RNI ≥ 2.71 [RR = 1.64 (95% CI: 1.41–1.90)], body surface ≥ 1.98 [RR = 0.81 (95% CI: 0.68–0.97)] and donor age ≥ 54 years [RR = 1.28 (95% CI: 1.11–1.48)], male gender [RR = 1.19(95% CI: 1.03–1.37)], dobutamine use [RR = 0.54 (95% CI: 0.36–0.82)] and intubation ≥ 6 days [RR = 1.16 (95% CI: 1.10–1.34)] affected the negative outcome rate.

Conclusions

The current study confirms that both donor and recipient characteristics must be considered in post-transplant outcomes and prognostic scores. Our data demonstrated that recipient characteristics have a greater impact on post-transplant outcomes than donor characteristics. This new concept makes liver transplant teams to rethink about the limits in a MELD allocation system, with many teams competing with each other. The results suggest that although we have some concerns about the donors features, the recipient factors were heaviest predictors for bad outcomes.     

The fermentation of sugarcane molasses by Dekkera bruxellensis and the mobilization of reserve carbohydrates

The yeast Dekkera bruxellensis is considered to be very well adapted to industrial environments, in Brazil, USA, Canada and European Countries, when different substrates are used in alcoholic fermentations. Our previous study described its fermentative profile with a sugarcane juice substrate. In this study, we have extended its physiological evaluation to fermentation situations by using sugarcane molasses as a substrate to replicate industrial working conditions. The results have confirmed the previous reports of the low capacity of D. bruxellensis cells to assimilate sucrose, which seems to be the main factor that can cause a bottleneck in its use as fermentative yeast. Furthermore, the cells of D. bruxellensis showed a tendency to deviate most of sugar available for biomass and organic acids (lactic and acetic) compared with Saccharomyces cerevisiae, when calculated on the basis of their respective yields. As well as this, the acetate production from molasses medium by both yeasts was in marked contrast with the previous data on sugarcane juice. Glycerol and ethanol production by D. bruxellensis cells achieved levels of 33 and 53 % of the S. cerevisiae, respectively. However, the ethanol yield was similar for both yeasts. It is worth noting that this yeast did not accumulate trehalose when the intracellular glycogen content was 30 % lower than in S. cerevisiae. The lack of trehalose did not affect yeast viability under fermentation conditions. Thus, the adaptive success of D. bruxellensis under industrial fermentation conditions seems to be unrelated to the production of these reserve carbohydrates.     

Allozymatic divergence between border populations of two cryptic species of the Drosophila buzzatii cluster species (Diptera: Drosophilidae)

Drosophila antonietae and Drosophila gouveai are allopatric, cactophilic, cryptic and endemic of South America species, which aedeagus morphology is considered the main diagnostic character. In this work, single close populations from the edge distributions of each species, located in an “introgressive corridor”, were analyzed regarding temporal isozenzymatic genetic variability. Isocitrate dehydrogenase (Idh) appeared as a diagnostic locus between D. antonieate and D. gouveai because each population was fixed for different alleles. Moreover, several polymorphic loci showed accentuated divergence in the allele frequency, as evidenced by Nei’s I (0.3188) and D (1.1432), and also by Reynolds’ genetic distance and identity (1.3207 and 0.7331, respectively). Our results showed that, in spite of the very similar external morphology, related evolutionary histories, close distributions, and events of introgression in the studied area, these cryptic species have high allozymatic differentiation, and this is discussed here.     

Polymorphism of Microsatellite Markers in Papaya (<Emphasis Type="Italic">Carica papaya</Emphasis> L.)

A set of 81 new microsatellite markers for Carica papaya L. previously identified by data mining using freely available sequence information from Genbank were tested for polymorphism using 30 germplasm accessions from the Papaya Germplasm Bank (PGM) at Embrapa Mandioca e Fruticultura Tropical (CNPMF) and 18 landraces. The data were used to estimate pairwise genetic distances between the genotypes. A neighbor-joining based dendrogram was used to define clusters and infer possible genetic structuring of the collection. Most microsatellites were polymorphic (73%), with an observed number of alleles per locus ranging from one to eleven. The levels of observed and expected heterozygosity for 51 polymorphic loci varied from 0.00 to 0.85 and from 0.08 to 0.82, averaging 0.19 and 0.59, respectively. Forty-four percent of microsatellites showed polymorphism information content (PIC) higher than 0.50. The compound microsatellites seem to be more informative than dinucleotide and trinucleotide repeats in average alleles per locus and PIC. Among dinucleotides, AG/TC or GA/CT repeat motifs exhibited more informativeness than TA/AT, GT/CA and TG/AC repeat motifs. The neighbor-joining analysis based on shared allele distance could differentiate all the papaya accessions and landraces as well as differences in their genetic structure. This set of markers will be useful for examining parentage, inbreeding and population structure in papaya.     

A missense mutation in the vacuolar protein GOLD36 causes organizational defects in the ER and aberrant protein trafficking in the plant secretory pathway

A central question in cell biology is how the identity of organelles is established and maintained. Here, we report on GOLD36, an EMS mutant identified through a screen for partial displacement of the Golgi marker, ST‐GFP, to other organelles. GOLD36 showed partial distribution of ST‐GFP into a modified endoplasmic reticulum (ER) network, which formed bulges and large skein‐like structures entangling Golgi stacks. GOLD36 showed defects in ER protein export as evidenced by our observations that, besides the partial retention of Golgi markers in the ER, the trafficking of a soluble bulk‐flow marker to the cell surface was also compromised. Using a combination of classical mapping and next‐generation DNA sequencing approaches, we linked the mutant phenotype to a missense mutation of a proline residue in position 80 to a leucine residue in a small endomembrane protein encoded by the gold36 locus ( At1g54030 ). Subcellular localization analyses indicated that GOLD36 is a vacuolar protein and that its mutated form is retained in the ER. Interestingly also, a gold36 knock‐out mutant mirrored the GOLD36 subcellular phenotype. These data indicate that GOLD36 is a protein destined to post‐ER compartments and suggest that its export from the ER is a requirement to ensure steady‐state maintenance of the organelle’s organization and functional activity in relation to other secretory compartments. We speculate that GOLD36 may be a factor that is necessary for ER integrity because of its ability to limit deleterious effects of other secretory proteins on the ER.