Ethnicity can predict GLRA1 genotypes in hyperekplexia.

OBJECTIVES: Hyperekplexia is predominantly caused by mutations in the α-1 subunit of the inhibitory glycine receptor (GLRA1). Three quarters of cases show autosomal-recessive inheritance. METHODS: We carefully ascertained reports of ethnicity from our hyperekplexia research cohort. These were compared with all published cases of hyperekplexia with an identified genetic cause. Ethnicities were subgrouped as Caucasian, Asian, Arabic, Turkish, Jewish or Afro-American. RESULTS: We report the ethnicity of 90 cases: 56 cases from our service augmented by 34 cases from the literature. Homozygous deletions of exons 1 to 7 are predominantly seen in people with Turkish backgrounds (n=16/17, p<0.001). In contrast, the dominant point mutation R271 is seen in people of Asian, Caucasian and African-American heritage (n=19) but not in people with Arab or Turkish ethnicities (p<0.001). CONCLUSIONS: Self-declared ethnicity can predict gene-screening outcomes. Cultural practices influence the inheritance patterns and a Caucasian founder is postulated for R271 mutations.

Genetic epilepsy with febrile seizures plus: definite and borderline phenotypes.

Generalised epilepsy with febrile seizures plus (GEFS+) is the most studied familial epilepsy syndrome. However, characteristics of UK families have not previously been reported. Among the first 80 families recruited to our families study, four broad subphenotypes were identified: families with classical GEFS+; families with borderline GEFS+; families with unclassified epilepsy; and families with an alternative syndromal diagnosis. Borderline GEFS+ families shared many characteristics of classical GEFS+ families-such as prominent febrile seizures plus and early onset febrile seizures-but included more adults with focal epilepsies (rather than the idiopathic generalised epilepsies predominating in GEFS+) and double the prevalence of migraine. Thus the authors believe that a novel and robust familial epilepsy phenotype has been identified. Subcategorising families with epilepsy is helpful in targeting both clinical and research resources. Most families with GEFS+ have no identified causal mutation, and so predicting genetic homogeneity by identifying endophenotypes becomes more important.

Identifying and prioritising epilepsy treatment uncertainties.

OBJECTIVE: To identify and prioritise uncertainties regarding epilepsy treatment from people with epilepsy, their carers and epilepsy clinicians. BACKGROUND: Failure to acknowledge and address genuine treatment uncertainties has caused unnecessary iatrogenic harm. The authors define an uncertainty as a question that cannot be sufficiently answered by a systematic review of the literature. The database of the uncertainties of the effects of treatment (DUETs) is a collection of 'known unknowns' that enables patient-prioritised research. DESIGN AND PARTICIPANTS: The authors organised five separate focus groups (two consisting of clinicians, three of patients and carers) to garner questions on epilepsy treatment uncertainties; these yielded 398 potential research questions. Participants were asked to rank the questions in terms of importance. The authors then performed a thematic analysis. RESULTS: Patients rated questions concerning cognitive drug side effects, managing the consequences of side effects and improving public awareness about the treatment of epilepsy through improved services as most important. For clinicians, the most important themes were treatment programmes for non-epileptic attack disorder (NEAD), concerns about side effects in utero and uncertainties regarding prescribing in pregnancy. CONCLUSIONS: Patient uncertainties were often focussed on very practical considerations-how to take prescribed medication, access to services and how to minimise drug side effects. Clinicians' questions were also practical but clustered around 'the challenging consultation'-for example, NEAD, sudden unexplained death in epilepsy and prescribing in pregnancy. The authors have published the research questions on NHS Evidence and are working with them to identify those questions which represent genuine uncertainties. The authors encourage other clinicians to seek patient and carers' priorities in order to shape their research agenda.

Differential localization of GABAA receptor subunits within the substantia nigra of the human brain: an immunohistochemical study.

