Nanda A. Singh

A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy

Stargardt disease (STGD, also known as fundus flavimaculatus; FFM) is an autosomal recessive retinal disorder characterized by a juvenile-onset macular dystrophy, alterations of the peripheral retina, and subretinal deposition of lipofuscin-like material. A gene encoding an ATP-binding cassette (ABC) transporter was mapped to the 2-cM (centiMorgan) interval at 1p13-p21 previously shown by linkage analysis to harbour the STGD gene. This gene, ABCR, is expressed exclusively and at high levels in the retina, in rod but not cone photoreceptors, as detected by in situ hybridization. Mutational analysis of ABCR in STGD families revealed a total of 19 different mutations including homozygous mutations in two families with consanguineous parentage. These data indicate that ABCR is the causal gene of STGD/FFM.

A Candidate Gene Approach Identifies the CHRNA5-A3-B4 Region as a Risk Factor for Age-Dependent Nicotine Addiction

People who begin daily smoking at an early age are at greater risk of long-term nicotine addiction. We tested the hypothesis that associations between nicotinic acetylcholine receptor (nAChR) genetic variants and nicotine dependence assessed in adulthood will be stronger among smokers who began daily nicotine exposure during adolescence. We compared nicotine addiction—measured by the Fagerstrom Test of Nicotine Dependence—in three cohorts of long-term smokers recruited in Utah, Wisconsin, and by the NHLBI Lung Health Study, using a candidate-gene approach with the neuronal nAChR subunit genes. This SNP panel included common coding variants and haplotypes detected in eight α and three β nAChR subunit genes found in European American populations. In the 2,827 long-term smokers examined, common susceptibility and protective haplotypes at the CHRNA5-A3-B4 locus were associated with nicotine dependence severity (p = 2.0×10−5; odds ratio = 1.82; 95% confidence interval 1.39–2.39) in subjects who began daily smoking at or before the age of 16, an exposure period that results in a more severe form of adult nicotine dependence. A substantial shift in susceptibility versus protective diplotype frequency (AA versus BC = 17%, AA versus CC = 27%) was observed in the group that began smoking by age 16. This genetic effect was not observed in subjects who began daily nicotine use after the age of 16. These results establish a strong mechanistic link among early nicotine exposure, common CHRNA5-A3-B4 haplotypes, and adult nicotine addiction in three independent populations of European origins. The identification of an age-dependent susceptibility haplotype reinforces the importance of preventing early exposure to tobacco through public health policies.

Genotype/Phenotype Analysis of a Photoreceptor-Specific ATP-Binding Cassette Transporter Gene, ABCR, in Stargardt Disease

Mutation scanning and direct DNA sequencing of all 50 exons of ABCR were completed for 150 families segregating recessive Stargardt disease (STGD1). ABCR variations were identified in 173 (57%) disease chromosomes, the majority of which represent missense amino acid substitutions. These ABCR variants were not found in 220 unaffected control individuals (440 chromosomes) but do cosegregate with the disease in these families with STGD1, and many occur in conserved functional domains. Missense amino acid substitutions located in the amino terminal one-third of the protein appear to be associated with earlier onset of the disease and may represent misfolding alleles. The two most common mutant alleles, G1961E and A1038V, each identified in 16 of 173 disease chromosomes, composed 18.5% of mutations identified. G1961E has been associated previously, at a statistically significant level in the heterozygous state, with age-related macular degeneration (AMD). Clinical evaluation of these 150 families with STGD1 revealed a high frequency of AMD in first- and second-degree relatives. These findings support the hypothesis that compound heterozygous ABCR mutations are responsible for STGD1 and that some heterozygous ABCR mutations may enhance susceptibility to AMD.

