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Russell J Buono

Publications and source records attributed to Russell J Buono.

17 recordsLinked to original sources

Identification of three mouse mu-opioid receptor (MOR) gene (Oprm1) splice variants containing a newly identified alternatively spliced exon.

The mouse mu-opioid receptor gene, Oprm1, is recognized currently to contain 17 alternatively spliced exons that generate 24 splice variants encoding at least 11 morphine-binding isoforms of the receptor. Here, we identify three new MOR splice variants that contain a previously undescribed exon, exon 18, and provide evidence that they are expressed in two mouse strains. The transcripts containing the newly identified exon 18 encode two new putative mu-opioid receptor isoforms, MOR-1V and MOR-1W. In mouse Oprm1, exon 18 is located between the described exons 10 and 6. Exon 18 appears to be conserved in the rat genome between exons 4 and 7. A BLAST search of the non-redundant GenBank database suggests that human OPRM1 may also contain exon 18. Analysis of mouse brain mRNA by RT-PCR suggests that MOR-1Vii transcripts are expressed in all areas of the brain analyzed, whereas expression of MOR-1Vi transcripts was restricted to thalamus and striatum. MOR-1W transcripts are expressed most highly in the hypothalamus, thalamus and striatum. In summary, we have identified three brain expressed, alternatively spliced mouse MOR splice variants containing a novel exon and encoding new putative MOR isoforms, MOR-1V and MOR-1W.

Alternative Splicing↗

Identification and functional significance of polymorphisms in the mu-opioid receptor gene (Oprm) promoter of C57BL/6 and DBA/2 mice.

C57BL/6J and DBA/2J mice demonstrate differences in morphine preference when tested in a two-bottle choice paradigm. Quantitative trait loci (QTL) mapping suggested the proximal region of chromosome 10 was responsible for 41% of the observed genetic variance. The mu-opioid receptor (MOR) gene (Oprm) maps to this region and is a prime candidate for explaining the QTL. We hypothesized that variations in Oprm between these strains are responsible for differences in morphine preference. We identify five single nucleotide polymorphisms (SNPs) in the Oprm promoter; three within or near putative transcription factor binding sites. Promoter fragments were amplified from genomic DNA by polymerase chain reaction (PCR) and subcloned into luciferase reporter vectors. A significant difference in basal Oprm promoter activity was seen with C57BL/6 and DBA/2 approximately 1675 constructs in MOR-positive BE(2)-C cells, but not in MOR-negative Neuro-2a cells. In BE(2)-C cells, average DBA/2 approximately 1675 construct activity was 1.3-2.0x greater than average C57BL/6 activity suggesting that the SNPs might alter MOR expression in these two mouse strains. Significant differences in promoter activities between the two cell lines suggest that cell-type-specific transcription factors are involved. No significant differences in construct activity were found between untreated and morphine-treated BE(2)-C or Neuro-2a cells, suggesting that morphine does not regulate transcription of Oprm.

Animals↗

Role of genetics in the diagnosis and treatment of epilepsy.

Epilepsy is a heterogeneous group of multifactorial diseases, the vast majority determined by interactions between many genes and environmental factors; however, there are rare epilepsy syndromes that can be caused by a single gene mutation and are inherited according to classical mendelian genetic principles. Finding disease-causing genetic mutations in epilepsy has provided new opportunities for aiding diagnosis and developing therapies. Thus, the discovery of KCNQ2 mutations in benign familial neonatal convulsions, SCN1A mutations in severe myoclonic epilepsy of infancy and in generalized epilepsy with febrile seizures plus, and CHRA4 and CHRB2 mutations in autosomal-dominant nocturnal frontal lobe epilepsy, has led to the establishment of epilepsy as a disorder of ion channel function and, furthermore, has led to the introduction of genetic tests that are available clinically to aid in diagnosis and treatment. At the present time, clinical use of genetic testing in epilepsy is greatest in suspected cases of severe myoclonic epilepsy of infancy, generalized epilepsy with febrile seizures plus, atypical cases of benign familial neonatal convulsions and 'occult' cases of autosomal-dominant nocturnal frontal lobe epilepsy without a family history. Overall, clinical use is limited by the low number of documented disease-associated mutations and the uncertain clinical significance of many test results. Further elucidation of the relationship between gene mutations and channel function will add value to genetic testing in the future, as will better characterization of the association between gene mutations and clinical phenotypes.