Gamma-aminobutyric acid(A) (GABA(A)) receptors (GABA(A)R) are inhibitory heteropentameric chloride ion channels comprising a variety of subunits and are localized at postsynaptic sites within the central nervous system. In this study we present the first detailed immunohistochemical investigation on the regional, cellular, and subcellular localisation of alpha(1), alpha(2), alpha(3), beta(2,3), and gamma(2) subunits of the GABA(A)R in the human substantia nigra (SN). The SN comprises two major regions, the SN pars compacta (SNc) consisting of dopaminergic projection neurons, and the SN pars reticulata (SNr) consisting of GABAergic parvalbumin-positive projection neurons. The results of our single- and double-labeling studies demonstrate that in the SNr GABA(A) receptors contain alpha(1), alpha(3), beta(2,3), and gamma(2) subunits and are localized in a weblike network over the cell soma, dendrites, and spines of SNr parvalbumin-positive nonpigmented neurons. By contrast, GABA(A)Rs on the SNc dopaminergic pigmented neurons contain predominantly alpha(3) and gamma(2) subunits; however there is GABA(A)R heterogeneity in the SNc, with a small subpopulation (6.5%) of pigmented SNc neurons additionally containing alpha(1) and beta(2,3) GABA(A)R subunits. Also, in the SNr, parvalbumin-positive terminals are adjacent to GABA(A)R on the soma and proximal dendrites of SNr neurons, whereas linear arrangements of substance P-positive terminals are adjacent to GABA(A) receptors on all regions of the dendritic tree. These results show marked GABA(A)R subunit hetereogeneity in the SN, suggesting that GABA exerts quite different effects on pars compacta and pars reticulata neurons in the human SN via GABA(A) receptors of different subunit configurations.

Association of gephyrin and glycine receptors in the human brainstem and spinal cord: an immunohistochemical analysis.

Gephyrin is a postsynaptic clustering molecule that forms a protein scaffold to anchor inhibitory neurotransmitter receptors at the postsynaptic membrane of neurons. Gephyrin was first identified as a protein component of the glycine receptor complex and is also colocalized with several GABAA receptor subunits in rodent brain. We have studied the distribution of gephyrin and glycine receptor subunits in the human brainstem and spinal cord using immunohistochemistry at light and confocal laser scanning microscopy levels. This study demonstrates the novel localization of gephyrin with glycine receptors in the human brainstem and spinal cord. Colocalization of immunoreactivities for gephyrin and glycine receptor subunits was detected in the dorsal and ventral horns of the spinal cord, the hypoglossal nucleus and the medial vestibular nucleus of the medulla. The results clearly establish that gephyrin is ubiquitously distributed and is colocalized, with a large proportion of glycine receptor subunits in the human brainstem and spinal cord. We therefore suggest that gephyrin functions as a clustering molecule for major subtypes of glycine receptors in the human CNS.

Distribution of gephyrin in the human brain: an immunohistochemical analysis.

Gephyrin is an ubiquitously expressed protein that, in the central nervous system, generates a protein scaffold to anchor inhibitory neurotransmitter receptors in the postsynaptic membrane. It was first identified as a protein component of the glycine receptor complex. Recent studies have demonstrated that gephyrin is colocalized with several subtypes of GABA(A) receptors and is part of postsynaptic GABA(A) receptor clusters. Here, we describe a study of the regional and cellular distribution of gephyrin in the human brain, determined by immunohistochemical localisation at the light and confocal laser scanning microscopic levels. At the regional level, gephyrin immunoreactivity was observed in most of the major brain regions examined. The most intense staining was in the cerebral cortex, hippocampus and caudate-putamen, in various brainstem nuclei with more moderate levels in the thalamus and cerebellum. At the cellular level gephyrin immunoreactivity was present on the plasma membranes of the soma and dendrites of pyramidal neurons throughout the various cortical regions examined. In the hippocampus, intense staining was observed on the granule cells of the dentate gyrus, and neurons of the CA1 and CA3 regions showed intense punctate gephyrin staining on their apical dendrites and cell bodies. Gephyrin immunoreactivity was also observed on neurons in the thalamus, globus pallidus and substantia nigra. In the putamen intense labelling of the striosomes was observed; most of the medium-sized neurons in the caudate-putamen were weakly labelled and many large neurons of the striatum were conspicuously stained. Many of the brainstem nuclei, notably the dorsal motor nucleus of the vagus, hypoglossal nucleus, trigeminal nucleus and inferior olive were all labelled with gephyrin. The spinal cord also showed high levels of gephyrin immunoreactivity. Our results demonstrate that the anchoring protein gephyrin is ubiquitously present in the human brain. We therefore suggest that gephyrin may have a central organizer role in assembling and stabilizing inhibitory postsynaptic membranes in human brain and is similar in function to those observed in the rodent brain. These findings contribute towards elucidating the role of gephyrin in the human brain.