A Role of SCN9A in Human Epilepsies, As a Cause of Febrile Seizures and As a Potential Modifier of Dravet Syndrome

A follow-up study of a large Utah family with significant linkage to chromosome 2q24 led us to identify a new febrile seizure (FS) gene, SCN9A encoding Nav1.7. In 21 affected members, we uncovered a potential mutation in a highly conserved amino acid, p.N641Y, in the large cytoplasmic loop between transmembrane domains I and II that was absent from 586 ethnically matched population control chromosomes. To establish a functional role for this mutation in seizure susceptibility, we introduced the orthologous mutation into the murine Scn9a ortholog using targeted homologous recombination. Compared to wild-type mice, homozygous Scn9a N641Y/N641Y knockin mice exhibit significantly reduced thresholds to electrically induced clonic and tonic-clonic seizures, and increased corneal kindling acquisition rates. Together, these data strongly support the SCN9A p.N641Y mutation as disease-causing in this family. To confirm the role of SCN9A in FS, we analyzed a collection of 92 unrelated FS patients and identified additional highly conserved Nav1.7 missense variants in 5% of the patients. After one of these children with FS later developed Dravet syndrome (severe myoclonic epilepsy of infancy), we sequenced the SCN1A gene, a gene known to be associated with Dravet syndrome, and identified a heterozygous frameshift mutation. Subsequent analysis of 109 Dravet syndrome patients yielded nine Nav1.7 missense variants (8% of the patients), all in highly conserved amino acids. Six of these Dravet syndrome patients with SCN9A missense variants also harbored either missense or splice site SCN1A mutations and three had no SCN1A mutations. This study provides evidence for a role of SCN9A in human epilepsies, both as a cause of FS and as a partner with SCN1A mutations.

Human neuronal acetylcholine receptor A5-A3-B4 haplotypes are associated with multiple nicotine dependence phenotypes.

INTRODUCTION Previous research revealed significant associations between haplotypes in the CHRNA5-A3-B4 subunit cluster and scores on the Fagerström Test for Nicotine Dependence among individuals reporting daily smoking by age 17. The present study used subsamples of participants from that study to investigate associations between the CHRNA5-A3-B4 haplotypes and an array of phenotypes not analyzed previously (i.e., withdrawal severity, ability to stop smoking, and specific scales on the Wisconsin Inventory of Smoking Dependence Motives (WISDM-68) that reflect loss of control, strong craving, and heavy smoking. METHODS Two cohorts of current or former smokers (N = 886) provided both self-report data and DNA samples. One sample (Wisconsin) comprised smokers making a quit smoking attempt, which permitted the assessment of withdrawal and relapse during the attempt. The other sample (Utah) comprised participants studied for risk factors for nicotine dependence and chronic obstructive pulmonary disease and included individuals originally recruited in the Lung Health Study. RESULTS The CHRNA5-A3-B4 haplotypes were significantly associated with the targeted WISDM-68 scales (Tolerance, Craving, Loss of Control) in both samples of participants but only among individuals who began smoking early in life. The haplotypes were significantly associated with relapse likelihood and withdrawal severity, but these associations showed no evidence of an interaction with age at daily smoking. DISCUSSION The CHRNA5-A3-B4 haplotypes are associated with a broad range of nicotine dependence phenotypes, but these associations are not consistently moderated by age at initial smoking.

The rod photoreceptor ATP-binding cassette transporter gene, ABCR, and retinal disease: from monogenic to multifactorial

The ABCR gene encodes a rod photoreceptor specific ATP-binding cassette transporter. Mutations in ABCR are associated with at least four inherited retinal dystrophies: Stargardt disease, Fundus Flavimaculatus, cone-rod dystrophy, and retinitis pigmentosa. A statistically significant increase in heterozygous ABCR alterations has been identified in patients with age-related macular degeneration (AMD). A pedigree is described which manifests both Stargardt disease and AMD in which an ABCR mutation cosegregates with both disease phenotypes. These data from this case report support the hypothesis that ABCR is a dominant susceptibility locus for AMD. Recent work regarding ABCR is reviewed and a model is presented in which decreased ABCR function correlates with severity of retinal disease.

Mouse models of human KCNQ2 and KCNQ3 mutations for benign familial neonatal convulsions show seizures and neuronal plasticity without synaptic reorganization