Biomarkers↗

Challenges and opportunities in the application of pharmacogenetics to antiepileptic drug therapy.

The recent surge of interest in pharmacogenetics has provoked considerable thought regarding its relevance to antiepileptic drug (AED) therapy. Initial studies have focused on genes whose products play a putatively important role in AED pharmacology, particularly drug transporter proteins, drug metabolizing enzymes and ion channel subunits. However, there is a lack of good correspondence between results from different laboratories, and more recent findings are awaiting attempts at confirmation. Thus, there are currently no AED treatment guidelines that are informed by pharmacogenetic data. In order to begin to have clinical impact, standards specific to the conduct of future AED studies must be established. Of particular importance are the need for accurate epilepsy classification, appropriate AED selection and clear and objective assessment outcome measures. In addition, general standards for analysis and interpretation of genetic association data must be better codified and applied consistently across studies. Finally, extensive clinical research networks must be formulated and large numbers of well characterized patients must be recruited. Further development of these critical factors will optimize chances for overcoming current challenges posed by AED pharmacogenetic research and ultimately allow the realization of improved, more rational therapeutic strategies.

Animals↗

Polygenic epilepsy.

Current perspective on the etiology of common forms of epilepsy reflects clinical and preclinical data that document a complex determinism involving multiple gene variations and environmental influences. This multifactorial architecture precludes effective use of conventional types of genetic analysis and requires application of alternative methods. Presently, such methods are based on candidate gene studies, and it is likely that these approaches will continue to be useful in the near term. Long-range strategies will combine traditional linkage methods with single nucleotide polymorphism gene maps and will seek to collect data on a genomic scale in the search for gene variation that influences both the development of epilepsy and the response to antiepileptic medication. Such studies will require large numbers of patients and controls and also valid, standardized clinical criteria for parsing patients into phenotypic categories. Animal models will continue to provide clues regarding potentially important candidate genes and can also be used in a reverse-translational manner to confirm and study the effects of gene variation that is identified in humans. In the end, genetic research approaches to common forms of human epilepsy will enhance understanding of underlying neurobiological mechanisms, and through this understanding will come new treatment options, new molecular markers to improve current diagnostic criteria, and new genetic tests for predicting drug efficacy in patients with epilepsy.

Animals↗

No association between common variations in the neuronal nicotinic acetylcholine receptor alpha2 subunit gene (CHRNA2) and bipolar I disorder.

The neuronal nicotinic acetylcholine receptor alpha2 subunit gene (CHRNA2) maps to the bipolar susceptibility locus on chromosome 8p21-22. Given the biological role of the neuronal nicotinic acetylcholine receptors and the substantial comorbidity of nicotine dependence in psychiatric disorders, the CHRNA2 gene is a plausible candidate gene for bipolar disorder (BPD). We tested the hypothesis that variations in the CHRNA2 gene confer susceptibility to bipolar I disorder in a case-control association study. Genotypes of one amino acid substitution polymorphism (Ala125Thr) and five non-coding variations across the CHRNA2 gene were obtained from 345 unrelated bipolar I patients and 273 control samples. Genotypes and allele frequencies were compared between groups using chi-square contingency analysis. Linkage disequilibrium (LD) between markers was calculated, and estimated haplotype frequencies were compared between groups. We observed no statitistically significant difference in genotype and allele frequencies for all six variations between bipolar patients and controls, but we did demonstrate strong LD throughout the gene. Haplotype analysis showed that no combinations of alleles were associated with illness. Our results suggest that common variations in the CHRNA2 gene are unlikely to confer susceptibility to BPD in this sample. Further studies are required to elucidate the susceptibility locus for BPD on chromosome 8p21-22.

Bipolar Disorder↗

Lack of association between single nucleotide polymorphisms in the corticotropin releasing hormone receptor 1 (CRHR1) gene and alcohol dependence.

