Molecular Characterization of Portuguese Patients with Hereditary Cerebellar Ataxia.

Hereditary cerebellar ataxia (HCA) comprises a clinical and genetic heterogeneous group of neurodegenerative disorders characterized by incoordination of movement, speech, and unsteady gait. In this study, we performed whole-exome sequencing (WES) in 19 families with HCA and presumed autosomal recessive (AR) inheritance, to identify the causal genes. A phenotypic classification was performed, considering the main clinical syndromes: spastic ataxia, ataxia and neuropathy, ataxia and oculomotor apraxia (AOA), ataxia and dystonia, and ataxia with cognitive impairment. The most frequent causal genes were associated with spastic ataxia (SACS and KIF1C) and with ataxia and neuropathy or AOA (PNKP). We also identified three families with autosomal dominant (AD) forms arising from de novo variants in KIF1A, CACNA1A, or ATP1A3, reinforcing the importance of differential diagnosis (AR vs. AD forms) in families with only one affected member. Moreover, 10 novel causal-variants were identified, and the detrimental effect of two splice-site variants confirmed through functional assays. Finally, by reviewing the molecular mechanisms, we speculated that regulation of cytoskeleton function might be impaired in spastic ataxia, whereas DNA repair is clearly associated with AOA. In conclusion, our study provided a genetic diagnosis for HCA families and proposed common molecular pathways underlying cerebellar neurodegeneration.

CD44v6 High Membranous Expression Is a Predictive Marker of Therapy Response in Gastric Cancer Patients.

In gastric cancer (GC), biomarkers that define prognosis and predict treatment response remain scarce. We hypothesized that the extent of CD44v6 membranous tumor expression could predict prognosis and therapy response in GC patients. Two GC surgical cohorts, from Portugal and South Korea (n = 964), were characterized for the extension of CD44v6 membranous immuno-expression, clinicopathological features, patient survival, and therapy response. The value of CD44v6 expression in predicting response to treatment and its impact on prognosis was determined. High CD44v6 expression was associated with invasive features (perineural invasion and depth of invasion) in both cohorts and with worse survival in the Portuguese GC cohort (HR 1.461; 95% confidence interval 1.002-2.131). Patients with high CD44v6 tumor expression benefited from conventional chemotherapy in addition to surgery (p < 0.05), particularly those with heterogeneous CD44v6-positive and -negative populations (CD44v6_3+) (p < 0.007 and p < 0.009). Our study is the first to identify CD44v6 high membranous expression as a potential predictive marker of response to conventional treatment, but it does not clarify CD44v6 prognostic value in GC. Importantly, our data support selection of GC patients with high CD44v6-expressing tumors for conventional chemotherapy in addition to surgery. These findings will allow better stratification of GC patients for treatment, potentially improving their overall survival.

A genetic interaction of NRXN2 with GABRE, SYT1 and CASK in migraine patients: a case-control study.

BACKGROUND: Migraine is a multifactorial disorder that is more frequent (two to four times) in women than in men. In recent years, our research group has focused on the role of neurotransmitter release and its regulation. Neurexin (NRXN2) is one of the components of the synaptic vesicle machinery, responsible for connecting intracellular fusion proteins and synaptic vesicles. Our aim was to continue exploring the role and interaction of proteins involved in the control and promotion of neurotransmission in migraine susceptibility. METHODS: A case-control study was performed comprising 183 migraineurs (148 females and 35 males) and 265 migraine-free controls (202 females and 63 males). Tagging single nucleotide polymorphisms of NRXN2 were genotyped to assess the association between NRXN2 and migraine susceptibility. The χ2 test was used to compare allele frequencies in cases and controls and odds ratios were estimated with 95% confidence intervals. Haplotype frequencies were compared between groups. Gene-gene interactions were analysed using the Multifactor Dimensionality Reduction v2.0. RESULTS: We found a statistically significant interaction model (p = 0.009) in the female group between the genotypes CG of rs477138 (NRXN2) and CT of rs1158605 (GABRE). This interaction was validated by logistic regression, showing a significant risk effect [OR = 4.78 (95%CI: 1.76-12.97)] after a Bonferroni correction. Our data also supports a statistically significant interaction model (p = 0.011) in the female group between the GG of rs477138 in NRXN2 and, the rs2244325's GG genotype and rs2998250's CC genotype of CASK. This interaction was also validated by logistic regression, with a protective effect [OR = 0.08 (95%CI: 0.01-0.75)]. A weak interaction model was found between NRXN2-SYT1. We have not found any statistically significant allelic or haplotypic associations between NRXN2 and migraine susceptibility. CONCLUSIONS: This study unravels, for the first time, the gene-gene interactions between NRXN2, GABRE - a GABAA-receptor - and CASK, importantly it shows the synergetic effect between those genes and its relation with migraine susceptibility. These gene interactions, which may be a part of a larger network, can potentially help us in better understanding migraine aetiology and in development of new therapeutic approaches.

Histone deacetylase knockouts modify transcription, CAG instability and nuclear pathology in Huntington disease mice.

