Motor phenotype of LRRK2 G2019S carriers in early-onset Parkinson disease

Objective: To determine the motor phenotype of LRRK2 G2019S mutation carriers. LRRK2 mutation carriers were previously reported to manifest the tremor dominant motor phenotype, which has been associated with slower motor progression and less cognitive impairment compared with the postural instability and gait difficulty (PIGD) phenotype. Design: Cross-sectional observational study. Setting: Thirteen movement disorders centers. Participants: Nine hundred twenty-five early-onset Parkinson disease cases defined as age at onset younger than 51 years. Main Outcome Measures: LRRK2 mutation status and Parkinson disease motor phenotype: tremor dominant or PIGD. Demographic information, family history of Parkinson disease, and the Unified Parkinson's Disease Rating Scale score were collected on all participants. DNA samples were genotyped for LRRK2 mutations (G2019S, I2020T, R1441C, and Y1699C). Logistic regression was used to examine associations of G2019S mutation status with motor phenotype adjusting for disease duration, Ashkenazi Jewish ancestry, levodopa dose, and family history of Parkinson disease. Results: Thirty-four cases (3.7%) (14 previously reported) were G2019S carriers. No other mutations were found. Carriers were more likely to be Ashkenazi Jewish (55.9% vs 11.9%; P < .001) but did not significantly differ in any other demographic or disease characteristics. Carriers had a lower tremor score (P = .03) and were more likely to have a PIGD phenotype (92.3% vs 58.9%; P = .003). The association of the G2019S mutation with PIGD phenotype remained after controlling for disease duration and Ashkenazi Jewish ancestry (odds ratio, 17.7; P < .001). Conclusion: Early-onset Parkinson disease G2019S LRRK2 carriers are more likely to manifest the PIGD phenotype, which may have implications for disease course

Genome-Wide association study identifies candidate genes for Parkinson's disease in an Ashkenazi Jewish population

To date, nine Parkinson disease (PD) genome-wide association studies in North American, European and Asian populations have been published. The majority of studies have confirmed the association of the previously identified genetic risk factors, SNCA and MAPT, and two studies have identified three new PD susceptibility loci/genes (PARK16, BST1 and HLA-DRB5). In a recent meta-analysis of datasets from five of the published PD GWAS an additional 6 novel candidate genes (SYT11, ACMSD, STK39, MCCC1/LAMP3, GAK and CCDC62/HIP1R) were identified. Collectively the associations identified in these GWAS account for only a small proportion of the estimated total heritability of PD suggesting that an 'unknown' component of the genetic architecture of PD remains to be identified. We applied a GWAS approach to a relatively homogeneous Ashkenazi Jewish (AJ) population from New York to search for both 'rare' and 'common' genetic variants that confer risk of PD by examining any SNPs with allele frequencies exceeding 2%. We have focused on a genetic isolate, the AJ population, as a discovery dataset since this cohort has a higher sharing of genetic background and historically experienced a significant bottleneck. We also conducted a replication study using two publicly available datasets from dbGaP. The joint analysis dataset had a combined sample size of 2,050 cases and 1,836 controls. We identified the top 57 SNPs showing the strongest evidence of association in the AJ dataset (p < 9.9 × 10-5). Six SNPs located within gene regions had positive signals in at least one other independent dbGaP dataset: LOC100505836 (Chr3p24), LOC153328/SLC25A48 (Chr5q31.1), UNC13B (9p13.3), SLCO3A1(15q26.1), WNT3(17q21.3) and NSF (17q21.3). We also replicated published associations for the gene regions SNCA (Chr4q21; rs3775442, p = 0.037), PARK16 (Chr1q32.1; rs823114 (NUCKS1), p = 6.12 × 10-4), BST1 (Chr4p15; rs12502586, p = 0.027), STK39 (Chr2q24.3; rs3754775, p = 0.005), and LAMP3 (Chr3; rs12493050, p = 0.005) in addition to the two most common PD susceptibility genes in the AJ population LRRK2 (Chr12q12; rs34637584, p = 1.56 × 10-4) and GBA (Chr1q21; rs2990245, p = 0.015). We have demonstrated the utility of the AJ dataset in PD candidate gene and SNP discovery both by replication in dbGaP datasets with a larger sample size and by replicating association of previously identified PD susceptibility genes. Our GWAS study has identified candidate gene regions for PD that are implicated in neuronal signalling and the dopamine pathway

Genetic Drivers of Kidney Defects in the DiGeorge Syndrome

Background The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. Methods We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. Results We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P=4.5×10(-14)). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. Conclusions We identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver. (Funded by the National Institutes of Health and others.)