While the physiological mechanisms that contribute to the development of alcohol dependence remain unclear, a number of recent studies have indicated a role for the corticotropin releasing hormone receptor 1 (CRHR 1) in modulating the response of the central nervous system to ethanol. Based on these data, the present study aims to identify associations between variations in the CRHR 1 gene and alcohol dependence in a population of individuals of European ancestry. In order to identify such putative associations, five single nucleotide polymorphisms (SNPs) in the CRHR 1 gene were analyzed in 120 alcohol dependent and 180 control subjects. In comparing both allele and genotype frequencies at these five loci between alcohol dependent and control populations, no significant associations between variations in the CRHR 1 gene and alcohol dependence were detected. The results of this study suggest that polymorphisms in the CRHR 1 gene are not major risk factors for the development of alcohol dependence in persons of European ancestry.

Alcoholism↗

Recruitment rates and fear of phlebotomy in pediatric patients in a genetic study of epilepsy.

This study examined participation rates and reasons for refusal in a genetic study of human epilepsy. The study enrolled children with epilepsy and their parents, and required signing informed consent, verbalizing assent, and giving a peripheral blood sample. One hundred sixty-eight children met inclusion criteria; 137 agreed to enroll (82%), and 31 refused (18%). Sixteen of thirty-one patients (52%) who refused cited fear of phlebotomy as the reason for refusal. All patients refusing due to fear of phlebotomy did not require blood tests for clinical purposes. As fear of phlebotomy is the primary reason for study refusal, obtaining DNA samples from a buccal swab or mouthwash protocol may be an alternative for some studies, although there are limitations to these methods. Further analysis of the factors influencing decisions to decline study enrollment is warranted. These data will help in the design of future genetic studies and may increase future participation rates.

Adolescent↗

The relationship between the pharmacology of antiepileptic drugs and human gene variation: an overview.

Individual differences in clinical responsiveness to antiepileptic drugs are due to a complex interaction between environmental factors and genetic variation. Considerable interest has arisen in exploiting advances in molecular genetics to improve drug therapy for epilepsy and many other diseases; however, practical application of pharmacogenetics has been difficult to realize. Attempts to define gene variants that are associated with therapeutic (or adverse) effects of antiepileptic drugs rely currently on the prior identification of candidate genes and the subsequent evaluation of the distribution of allelic variants between individuals who have a "good" versus a "poor" clinical response. Many factors can adversely affect interpretation of such data, and careful consideration must be given to the design of genetic association studies involving candidate genes. Candidate genes may be identified in a number of ways; however, for studies of drugs, application of knowledge derived from basic pharmacology can suggest focused and testable hypotheses that are based on the fundamental principles of drug action. Thus, studies of genetic variation as they relate to proteins involved in antiepileptic drug kinetics and dynamics will identify key polymorphisms in endogenous molecules that determine degrees of drug efficacy and toxicity. Delineation of these effects in the coming years will promote enhanced success in the treatment of epilepsy.

Anticonvulsants↗

Confirmation of a major QTL influencing oral morphine intake in C57 and DBA mice using reciprocal congenic strains.

C57BL/6 (B6) and DBA/2 (D2) mice exhibit disparate behavior when tested for voluntary morphine intake in a two-bottle choice drinking paradigm with B6 mice consuming 10 times more drug than D2 mice. Previous genetic mapping studies identified a locus, Mop2, on the proximal part of chromosome 10 that explained over half of the genetic variance in this mouse model of opioid self-administration. We constructed a set of reciprocal congenic strains between B6 and D2 mice in which the proximal portion of chromosome 10 has been introgressed from one strain onto the background of the other. We tested mice from this pair of reciprocal strains together with progenitor B6 and D2 mice in a two-bottle choice drinking paradigm with morphine and quinine. The results showed that introgression of chromosome 10 alleles from the B6 strain onto a D2 genetic background increased voluntary morphine intake four-fold compared to progenitor D2 mice. Preference for morphine was also increased significantly in D2.B6-Mop2 mice compared to progenitor D2 mice. Conversely, introgression of chromosome 10 alleles from the D2 strain onto a B6 genetic background decreased morphine intake by half compared to progenitor B6 mice in B6.D2 -Mop2 mice; however, high morphine preference was maintained in this congenic strain most likely due to strong quinine aversion. When quinine was eliminated from the control bottle, morphine preference in B6.D2-Mop2 mice was decreased significantly relative to B6 and D2.B6-Mop2 mice. Overall, these data confirm the existence of a gene(s) on chromosome 10 proximal to D10Mit124 that has a strong influence on the difference in morphine drinking behavior between B6 and D2 mice.

Alleles↗

Association analysis of CHMP1.5 genetic variation and bipolar disorder.

