Fragile X syndrome: An overview and update of the FMR1 gene.

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability and the leading form of the monogenic cause of autism. Fragile X mental retardation type 1 (FMR1) gene premutation is the first single-gene cause of primary ovarian failure (Fragile X-associated primary ovarian insufficiency [FXPOI]) and one of the most common causes of ataxia (fragile X-associated tremor/ataxia syndrome [FXTAS]), multiple additional phenotypes such as fibromyalgia, hypothyroidism, migraine headaches, sleep disturbances, sleep apnea, restless legs syndrome, central pain syndrome, neuropathy and neuropsychiatric alterations has been described. Clinical involvement in men and women carrying the FMR1 premutation currently constitutes a real health problem in the society that should be taken into account. It is important to highlight that while in FXS there is a loss-of-function of the FMR1 gene, in premutation associated disorders there is a gain of FMR1 mRNA function. To date, the tremendous progress achieved in the understanding of the pathophysiology of FXS, has led to the development of several targeted therapies aimed at preventing or improving the neurological manifestations of the disease. This review is an update of the diseases associated with the FMR1 gene.

Clinical implication of FMR1 intermediate alleles in a Spanish population.

FMR1 premutation carriers (55-200 CGGs) are at risk of developing Fragile X-associated primary ovarian insufficiency as well as Fragile X-associated tremor/ataxia syndrome. FMR1 premutation alleles are also associated with a variety of disorders, including psychiatric, developmental, and neurological problems. However, there is a major concern regarding clinical implications of smaller CGG expansions known as intermediate alleles (IA) or gray zone alleles (45-54 CGG). Although several studies have hypothesized that IA may be involved in the etiology of FMR1 premutation associated phenotypes, this association still remains unclear. The aim of this study was to provide new data on the clinical implications of IA. We reviewed a total of 17 011 individuals: 1142 with primary ovarian insufficiency, 478 with movement disorders, 14 006 with neurodevelopmental disorders and 1385 controls. Similar IA frequencies were detected in all the cases and controls (cases 1.20% vs controls 1.39%, P = .427). When comparing the allelic frequencies of IA ≥ 50CGGs, a greater, albeit not statistically significant, number of alleles were detected in all the cohorts of patients. Therefore, IA below 50 CGGs should not be considered as risk factors for FMR1 premutation-associated phenotypes, at least in our population. However, the clinical implication of IA ≥ 50CGGs remains to be further elucidated.

Social anxiety and autism spectrum traits among adult FMR1 premutation carriers.

Behavioral symptoms and traits have been proposed as early markers in neurodegenerative diseases. The aim of this study was to evaluate social anxiety and autism in FMR1 premutation carriers using the Social Phobia Inventory (SPIN) and the Autism-Spectrum Quotient (AQ) questionnaires. Fifty-nine premutation carriers were compared with 50 controls. The SPIN test showed statistically significant differences between female but not male carriers. The AQ questionnaire found statistically significant differences between premutation carriers and controls in the total AQ as well as in the social skills and attention switching subdomains. A gender effect was only observed for the social skills subdomain. Spearman's correlation analysis revealed a moderately positive correlation with the total AQ scores as well as the social skills and communication subdomains. Our results show that fragile X-associated tremor/ataxia syndrome (FXTAS) patients have higher AQ scores. Moreover, this is the first study to find statistically significant differences between FXTAS and no-FXTAS premutation carriers in the communication and the imagination subdomains, suggesting that FXTAS patients present a broader autistic phenotype than premutation carriers without FXTAS. Based on our results, a wide range of behavioral/psychiatric traits should be included within the broader phenotypic presentation of individuals with the FMR1 premutation.

Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation.

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

Elements of functional genital asymmetry in the cow.