Somatic expansion of the Huntington's disease (HD) CAG repeat drives the rate of a pathogenic process ultimately resulting in neuronal cell death. Although mechanisms of toxicity are poorly delineated, transcriptional dysregulation is a likely contributor. To identify modifiers that act at the level of CAG expansion and/or downstream pathogenic processes, we tested the impact of genetic knockout, in HttQ111 mice, of Hdac2 or Hdac3 in medium-spiny striatal neurons that exhibit extensive CAG expansion and exquisite disease vulnerability. Both knockouts moderately attenuated CAG expansion, with Hdac2 knockout decreasing nuclear huntingtin pathology. Hdac2 knockout resulted in a substantial transcriptional response that included modification of transcriptional dysregulation elicited by the HttQ111 allele, likely via mechanisms unrelated to instability suppression. Our results identify novel modifiers of different aspects of HD pathogenesis in medium-spiny neurons and highlight a complex relationship between the expanded Htt allele and Hdac2 with implications for targeting transcriptional dysregulation in HD.

Going Deep into Synaptic Vesicle Machinery Genes and Migraine Susceptibility - A Case-Control Association Study.

OBJECTIVE: A number of observations, including among our study population, have implicated variants in the syntaxin-1A, a component of the synaptic vesicles, in migraine susceptibility. Therefore, we hypothesize that variants in other components of the vesicle machinery are involved in migraine. BACKGROUND: Migraine is a common and complex neurologic disorder that affects approximately 15-18% of the general population. The exact cause of migraine is unknown; however, genetic studies have made possible substantial progress toward the identification of underlying molecular pathways. Neurotransmitters have been for long considered to have a key role in migraine pathophysiology; so we investigated common variants in genes involved in the synaptic vesicle machinery and their impact in migraine susceptibility. METHODS: We performed a case-control study comprising 188 unrelated patients with headache and 286 healthy controls in a population from the north of Portugal. Benefiting from the presence of linkage disequilibrium, we selected and genotyped 119 tagging single-nucleotide polymorphisms in 18 genes. RESULTS: We found significant associations between single-nucleotide variants and migraine in 7 genes, SYN1, SYN2, SNAP25, VAMP2, STXBP1, STXBP5, and UNC13A, either conferring an increased risk or protection of migraine. Due to SYN1 X-chromosomal location, we performed the statistical analysis separated by gender and, in the female group, the C allele of rs5906435 increased the risk for migraine susceptibility (P = .021; OR = 1.69; 95% CI: 1.21-2.34). In contrast, the TT genotype of the same variant emerged as a potential protective factor (P = .003; OR = 0.45; 95% CI: 0.27-0.74). The SYN2 analysis supported the rs3773364's G allele (P = .014) as a risk factor for migraine, and although not statistically significant after correction, the AG genotype (P = .006; OR = 1.86; 95% CI: 1.20-2.90) reinforced the allelic findings. Additionally, we found the SNAP25-rs363039's CT genotype (P = .001; OR = 2.14; 95% CI: 1.36-3.34), the STXBP5-rs1765028's T allele (P = .041; OR = 1.46; 95% CI: 1.13-1.90), and the UNC13B-rs7851161's TT genotype (P = .001; OR = 2.14; 95% CI: 1.36-3.34) as statistically significant risk factors for migraine liability. VAMP2-rs1150's G allele revealed a risk association to migraine, not statistically significant after correction (P = .068). Additionally, we found haplotypes in SYN1, SYN2, STXBP1, and UNC13B to be associated with migraine. CONCLUSIONS: Overall, this study provides a new insight into migraine liability, identifying possible starting points for functional studies.

Heritability and genetic variance of dementia with Lewy bodies.

Recent large-scale genetic studies have allowed for the first glimpse of the effects of common genetic variability in dementia with Lewy bodies (DLB), identifying risk variants with appreciable effect sizes. However, it is currently well established that a substantial portion of the genetic heritable component of complex traits is not captured by genome-wide significant SNPs. To overcome this issue, we have estimated the proportion of phenotypic variance explained by genetic variability (SNP heritability) in DLB using a method that is unbiased by allele frequency or linkage disequilibrium properties of the underlying variants. This shows that the heritability of DLB is nearly twice as high as previous estimates based on common variants only (31% vs 59.9%). We also determine the amount of phenotypic variance in DLB that can be explained by recent polygenic risk scores from either Parkinson's disease (PD) or Alzheimer's disease (AD), and show that, despite being highly significant, they explain a low amount of variance. Additionally, to identify pleiotropic events that might improve our understanding of the disease, we performed genetic correlation analyses of DLB with over 200 diseases and biomedically relevant traits. Our data shows that DLB has a positive correlation with education phenotypes, which is opposite to what occurs in AD. Overall, our data suggests that novel genetic risk factors for DLB should be identified by larger GWAS and these are likely to be independent from known AD and PD risk variants.

Genetic Contributors to Intergenerational CAG Repeat Instability in Huntington's Disease Knock-In Mice.