OBJECTIVES: The18p11.2 region surrounding the G-olf gene has been linked in several independent studies to bipolar disorder and schizophrenia, yet association studies between G-olf genetic variations and bipolar disorder have been negative. We hypothesized that the linkage in this region might be due to a gene in close physical proximity to G-olf, and we examined variations in the CHMP1.5 gene within intron 5 of G-olf for association with bipolar disorder. METHODS: Two single-nucleotide polymorphisms, rs1786581 and rs1249624, were analyzed for association with bipolar disorder in 402 unrelated bipolar individuals and 181 unrelated controls. Genotyping was performed via pyrosequencing and restriction fragment length polymorphism analysis; results were compared by chi2 contingency analysis. RESULTS: No evidence was found for association of either allele at rs1249624 with bipolar disorder (chi2=1.25, degrees of freedom=1, P=0.26); however, a trend towards association with the 'T' allele at rs1786581 and with the 'T/T' 1786581/1249624 haplotype was observed. The chi2 for the haplotype was 7.16, (degrees of freedom=3, P=0.067) and for rs1786581 chi2=3.56, degrees of freedom=1, P=0.060; these differences are not statistically significant. CONCLUSIONS: Variation in the CHMP1.5 gene does not appear to be associated with bipolar disorder. A systematic assessment of genetic variation in the region using association studies will be necessary.

3' Untranslated Regions↗

A functional prodynorphin promoter polymorphism and opioid dependence.

OBJECTIVES: The prodynorphin gene (PDYN) promoter has a repeat polymorphism that is functionally important in association with substance abuse. We examined this polymorphism for association in our sample of 168 opioid-dependent patients and 122 ethnically and geographically matched controls. METHODS: Patients were selected from university-affiliated residential and non-residential addiction treatment programs in the Philadelphia area. A sample of blood was drawn from consenting individuals and genomic DNA was isolated. Polymerase chain reaction amplification of the PDYN promoter was performed and various genotypes were determined on the basis of differing sizes of the polymerase chain reaction products. The genotype and allele data were analyzed by Fisher's exact test. RESULT: A significant difference in genotype (P<0.0006) and allele (P<10) frequencies was found between the African American and European American populations. We did not detect any significant difference in genotype or allele frequencies between the patients and controls within the European American ethnic group. However, we detected a weak association (P=0.013) when we compared allele frequencies of patients and controls in the African American population. CONCLUSIONS: These data suggest that the PDYN repeat polymorphism should be studied in additional opioid-dependent populations.

Enkephalins↗

Fine mapping of a seizure susceptibility locus on mouse Chromosome 1: nomination of Kcnj10 as a causative gene.

Previous quantitative trait loci (QTL) mapping studies document that the distal region of mouse Chromosome (Chr) 1 contains a gene(s) that is in large part responsible for the difference in seizure susceptibility between C57BL/6 (B6) (relatively seizure-resistant) and DBA/2 (D2) (relatively seizure-sensitive) mice. We now confirm this seizure-related QTL ( Szs1) using reciprocal, interval-specific congenic strains and map it to a 6.6-Mb segment between Pbx1 and D1Mit150. Haplotype conservation between strains within this segment suggests that Szs1 may be localized more precisely to a 4.1-Mb critical interval between Fcgr3 and D1Mit150. We compared the coding region sequences of candidate genes between B6 and D2 mice using RT-PCR, amplification from genomic DNA, and database searching and discovered 12 brain-expressed genes with SNPs that predict a protein amino acid variation. Of these, the most compelling seizure susceptibility candidate is Kcnj10. A survey of the Kcnj10 SNP among other inbred mouse strains revealed a significant effect on seizure sensitivity such that most strains possessing a haplotype containing the B6 variant of Kcnj10 have higher seizure thresholds than those strains possessing the D2 variant. The unique role of inward-rectifying potassium ion channels in membrane physiology coupled with previous strong association between ion channel gene mutations and seizure phenotypes puts even greater focus on Kcnj10 in the present model. In summary, we confirmed a seizure-related QTL of large effect on mouse Chr 1 and mapped it to a finely delimited region. The critical interval contains several candidate genes, one of which, Kcnj10, exhibits a potentially important polymorphism with regard to fundamental aspects of seizure susceptibility.