Asymmetry in the cow affects ovarian function and pregnancy. In this work we studied ovarian and uterine asymmetry. Synchronised animals, in which in vitro-produced embryos (n=30-60) had been transferred on Day 5 to the uterine horn ipsilateral to the corpus luteum (CL), were flushed on Day 8. Ovulatory follicle diameter, oestrus response and total protein flushed did not differ between sides. However, a corpus luteum in the right ovary led to plasma progesterone concentrations that were higher than when it was present in the left ovary. Fewer embryos were recovered from the left than the right horn. Among 60 uterine proteins identified by difference gel electrophoresis, relative abundance of nine (acyl-CoA dehydrogenase, very long chain; twinfilin, actin-binding protein, homologue 1; enolase 1; pyruvate kinase isozymes M1/M2 (rabbit); complement factor B Bb fragment ; albumin; fibrinogen gamma-B chain; and ezrin differed (P<0.05) between horns. Glucose concentration was higher, and fructose concentration lower, in the left horn. In a subsequent field trial, pregnancy rates after embryo transfer did not differ between horns (51.0±3.6, right vs 53.2±4.7, left). However, Day 7 blood progesterone concentrations differed (P=0.018) between pregnant and open animals in the left (15.9±1.7 vs 8.3±1.2) but not in the right horn (12.4±1.3 vs 12.4±1.2). Progesterone effects were independent of CL quality (P=0.55). Bilateral genital tract asymmetry in the cow affects progesterone, proteins and hexoses without altering pregnancy rates.

Microenvironment M1/M2 macrophages and tumoral progression vary within C57BL/6 mice from same substrain in prostate cancer model.

Cancer mice models are critical for immune-oncology research; they provide conditions to explore tumor immunoenviroment aiming to advance knowledge and treatment development. Often, research groups breed their own mice colonies. To assess the effect of C57BL/6 mice breeding nuclei in prostate cancer development and intratumoral macrophage populations, an isotransplantation experiment was performed. C57BL/6J mice from two breeding nuclei (nA and nB) were employed for prostate adenocarcinoma TRAMP-C1 cell implantation; tumor growth period and intratumoral macrophage profile were measured. BL/6nB mice (54%) showed tumor implantation after 69-day growth period while BL/6nA implantation reached 100% across tumor growth period (28 days). No difference in total macrophage populations was observed between groups within several tumoral regions; significantly higher M2 macrophage profile was observed in tumor microenvironments from both mice groups. Nevertheless, BL/6nB tumors showed around twice the population of M1 profile (11-27%) than BL6nA (4-15%) and less non-polarized macrophages. The M1:M2 average ratio was 1:8 for group A and 1:4 for B. Our results demonstrate different tumor progression and intratumoral macrophage populations among mice from the same substrain. Data obtained in this study shows the relevance of animal source renewal for better control of murine cancer model variables.

Biallelic variants in genes previously associated with dominant inheritance: CACNA1A, RET and SLC20A2.

A subset of families with co-dominant or recessive inheritance has been described in several genes previously associated with dominant inheritance. Those recessive families displayed similar, more severe, or even completely different phenotypes to their dominant counterparts. We report the first patients harboring homozygous disease-related variants in three genes that were previously associated with dominant inheritance: a loss-of-function variant in the CACNA1A gene and two missense variants in the RET and SLC20A2 genes, respectively. All patients presented with a more severe clinical phenotype than the corresponding typical dominant form. We suggest that co-dominant or recessive inheritance for these three genes could explain the phenotypic differences from those documented in their cognate dominant phenotypes. Our results reinforce that geneticists should be aware of the possible different forms of inheritance in genes when WES variant interpretation is performed. We also evidence the need to refine phenotypes and inheritance patterns associated with genes in order to avoid failures during WES analysis and thus, raising the WES diagnostic capacity in the benefit of patients.

Spectrum of clinical heterogeneity of ß-tubulin TUBB5 gene mutations.

Microcephaly is a rare condition in which the occipitofrontal circumference in a child is more than two standard deviations below the mean of children of the same age and gender. It is mainly caused by genetic abnormalities that interfere with the growth of the cerebral cortex during early months of fetal development. We present a case of a 12 years old patient with microcephaly. To identify a possible genetic origin of the phenotype, we performed array CGH and exome sequencing in the patient. Exome sequencing revealed the presence of a de novo missense mutation in the TUBB5 gene (E401K). Mutations in the TUBB5 are mainly responsible for microcephaly but the clinical spectrum is wide, from patients with severe developmental delay, and the presence of different brain malformations, to patients with only slightly cognitive impairment and normal motor development. Our patient shows a milder phenotype than other patients carrying the same mutation. These differences in the clinical features suggest that other factors, presumably genetic or epigenetic, could be modulating clinical expressivity of TUBB5. It is therefore evident that more functional studies are needed to understand the pathology that underlies the clinical spectrum of tubulin associated disease states.