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in exon 1 of the HTT gene. Longer repeat sizes are associated with increased disease penetrance and earlier ages of onset. Intergenerationally unstable transmissions are common in HD families, partly underlying the genetic anticipation seen in this disorder. HD CAG knock-in mouse models also exhibit a propensity for intergenerational repeat size changes. In this work, we examine intergenerational instability of the CAG repeat in over 20,000 transmissions in the largest HD knock-in mouse model breeding datasets reported to date. We confirmed previous observations that parental sex drives the relative ratio of expansions and contractions. The large datasets further allowed us to distinguish effects of paternal CAG repeat length on the magnitude and frequency of expansions and contractions, as well as the identification of large repeat size jumps in the knock-in models. Distinct degrees of intergenerational instability were observed between knock-in mice of six background strains, indicating the occurrence of trans-acting genetic modifiers. We also found that lines harboring a neomycin resistance cassette upstream of Htt showed reduced expansion frequency, indicative of a contributing role for sequences in cis, with the expanded repeat as modifiers of intergenerational instability. These results provide a basis for further understanding of the mechanisms underlying intergenerational repeat instability.

Scintigraphic assessment of radio-aerosol pulmonary deposition with the acapella positive expiratory pressure device and various nebulizer configurations.

BACKGROUND: The Acapella device produces high-frequency oscillations and positive expiratory pressure to promote bronchial secretion clearance. Its performance during aerosol delivery has not been described. We evaluated the effect of nebulizer and Acapella configuration on pulmonary deposition of radio-tagged aerosol in healthy subjects. METHODS: Ten healthy male subjects (mean age 24.4 ± 2.2 y) participated in a crossover study that compared pulmonary delivery of 4 mL of technetium-99m-labeled diethylene triamine penta-acetic acid (25 mCi) and 0.9% saline solution via jet nebulizer. We tested 3 configurations: nebulizer attached to the distal end of the Acapella; nebulizer placed between the mouthpiece and the Acapella; and nebulizer alone (control). With scintigraphy we measured radio-aerosol deposition in 6 lung regions: upper, middle, lower, central, intermediate, and peripheral. RESULTS: Deposition was similar between the right and left lungs, with no significant differences between device configurations. Lung deposition was less with the nebulizer attached to the Acapella than with nebulizer between the mouthpiece and the Acapella (P = .001, for both lungs) or without the Acapella (P = .003 and P = .001 for the right and left lungs, respectively). There was no significant difference between the setup without Acapella and the setup with the nebulizer between the mouthpiece and the Acapella (P = .001, for both lungs). On the vertical axis, deposition was lower with the nebulizer attached to the distal end of the Acapella than with the nebulizer between the mouthpiece and the Acapella (upper region P < .001, middle region P = .001, lower region P = .003), and lower with the nebulizer attached to the distal end of the Acapella than with the setup without Acapella (upper and middle region both P = .001, lower region P = .002), with up to a 3-fold difference in the middle and lower regions. On the central-peripheral axis, deposition was lower with the nebulizer attached to the distal end of the Acapella than with the nebulizer between the mouthpiece and the Acapella (central region P < .001, peripheral region P < .001), and lower with the nebulizer attached to the distal end of the Acapella than with the setup without Acapella (central and peripheral regions both P = .002), with differences of 3-4-fold between the central and peripheral regions. CONCLUSIONS: Placing the nebulizer distal to the Acapella, as recommended by the manufacturer, decreased intrapulmonary deposition, compared to placing the nebulizer between the Acapella and the patient airway, or delivering aerosol without the Acapella in the circuit. (ClinicalTrials.gov NCT01102166).

Interaction between γ-aminobutyric acid A receptor genes: new evidence in migraine susceptibility.

Migraine is a common neurological episodic disorder with a female-to-male prevalence 3- to 4-fold higher, suggesting a possible X-linked genetic component. Our aims were to assess the role of common variants of gamma-aminobutyric acid A receptor (GABAAR) genes, located in the X-chromosome, in migraine susceptibility and the possible interaction between them. An association study with 188 unrelated cases and 286 migraine-free controls age- and ethnic matched was performed. Twenty-three tagging SNPs were selected in three genes (GABRE, GABRA3 and GABRQ). Allelic, genotypic and haplotypic frequencies were compared between cases and controls. We also focused on gene-gene interactions. The AT genotype of rs3810651 of GABRQ gene was associated with an increased risk for migraine (OR: 4.07; 95% CI: 1.71-9.73, p=0.002), while the CT genotype of rs3902802 (OR: 0.41; 95% CI: 0.21-0.78, p=0.006) and GA genotype of rs2131190 of GABRA3 gene (OR: 0.53; 95% CI: 0.32-0.88, p=0.013) seem to be protective factors. All associations were found in the female group and maintained significance after Bonferroni correction. We also found three nominal associations in the allelic analyses although there were no significant results in the haplotypic analyses. Strikingly, we found strong interactions between six SNPs encoding for different subunits of GABAAR, all significant after permutation correction. To our knowledge, we show for the first time, the putative involvement of polymorphisms in GABAAR genes in migraine susceptibility and more importantly we unraveled a role for novel gene-gene interactions opening new perspectives for the development of more effective treatments.