A genomewide linkage study of age at onset in schizophrenia.

There is strong evidence for a genetic contribution to age at onset of schizophrenia, which probably involves both susceptibility loci for schizophrenia and modifying loci acting independent of disease risk. We sought evidence of linkage to loci that influence age at onset of schizophrenia in a sample of 94 affected sibling pairs with DSM-IV schizophrenia or schizoaffective disorder, and age at first psychiatric contact of 45 years or less. Individuals were genotyped for 229 microsatellite markers spaced at approximately 20 cM intervals throughout the genome. Loci contributing to age at onset were sought by a quantitative maximum-likelihood multipoint linkage analysis using MAPMAKER/SIBS. A nonparametric multipoint analysis was also performed. The genomewide significance of linkage results was assessed by simulation studies. There were six maximum-likelihood LOD score peaks of 1.5 or greater, the highest being on chromosome 17q (LOD = 2.54; genomewide P = 0.27). This fulfils Lander and Kruglyak's [1995: Nat Genet 11:241-247] criteria for suggestive linkage in that it would be expected to occur once or less (0.3 times) per genome scan. However, this finding should be treated with caution because the LOD score appeared to be almost solely accounted for by the pattern of ibd sharing at one marker (D17S787), with virtually no evidence of linkage over flanking markers. None of the linkage results achieved genomewide statistical significance, but the LOD score peak on chromosome 13q (LOD = 1.68) coincided with the region showing maximum evidence for linkage in the study by Blouin et al. [1998: Nat Genet 20:70-73] of categorical schizophrenia.

GABA(A) receptor subunit and gephyrin protein changes differ in the globus pallidus in Huntington's diseased brain.

Immunoreactivity of GABA(A) receptor subunits and the receptor anchoring protein gephyrin was investigated in the human globus pallidus using antibodies raised against the alpha(1) and gamma(2) subunits of the GABA(A) receptor complex and gephyrin. The results revealed increased GABA(A) receptor subunit immunoreactivity and unchanged levels of gephyrin immunoreactivity in Huntington's diseased (HD) globus pallidus (GP). The results demonstrate that gephyrin immunoreactivity did not change in unison with GABA(A) receptor changes in HD, suggesting that the receptor anchoring protein gephyrin is unaltered and maintains a stable lattice structure in the face of GABA(A) receptor changes in HD.

An unusual case of hyperekplexia.

Hyperekplexia is a rare paroxysmal disorder characterized by exaggerated startle response, hypertonia during infancy and a transient increase in tone following startle attacks. We report an unusual case of hyperekplexia in a young man. In addition to common symptoms of the condition, we found generalized spasticity persisting beyond infancy, and facial and skeletal dysmorphism. Because of an unsteady gait with frequent falls and raised serum creatine kinase levels, a congenital myopathy had been suspected in the past and an abnormal muscle biopsy had been documented. We diagnosed hyperekplexia at the age of 21 years on clinical grounds and following the response to pharmacological treatment. A mutation in the alpha1 subunit of the glycine receptor confirmed the diagnosis. A repeated needle muscle biopsy demonstrated mild myopathic changes, which we considered to be secondary to increased muscle tone. This case highlights the diagnostic difficulties of hyperekplexia, particularly in sporadic cases with unusual presentation.