Hypermethioninaemia due to methionine adenosyltransferase I/III (MAT I/III) deficiency: diagnosis in an expanded neonatal screening programme.

The Expanded Newborn Screening Program (MS/MS) in the region of Galicia (NW Spain) was initiated in 2000 and includes the measurement of methionine levels in dried blood spots. Between June 2000 and June 2007, 140 818 newborns were analysed, and six cases of persistent hypermethioninaemia were detected: one homocystinuria due to cystathionine ß-synthase (CßS) deficiency, and five methionine adenosyltransferase I/III (MAT I/III) deficiencies. The five cases of MAT I/III deficiency represent an incidence of 1/28 163 newborns. In these five patients, methionine levels in dried blood spots ranged from 50 to 147 µmol/L. At confirmation of the persistence of the hypermethioninaemia in a subsequent plasma sample, plasma methionine concentrations were moderately elevated in 4 of the 5 patients (mean 256 µmol/L), while total homocysteine (tHcy) was normal; the remaining patient showed plasma methionine of 573 µmol/L and tHcy of 22.8 µmol/L. All five patients were heterozygous for the same dominant mutation, R264H in the MAT1A gene. With a diet not exceeding recommended protein requirements for their age, all patients maintained methionine levels below 300 µmol/L. Currently, with a mean of 2.5 years since diagnosis, the patients are asymptomatic and show developmental quotients within the normal range. Our results show a rather high frequency of hypermethioninaemia due to MAT I/III deficiency in the Galician neonatal population, indicating a need for further studies to evaluate the impact of persistent isolated hypermethioninaemia in neonatal screening programmes.

Impaired Mitochondrial Function and Dynamics in the Pathogenesis of FXTAS.

Mitochondrial involvement plays an important role in neurodegenerative diseases. At least one-third of adult carriers of a FMR1 premutation (55-200 CGG repeats) are at risk of presenting an adult-onset neurodegenerative disorder known as fragile X-associated tremor/ataxia syndrome (FXTAS). In an attempt to provide new insights into the mechanisms involved in the pathogenesis of FXTAS, we characterized mitochondrial function and dynamics by the assessment of oxidative respiratory chain function, mitochondrial content, oxidative stress levels, and mitochondrial network complexity. Regarding mitochondrial function, we found that mitochondrial respiratory capacity is compromised in skin fibroblasts whereas in blood, no differences were observed between the FXTAS and control groups. Furthermore, fibroblasts from FXTAS patients presented altered mitochondrial architecture, with more circular and less interconnected mitochondria being observed. Mitochondrial function and dynamics deregulation and characteristic of neurological disorders are present in FXTAS patients. These features might be limiting temporal and spatial bioenergetics cells supply and thus contributing to disease pathogenesis.

A new mass spectrometry-based method for the quantification of histones in plasma from septic shock patients.

The aim of this study was to develop a novel method to detect circulating histones H3 and H2B in plasma based on multiple reaction monitoring targeted mass spectrometry and a multiple reaction monitoring approach (MRM-MS) for its clinical application in critical bacteriaemic septic shock patients. Plasma samples from 17 septic shock patients with confirmed bacteraemia and 10 healthy controls were analysed by an MRM-MS method, which specifically detects presence of histones H3 and H2B. By an internal standard, it was possible to quantify the concentration of circulating histones in plasma, which were significantly higher in patients, and thus confirmed their potential as biomarkers for diagnosing septic shock. After comparing surviving patients and non-survivors, a correlation was found between higher levels of circulating histones and unfavourable outcome. Indeed, histone H3 proved a more efficient and sensitive biomarker for septic shock prognosis. In conclusion, these findings suggest the accuracy of the MRM-MS technique and stable isotope labelled peptides to detect and quantify circulating plasma histones H2B and H3. This method may be used for early septic shock diagnoses and for the prognosis of fatal outcomes.

Molecular characterization and phylogenetic study of Maedi Visna and Caprine Arthritis Encephalitis viral sequences in sheep and goats from Spain.