Evaluation of the cost and effectiveness of diverse recruitment methods for a genetic screening study

Purpose: Recruitment of participants from diverse backgrounds is crucial to the generalizability of genetic research, but has proven challenging. We retrospectively evaluated recruitment methods used for a study on return of genetic results. Methods: The costs of study design, development, and participant enrollment were calculated, and the characteristics of the participants enrolled through the seven recruitment methods were examined. Results: A total of 1118 participants provided consent, a blood sample, and questionnaire data. The estimated cost across recruitment methods ranged from $579 to$1666 per participant and required a large recruitment team. Recruitment methods using flyers and staff networks were the most cost-efficient and resulted in the highest completion rate. Targeted sampling that emphasized the importance of Latino/a participation, utilization of translated materials, and in-person recruitments contributed to enrolling a demographically diverse sample. Conclusions: Although all methods were deployed in the same hospital or neighborhood and shared the same staff, each recruitment method was different in terms of cost and characteristics of the enrolled participants, suggesting the importance of carefully choosing the recruitment methods based on the desired composition of the final study sample. This analysis provides information about the effectiveness and cost of different methods to recruit adults for genetic research

Caracterización farmacológica y estudio de la relación estructura - Función del receptor nicotínico alfa 9

En este trabajo se presenta una extensa caracterización del receptor colinérgico nicotínico α9, utilizando el sistema de expresión heteróloga de ovocitos de Xenopus laevis. Durante la mayor parte del desarrollo del mismo se tuvo como hipótesis de trabajo que α9 sería la única subunidad integrante del receptor colinérgico nativo de las células ciliadas externas de la cóclea y de las células tipo II del aparato vestibular. Este receptor es responsable de la inhibición de la actividad de dichas células, mediada por la inervación colinérgica eferente coclear y vestibular. En 2001 se identificó y clonó una nueva subunidad de receptores nicotínicos, α1O. Esto condujo a modificar la hipótesis de trabajo, para incluir la posibilidad de un receptor heteromérico α9αl0 en las células ciliadas, pero sin excluir la posible existencia del receptor homomérico α9 en otros tipos celulares. La caracterización del receptor homomérico recombinante α9 incluyó el estudio de las propiedades farmacológicas del mismo. En particular, se demostró que a pesar que α9 es un miembro de la familia de genes de subunidades de receptores nicotínicos, el receptor no puede ser clasificado farmacologicamente como nicotínico o muscarínico y presenta un perfil farmacológico mixto, en coincidencia con lo reportado para el receptor nativo de las células ciliadas. Se mostró que tanto agonistas colinérgicos no específicos como la acetilcolina y el carbacol, agonistas nicotínicos como el metilcarbacol, la suberildicolina y el DMPP y agonistas muscarínicos como la oxotremorina - M, el metilfurtretonio y el McN-A-343 son capaces de activar al receptor α9. También la colina, un producto de degradación y precursor en la síntesis de la acetilcolina, activó al receptor α9. Notablemente, agonistas nicotínicos clásicos como la epibatidina, la nicotina y la citisina bloquearon al receptor α9 (IC50 : 1.6±0.l, 31.5±l.0 y 43.1±3.5 µM, respectivamente). Se estudió la sensibilidad de α9 a diversos antagonistas nicotínicos. En particular la metillicaconitina, un antagonista selectivo de receptores nicotínicos neuronales sensibles a la α-bungarotoxina, inhibió las respuestas de α9 a la acetilcolina en forma competitiva (IC50 : l.l±0.2 nM), siendo este el antagonista más potente de α9 hasta ahora reportado. Se demostró también que el receptor α9 es bloqueado tanto por agonistas como por antagonistas muscarínicos con el siguiente orden de potencia: atropina (IC50: l.0±0.1µM, ) ~ galamina (IC50: 1.5±0.2 µM) > pilocarpina (IC50: 76±9 µM) ~ muscarina (IC50: 84±6 µM) ~ betanecol (IC50: 105±14 µM), un rango de concentraciones comparable con las requeridas para el bloqueo producido por compuestos nicotínicos. Por otra parte, se demostró la inhibición de la respuesta a acetilcolina de los receptores α9 y α9α10 por morfina y péptidos opioides. Estos péptidos estarían presentes en la inervación eferente a las células ciliadas del aparato vestibular y modularían su actividad a través de su acción sobre α9αl0. Se demostró también que el receptor homomérico α9 es bloqueado por Ca++ extracelular (IC50: 100±10 µM a -70 mV), en forma dependiente del potencial de membrana. Este resultado es relevante para la fisiología de las células ciliadas. Mediante la construcción de una subunidad quimérica entre α9 y el receptor de serotonina de tipo 3A, se aportó evidencia de que el dominio aminoterminal de α9 es el determinante estructural de la interacción de α9 con agonistas y antagonistas. También se construyó una subunidad quimérica mediante el empalme de porciones de las subunidades α7 y α9. Los resultados obtenidos con las dos quimeras mencionadas, indican que los determinantes estructurales de la particular relación corriente - potencial de α9 (relacionada con la presencia de Ca++ extracelular), estarían confinados a la región que se extiende desde el comienzo del segundo segmento hidrófobo, supuestamente transmembranal, hasta el extremo carboxiterminal. Se identificó un residuo en el segmento hidrófobo M2 (Q261) involucrado en la rectificación de la corriente y en el bloqueo por el Ca++. En suma, el presente trabajo demuestra que el receptor α9 presenta una farmacología mixta nicotínica - muscarínica coincidente con la del receptor colinérgico de las células ciliadas. Esta coincidencia incluye también la sensibilidad de los receptores que contienen la subunidad α9 a péptidos opioides y a cationes divalentes. Por otro lado aporta evidencia de la importancia clave del dominio aminoterminal en determinar características farmacológicas de α9 y del segundo segmento hidrófobo en las propiedades del canal de los receptores α9 y α9α10.This work provides an extensive characterisation of the α9 nicotinic acetylcholine receptor, making use of the Xenopus laevis oocytes heterologous expression system. As a working hypothesis during most of the process of this work, the α9 protein was thought to be the only subunit present at the native acetylcholine receptors of cochlear outer hair cells and type II vestibular hair cells. These receptors mediate inhibition of the activity of these cells by cholinergic cochlear and vestibular efferent innervation. In 200l α10, a new nicotinic subunit, was identified and cloned. This led to the modification of the previous hypothesis, in order to include the possibility of an heteromeric α9α10 receptor present in hair cells, not excluding the occurrence of homomeric α9 receptors in other cell types. The characterisation of the homomeric recombinant α9 receptor included the study of its pharmacological properties. It is demonstrated that although α9 is a member of the nicotinic receptor subunit gene family, the receptor cannot be pharmacologically classified either as nicotinic or muscarinic, since it displays a mixed pharmacological profile, in coincidence with what has been reported for the hair cells native receptor. It is shown that the α9 receptor is activated by the non-specific cholinergic agonists acetylcholine, and carbachol, as well as by the nicotinic agonists methylcarbachol, suberyldicholine and DMPP and the muscarinic agonists oxotremorine-M, methylfurthretonium and McN-A-343. Moreover choline, both a degradation product and a precursor of the synthesis of acetylcholine activates α9. Remarkably nicotinic agonists such as epibatidine, nicotine and cytisine blocked the α9 receptor (IC50 : 1.6±0.l, 31.5±l.0 y 43.l±3.5 μM, respectively). The sensitivity of α9 to various nicotinic antagonists is reported. Methyllycaconitine, a specific antagonist for α-bungarotoxin-sensitive neuronal nicotinic receptors, inhibited α9 acetylcholine-evoked responses in a competitive manner (IC50: l.1±0.2 μM), thus being the most potent α9 antagonist identified so far. It is also demonstrated that the α9 receptor is blocked by both muscarinic agonists and antagonists with the following rank order of potencies: atropine (IC50: l.0±0.1 μM) ~ gallamine (IC50: 1.5±0.2 µM) > pilocarpine (IC50: 76±9 µM) ~ muscarine (IC50: 84±6 µM) ~ betanechol (IC50: 105±14 µM), a concentration range which is similar to that required for the block by nicotinic compounds. It is also demonstrated that morphine and opioid peptides inhibit acetylcholine-evoked responses in α9 and α9αl0 receptors. These peptides are present at the efferent innervation to vestibular hair cells and would modulate their activity through their action on α9αl0. It is also demonstrated that the α9 homomeric receptor is blocked by extracellular Ca++ (IC50: 100±10 µM a -70 mV) in a voltage-dependent manner, a result relevant to hair cell physiology. By means of the construction of a chimeric subunit between α9 and the type 3 serotonin receptor, evidence is provided that the α9 aminoterminal domain is the structural determinant of the α9 interaction with agonists and antagonists. A chimeric subunit was also constructed by splicing portions of the α7 and α9 subunits. Results obtained with the aforementioned chimeras, indicate that the structural determinants underlying the peculiar α9 current-voltage relationship, are confined within the region that extends from the beginning of the putative transmembrane second hydrophobic segment, to the carboxiterminus. A residue (Q26l) within the the hydrophobic region M2 was identified as being involved in the rectification properties and in the block by Ca++. In summary, the present work demonstrates that the α9 receptor displays a mixed nicotinic-muscarinic pharmacology that matches that of the cholinergic receptor of cochlear and vestibular hair cells. The similarities between the native and the recombinant receptors extends to the sensitivity of the α9-containing receptors to opioid peptides and divalent cations. Evidence indicating the key importance of the aminoterminal domain in determining the α9 receptor pharmacological characteristics and of the second hydrophobic segment in the channel properties of the α9 and α9α10 receptors are also presented.Fil:Verbitsky, Miguel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Functional expression and properties of a nicotinic alpha9/5-HT3A chimeric receptor