The childhood epilepsy syndrome of benign familial neonatal convulsions (BFNC) exhibits the remarkable feature of clinical remission within a few weeks of onset and a favourable prognosis, sparing cognitive abilities despite persistent expression of the mutant KCNQ2 or KCNQ3 potassium channels throughout adulthood. To better understand such dynamic neuroprotective plasticity within the developing brain, we introduced missense mutations that underlie human BFNC into the orthologous murine Kcnq2 (Kv7.2) and Kcnq3 (Kv7.3) genes. Mutant mice were examined for altered thresholds to induced seizures, spontaneous seizure characteristics, hippocampal histology, and M‐current properties of CA1 hippocampal pyramidal neurons. Adult Kcnq2A306T/+ and Kcnq3G311V/+ heterozygous knock‐in mice exhibited reduced thresholds to electrically induced seizures compared to wild‐type littermate mice. Both Kcnq2A306T/A306T and Kcnq3G311V/G311V homozygous mutant mice exhibited early onset spontaneous generalized tonic‐clonic seizures concurrent with a significant reduction in amplitude and increased deactivation kinetics of the neuronal M‐current. Mice had recurrent seizures into adulthood that triggered molecular plasticity including ectopic neuropeptide Y (NPY) expression in granule cells, but without hippocampal mossy fibre sprouting or neuronal loss. These novel knockin mice recapitulate proconvulsant features of the human disorder yet show that inherited M‐current defects spare granule cells from reactive changes in adult hippocampal networks. The absence of seizure‐induced pathology found in these epileptic mouse models parallels the benign neurodevelopmental cognitive profile exhibited by the majority of BFNC patients.

The role of NMDA receptor systems in neuropeptide responses to stimulants of abuse

High doses of stimulants of abuse, such as methamphetamine and cocaine, cause significant increases in the content of neurotension- and dynorphin-like immunoreactivity in the striatum and nucleus accumbens (approximately 200-600% of control) in the rat. These changes in neuropeptide content are caused by stimulation of dopamine D1 receptors and prevented by the glutamate NMDA receptor antagonist, MK 801. Stimulation of the NMDA receptor with N-methyl-D-aspartate results in increases in the neuropeptide levels like that caused by methamphetamine and cocaine. These findings demonstrate that stimulants of abuse profoundly influence neurotensin and dynorphin pathways associated with extrapyramidal and limbic structures by an interaction of activated dopamine D1 and glutamate NMDA receptors.

N-methyl-d-aspartate receptors mediate dopamone-induced changes in extrapyramidal and limbic dynorphin systems

The N-methyl-D-aspartate (NMDA)-type glutamate receptor was shown to mediate dopamine-induced dynorphin A (Dyn) changes in extrapyramidal and limbic structures. MK801, a potent noncompetitive antagonist of the NMDA receptor, blocked increases in striatal and nigral Dyn content following single and multiple administrations of methamphetamine (METH). Significant attenuation of the METH-induced increases occurred with MK801 doses of 0.1 mg/kg/dose with complete blockade at 2.5 mg/kg/dose. Similar to METH, NMDA itself caused significant increases in striatal and nigral Dyn content. The NMDA-induced increase in striatal Dyn content was blocked by coadministration of an intermediate dose of MK801. The Dyn system associated with the nucleus accumbens responded in a similar manner in that MK801 totally blocked the METH-induced increases; moreover, NMDA elevated the Dyn content in this structure. The inability of MK801 to alter the quinpirole-induced decrease in striatal Dyn content suggests that the NMDA receptor is not involved in the D2 receptor regulation of striatal Dyn systems.

Dynamic dopaminergic regulation of neuropeptide Y systems in discrete striatal and accumbens regions

In this study we evaluated the effects of multiple administrations of selective dopamine D1 and D2 receptor agonists and antagonists on striatal, nigral, accumbens, pallidal and cortical neuropeptide Y systems. Treatment with the D1 receptor agonist, SKF 38393, decreased, while that with the D1 receptor antagonist, SCH 23390, increased neuropeptide Y-like immunoreactivity in the globus pallidus and several regions within the caudate-putamen. SCH 23390 did not change accumbens neuropeptide Y-like immunoreactivity levels but SKF 38393 increased neuropeptide Y-like immunoreactivity levels in anterior and decreased neuropeptide Y-like immunoreactivity levels in the posterior nucleus accumbens. Interestingly, reductions in neuropeptide Y-like immunoreactivity content occurred in response to administrations of both D2 receptor agonist, quinpirole, or antagonist, sulpiride, in all identified regions of each structure at some time point. These data suggest that the neuropeptide Y systems studied may be regulated by selective activity at postsynaptic or presynaptic dopamine receptors. They further suggest that within structures such as the caudate-putamen and nucleus accumbens are multiple distinct neuropeptide Y systems which are uniquely influenced by dopamine receptors.