Small ruminant lentiviruses (SRLV) are widely spread in many countries, including Spain. However, little is known about the genetic characteristics of Spanish goat and sheep SRLV. In this study, segments from three genomic regions (pol, gag-p25 and LTR) were amplified using DNA isolated from three Spanish autochthonous sheep (one) and goats (two). Animals (one per flock) belonged to distantly located, single-species flocks (goat or sheep). Sequence analysis showed conservation of regions that are putatively relevant to viral survival. Sequences of Spanish goat and sheep SRLV were allocated into phylogenetic trees (phylograms) with known SRLV groups. The phylograms corresponding to the pol, gag-p25 and LTR regions analyzed presented a compatible topology. This showed that Spanish caprine and ovine SRLV sequences belonged to the A or D phylogenetic groups and were closer to sheep SRLV prototypes (A1 group) than to goat SRLV prototypes (B or C groups), according to the current classification [Shah, C., Boni, J., Huder, J.B., Vogt, H.R., Muhlherr, J., Zanoni, R., Miserez, R., Lutz, H., Schupbach, J., 2004a. Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade. Virology 319 (1), 12-26]. It was not possible to amplify in the three genetic regions the expected fragment in additional Spanish caprine and ovine SRLV proviral DNA sequences with the PCR primers used. This suggests that there is heterogeneity at the primer binding site among Spanish SRLV sequences. It also illustrates the need to develop diagnostic tests that are sensitive in local breeds.

[Intimate partner violence and family dysfunction among Mexican women seen a Primary Care Unit]. / Factores asociados a violencia doméstica en mujeres mexicanas vistas en primer nivel atención.

OBJECTIVE: To assess the prevalence and risk factors for intimate-partner violence (IPV) in women who attended a Family Medicine Unit in Tijuana, Mexico. METHODS: A total of 297 women were interviewed and evaluated using two validated scales: violence and severity index and family APGAR to assess family functioning and IPV respectively. RESULTS: The mean age (± SD) was 40.6±13.8 years, and 120 (40.4%) women had suffered IPV: 47 (15.8%) psychological violence; 31 (10.4%) sexual violence; 77 (25.9%) physical violence, and in 19 (6.4%) there were actions that threatened the lives of women. The most common causes of domestic violence were women who reported that their partner had been jealous, or suspicion from friends (37.4%). Twenty two (7.4%) of the women with domestic violence reported that they had sought help. CONCLUSIONS: The prevalence of IPV was high and associated with the education level of the couple and family functioning.

Deregulation of key signaling pathways involved in oocyte maturation in FMR1 premutation carriers with Fragile X-associated primary ovarian insufficiency.

FMR1 premutation female carriers are at risk for Fragile X-associated primary ovarian insufficiency (FXPOI). Insights from knock-in mouse model have recently demonstrated that FXPOI is due to an increased rate of follicle depletion or an impaired development of the growing follicles. Molecular mechanisms responsible for this reduced viability are still unknown. In an attempt to provide new data on the mechanisms that lead to FXPOI, we report the first investigation involving transcription profiling of total blood from FMR1 premutation female carriers with and without FXPOI. A total of 16 unrelated female individuals (6 FMR1 premutated females with FXPOI; 6 FMR1 premutated females without FXPOI; and 4 no-FXPOI females) were studied by whole human genome oligonucleotide microarray (Agilent Technologies). Fold change analysis did not show any genes with significant differential gene expression. However, functional profiling by gene set analysis showed large number of statistically significant deregulated GO annotations as well as numerous KEGG pathways in FXPOI females. These results suggest that the impairment of fertility in these females might be due to a generalized deregulation of key signaling pathways involved in oocyte maturation. In particular, the vasoendotelial growth factor signaling, the inositol phosphate metabolism, the cell cycle, and the MAPK signaling pathways were found to be down-regulated in FXPOI females. Furthermore, a high statistical enrichment of biological processes involved in cell death and survival were found deregulated among FXPOI females. Our results provide new strategic approaches to further investigate the molecular mechanisms and potential therapeutic targets for FXPOI not focused in a single gene but rather in the set of genes involved in these pathways.