Flow-induced ATP release in patient-specific arterial geometries--a comparative study of computational models.

The importance of the endothelium in the local regulation of blood flow is reflected by its influence on vascular tone by means of vasodilatory responses to many physiological stimuli. Regulatory pathways are affected by mass transport and wall shear stress (WSS), via mechanotransduction mechanisms. In the present work, we review the most relevant computational models that have been proposed to date, and introduce a general framework for modelling the responses of the endothelium to alteration in the flow, with a view to understanding the biomechanical processes involved in the pathways to endothelial dysfunction. Simulations are performed on two different patient-specific stenosed carotid artery geometries to investigate the influence of WSS and mass transport phenomena upon the agonist coupling response at the endothelium. In particular, results presented for two different models of WSS-dependent adenosine-5'-triphosphate (ATP) release reveal that existing paradigms may not account for the conditions encountered in vivo and may therefore not be adequate to model the kinetics of ATP at the endothelium.

Differential localization of gamma-aminobutyric acid type A and glycine receptor subunits and gephyrin in the human pons, medulla oblongata and uppermost cervical segment of the spinal cord: an immunohistochemical study.

Gephyrin is a multifunctional protein responsible for the clustering of glycine receptors (GlyR) and gamma-aminobutyric acid type A receptors (GABA(A)R). GlyR and GABA(A)R are heteropentameric chloride ion channels that facilitate fast-response, inhibitory neurotransmission in the mammalian brain and spinal cord. We investigated the immunohistochemical distribution of gephyrin and the major GABA(A)R and GlyR subunits in the human light microscopically in the rostral and caudal one-thirds of the pons, in the middle and caudal one-thirds of the medulla oblongata, and in the first cervical segment of the spinal cord. The results demonstrate a widespread pattern of immunoreactivity for GlyR and GABA(A)R subunits throughout these regions, including the spinal trigeminal nucleus, abducens nucleus, facial nucleus, pontine reticular formation, dorsal motor nucleus of the vagus nerve, hypoglossal nucleus, lateral cuneate nucleus, and nucleus of the solitary tract. The GABA(A)R alpha(1) and GlyR alpha(1) and beta subunits show high levels of immunoreactivity in these nuclei. The GABA(A)R subunits alpha(2), alpha(3), beta(2,3), and gamma(2) present weaker levels of immunoreactivity. Exceptions are intense levels of GABA(A)R alpha(2) subunit immunoreactivity in the inferior olivary complex and high levels of GABA(A)R alpha(3) subunit immunoreactivity in the locus coeruleus and raphe nuclei. Gephyrin immunoreactivity is highest in the first segment of the cervical spinal cord and hypoglossal nucleus. Our results suggest that a variety of different inhibitory receptor subtypes is responsible for inhibitory functions in the human brainstem and cervical spinal cord and that gephyrin functions as a clustering molecule for major subtypes of these inhibitory neurotransmitter receptors.

Posthumous diagnosis of long QT syndrome from neonatal screening cards.

BACKGROUND: Molecular autopsy in sudden unexplained death in the young (SUDY) victims cannot usually be performed if tissue suitable for DNA extraction is not retained at autopsy. OBJECTIVE: The purpose of this study was to assess the feasibility and clinical value of posthumous genetic testing for long QT syndrome (LQTS) using residual material from the neonatal screening (Guthrie) card in SUDY victims. METHODS: Twenty-one cases were investigated up to 13 years after death. Deaths occurred at <1 year in one, 1-18 years in 18, and 19-35 years in two patients. Guthrie cards were 3-39 years old. DNA was extracted, and amplicons corresponding to the coding regions of the LQTS genes 1, 2, 3, 5, and 6 underwent either denaturing high-performance liquid chromatography screening or direct DNA sequencing. RESULTS: Adequate DNA was extracted in every case, although repeated purification and amplification was often required. Rare variants were detected in six of 19 cases undergoing diagnostic screening. Four (21%) are considered to be pathological and have been used for family screening: R243C and H455Y in KCNQ1 in 12-year-old and 13-year-old boys, respectively, and Q81H and S621R in KCNH2 in 21-month and 28-year-old females, respectively. Variants of uncertain significance were R1047L in KCNH2 in a 2-year-old girl and S38G in KCNE1 in a 19-month-old boy. Point mutation tests for previously identified familial LQTS mutations revealed a positive result in both cases: E146K in KCNQ1 and exon 6-4del in KCNH2. CONCLUSION: Residual material from Guthrie cards collected for newborn metabolic screening can be used as a reliable source of DNA for the posthumous diagnosis of LQTS decades after SUDY, although purification and amplification of DNA is time intensive.