Amino Acid Sequence↗

Predicting outcome of initial treatment with carbamazepine in childhood focal epilepsy.

We developed a model to predict the outcome of treatment with carbamazepine in children with newly diagnosed focal epilepsy of presumed temporal lobe origin, using data available at the time of diagnosis. Eligible children observed at The Children's Hospital of Philadelphia during 1999 were identified. Data were abstracted on four potential predictor variables for carbamazepine success or failure. A total of 149 patients completed an adequate first antiepileptic trial. Carbamazepine was the initial drug used in 129 (87%) patients. Forty-one of these 129 patients (32%) had persistent seizures. Significant predictors of initial carbamazepine failure were as follows: early risk factor for epilepsy (risk ratio = 3.1 [95% confidence interval 1.6, 4.0]) and temporal lobe abnormality on magnetic resonance imaging scan (risk ratio = 3.1 [1.7, 4.2]). The outcome of the initial carbamazepine trial was correctly classified in up to 78% of patients. Accurate prediction of initial carbamazepine failure was as high as 0.67 (0.53, 0.79). Accurate prediction of initial carbamazepine success was as high as 0.87 (0.77, 0.94). In this study, standard clinical data were less than adequate for predicting response to the initial trial of carbamazepine, with prediction of carbamazepine failure being particularly difficult. Better markers of antiepileptic response and nonresponse are required to guide optimal therapy in patients with epilepsy.

Adolescent↗

Mouse strain variation in maximal electroshock seizure threshold.

Maximal electroshock seizure threshold (MEST) is a classical measure of seizure sensitivity with a wide range of experimental applications. We determined MEST in nine inbred mouse strains and one congenic strain using a procedure in which mice are given one shock per day with an incremental (1 mA) current increase in each successive trial until a maximal seizure (tonic hindlimb extension) is elicited. C57BL/6J and DBA/2J mice exhibited the highest and lowest MEST, respectively, with the values of other strains falling between these two extremes. The relative rank order of MEST values by inbred strain (highest to lowest) is as follows: C57BL/6J > CBA/J = C3H/HeJ > A/J > Balb/cJ = 129/SvIMJ = 129/SvJ > AKR/J > DBA/2J. Results of experiments involving a single electroconvulsive shock given to separate groups of mice at different current intensities suggest that determination of MEST by the method used is not affected by repeated sub-maximal seizures. Overall, results document a distinctive mouse strain distribution pattern for MEST. Additionally, low within strain variability suggests that environmental factors which affect quantification of MEST are readily controlled under the conditions of this study. We conclude that MEST represents a useful tool for dissecting the multifactorial nature of seizure sensitivity in mice.

Animals↗

Lack of association between variations in the brain-derived neurotrophic factor (BDNF) gene and temporal lobe epilepsy.

Recently, an association between the C240T polymorphism in the brain-derived neurotrophic factor (BDNF) gene and partial epilepsy was demonstrated in a Japanese population. In this study we attempted to replicate the initial finding in a patient-control population of European ancestry and in addition tested whether the functional Val66Met polymorphism is associated with temporal lobe epilepsy (TLE). Genotypes of 151 TLE patients and 189 controls did not differ significantly for either of the variations. Results suggest that neither of the studied polymorphisms are strong susceptibility factors for TLE in this sample of individuals of European ancestry.

Adult↗

No association between common variations in the human alpha 2 subunit gene (ATP1A2) of the sodium-potassium-transporting ATPase and idiopathic generalized epilepsy.

Quantitative trait loci studies in inbred mice have identified a locus on chromosome 1 (Szs1) of fundamental importance to seizure susceptibility. High-ranking candidate genes in this susceptibility region include KCNJ9, KCNJ10 and ATP1A2. We performed a systematic mutation scan of the coding region of the human ATP1A2 gene and performed a case-control association study with seven common markers. Genotypes were assessed in 152 idiopathic generalized epilepsy (IGE) patients of German ancestry and 111 healthy German controls for all seven polymorphisms. No significant differences were found in genotype or allele frequencies for any of the variations between the IGE patients and controls. No haplotypes were associated with IGE when compared to controls. Linkage disequilibrium was demonstrated throughout the gene. Results suggest that the polymorphisms we studied in the ATP1A2 gene do not represent major susceptibility factors for common forms of IGE.

DNA↗