We describe the functional properties of a nicotinic alpha9/serotonin subtype 3A (5HT3A) chimeric receptor expressed in Xenopus laevis oocytes. The chimera preserved ligand-binding properties of alpha9 and channel properties of 5HT3A. Thus, it responded to acetylcholine in a concentration-dependent manner with an EC50 of 70 microM but not to serotonin. It was blocked by methyllycaconitine, strychnine, atropine and nicotine, with the same rank order of potency as alpha9 receptors. The current-voltage relationship of currents through the alpha9/5HT3A chimera was similar to that of the 5HT3A receptors. These results are an evidence of functional coupling between the ligand-binding and the channel domains of the chimeric receptor.Fil: Verbitsky, Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Plazas, Paola Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

The alpha9 nicotinic acetylcholine receptor shares pharmacological properties with type A gammaaminobutyric acid, glycine, and type 3 serotonin receptors

ABSTRACT In the present study, we provide evidence that the ␣9 nicotinic acetylcholine receptor (nAChR) shares pharmacological properties with members of the Cys-loop family of receptors. Thus, the type A ␥-aminobutyric acid receptor antagonist bicuculline, the glycinergic antagonist strychnine, and the type 3 serotonin receptor antagonist ICS-205,930 block ACh-evoked currents in ␣9-injected Xenopus laevis oocytes with the following rank order of potency: strychnine Ͼ ICS-205,930 Ͼ bicuculline. Block by antagonists was reflected in an increase in the acetylcholine (ACh) EC 50 value, with no changes in agonist maximal response or Hill coefficient, which suggests a competitive type of block. Moreover, whereas neither ␥-aminobutyric acid nor glycine modified ACh-evoked currents, serotonin blocked responses to ACh in a concentration-dependent manner. The present results suggest that the ␣9 nAChR must conserve in its primary structure some residues responsible for ligand binding common to other Cys-loop receptors. In addition, it adds further evidence that the ␣9 nAChR and the cholinergic receptor present at the base of cochlear outer hair cells have similar pharmacological properties

Altered Hippocampal Transcript Profile Accompanies an Age-Related Spatial Memory Deficit in Mice

We have carried out a global survey of age-related changes in mRNA levels in the C57BL/6NIA mouse hippocampus and found a difference in the hippocampal gene expression profile between 2-month-old young mice and 15-month-old middle-aged mice correlated with an age-related cognitive deficit in hippocampal-based explicit memory formation. Middle-aged mice displayed a mild but specific deficit in spatial memory in the Morris water maze. By using Affymetrix GeneChip microarrays, we found a distinct pattern of age-related change, consisting mostly of gene overexpression in the middle-aged mice, suggesting that the induction of negative regulators in the middle-aged hippocampus could be involved in impairment of learning. Interestingly, we report changes in transcript levels for genes that could affect synaptic plasticity. Those changes could be involved in the memory deficits we observed in the 15-month-old mice. In agreement with previous reports, we also found altered expression in genes related to inflammation, protein processing, and oxidative stress