MicroRNA expression profiling in blood from fragile X-associated tremor/ataxia syndrome patients.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder associated with FMR1 gene premutation alleles (55-200 CGG repeats). Fragile X-associated tremor/ataxia syndrome clinical core features include action tremor, gait ataxia, cognitive deficits progressing to dementia, and frequently parkinsonism. Although the pathogenic molecular mechanism of FXTAS is not completely understood, the restriction of the phenotype to the FMR1 premutation range has given rise to a model based on a RNA toxic gain-of-function. Since the identification of the first microRNAs (miRNAs) and their role in normal development, several studies have associated them with neurodegenerative diseases such as Parkinson, Alzheimer and Huntington diseases, suggesting that they play a key role in brain development, as well as in its morphogenesis. Herein, we present the characterization of miRNA expression profiles in FXTAS male patients using deep sequencing-based technologies and microarray technology. Deep sequencing analysis evidenced 83 miRNAs that were significantly deregulated whereas microarray analysis showed 31. When comparing these results, 14 miRNAs were found deregulated in FXTAS patients. MiR-424 and miR-574-3p showed significant fold change adjusted P-values in both platforms in FXTAS patients. MiR-424 has been founded substantially and specifically enriched in human cerebral cortical white matter of Alzheimer disease patients, which, together with cerebral atrophy, is a prominent imaging finding in individuals with FXTAS. The study provides the first systematic evidence of differential miRNA expression changes in FXTAS blood samples. Although further studies are necessary to better characterize the miRNA function in FXTAS disorder, our results suggest that they might contribute to its pathogenesis.

The effect of chronic intraperitoneal infusion of bacterial endotoxin on exocrine pancreas function in rats.

CONCLUSION: This study demonstrated that LPS infusion can induce tissue lesions and impair the exocrine protein secretion of the pancreas in rats. BACKGROUND: The effect of chronic ip infusion of lipopolysaccharide (LPS) on the exocrine pancreas function was studies in rats. METHODS: Four milligrams per kilogram per day of Salmonella typhi LPS were infused intraperitoneally by means of surgically implanted osmotic pumps. Rats were studied after 7-d LPS infusion. RESULTS: Plasma fibrinogen and amylase activity increased significantly in LPS-treated rats when compared with control rats. Histological examination of the pancreas showed congestion, infiltration, and focal necrosis in LPS-treated rats. The pancreas wet weight, as well as DNA and total soluble protein contents were significantly increased in LPS-treated animals when compared with controls. The pancreas protein output was significantly decreased in pure pancreatic juice, whereas the pancreatic juice flow rate was significantly increased in LPS-treated animals, when compared with controls. Electrophoretic patterns showed a marked decrease in digestive enzyme contents, whereas there was an increased content of 15 kDa protein.

Biological activity of phenolic compounds. Hepatic cytochrome P-450, cytochrome b5, and NADPH cytochrome c reductase in chicks and rats fed phenolic monomers, polymers, and glycosides.

Eight experiments were conducted to determine effects of a phenolic polymer (Kraft wood lignin, Indulin), phenolic glycosides (cane molasses and wood molasses), and phenolic monomers (vanillin, vanillic acid, ferulic acid, and p-coumaric acid) on liver cytochromes P-450, cytochrome b5, and NADPH cytochrome c reductase in chicks and rats. Chicks fed 6.0% lignin had a higher (P less than 0.01) cytochromes P-450 content than did chicks fed 0% fiber, 6.0% wood cellulose (Solka Floc), or 6.0% arenaceous flour. NADPH cytochrome c reductase activity was not affected by treatment. Chicks fed 12.0% wood molasses had a higher (P less than 0.06) cytochromes P-450 level than did chicks fed 0% fiber or 6.0% wood molasses. Cane molasses incorporated at both 6.0 and 12.0% of the diet induced (P less than 0.05) cytochromes P-450 content over those of control-fed birds. Chicks fed 6.0% lignin, with or without antibiotic (bacitracin:neomycin sulfate, 2:1), had a higher (P less than 0.01) cytochromes P-450 level than did chicks fed control diets, with or without antibiotic. Additionally, chicks fed 6.0% lignin had lower (P less than 0.01) intestinal diaminopimelic acid (DAP) levels than did chicks fed 0% fiber. Rats fed 0% fiber, 6.0% wood cellulose, 6.0% arenaceous flour, or 6.0% lignin exhibited no difference in cytochrome level or activity among treatments. Chicks fed 0.5% vanillin, 0.5% vanillic acid, 0.5% ferulic acid, or 0.5% p-coumaric acid had comparable cytochromes level and activity compared with chicks fed no phenolics. Chicks fed 0.5% p-coumaric acid had lower (P less than 0.05) rates of gain than did chicks fed control or other phenolic-containing diets. Rats fed these phenolics had similar cytochromes P-450 content among treatments.