Epilepsy genetics: clinical beginnings and social consequences.

The approach to epilepsy care has transformed in the last 30 years, with more and better anti-epileptic medications, improved cerebral imaging and increased surgical options. Alongside this, developments in neuroscience and molecular genetics have furthered the understanding of epileptogenesis. Future developments in pharmacogenomics hold the promise of antiepileptic drugs matched to specific genotypes. Despite this rapid progress, one-third of epilepsy patients remain refractory to medication, with their seizures impacting upon day-to-day activity, social well-being, independence, economic output and quality of life. International genome collaborations, such as HapMap and the Welcome Trust Case-Control Consortium single nucleotide polymorphism (SNP) mapping project have identified common genetic variations in diseases of major public health importance. Such genetic signposts should help to identify at-risk populations with a view to producing more effective pharmaceutical treatments. Neurological disorders, despite comprising one-fifth of UK acute medical hospital admissions, are surprisingly under-represented in these projects. Epilepsy is the commonest serious neurological disorder worldwide. Although physically, psychologically, socially and financially disabling, it rarely receives deserved attention from physicians, scientists and governmental bodies. As outlined in this article, research into epilepsy genetics presents unique challenges. These help to explain why the identification of its complex genetic traits has lagged well behind other disciplines, particularly the efforts made in neuropsychiatric disorders. Clinical beginnings must underpin any genetic understanding in epilepsy. Success in identifying genetic traits in other disorders does not make the automatic case for genome-wide screening in epilepsy, but such is a desired goal. The essential clinical approach of accurately phenotyping, diagnosing and interpreting the dynamic nature of epilepsy remains fundamental to harvesting its potential translational outcomes.

Functional variants of antioxidant genes in smokers with COPD and in those with normal lung function.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is predominantly the consequence of chronic smoking exposure, but its development may be influenced by genetic variants that affect lung remodelling, inflammation, and defence from oxidant stress. A study was undertaken to determine whether genetic variants within genes encoding the antioxidant enzymes superoxide dismutase (SOD) and catalase may be associated with the development of impaired lung function. METHODS: In a case-control study, the allele and genotype frequencies of functional polymorphisms from SOD1 (CuZnSOD), SOD2 (MnSOD), SOD3 (extracellular SOD), and catalase (CAT) were compared in chronic smokers with normal lung function (resistant smokers) and in those with COPD. RESULTS: Significantly higher frequencies of the G allele and CG/GG genotype of the 213 SOD3 polymorphism were found in resistant smokers (odds ratios (ORs) 4.3 (95% CI 1.5 to 13.3) and 4.2, 95% CI 1.4 to 13.3), Bonferroni corrected p = 0.02 and p = 0.02, respectively) than in those with COPD. There were no differences between the COPD and resistant smokers for the SOD1, SOD2, or CAT polymorphisms tested. CONCLUSIONS: The 213Gly variant of the SOD3 gene may, through antioxidant or anti-inflammatory effects, confer a degree of resistance in some smokers to the development of COPD.

Near-miss SIDS due to Brugada syndrome.

A 19 day old infant was successfully resuscitated from ventricular fibrillation. The 12 lead ECG was normal, with a normal QT interval, and remains so over three years follow up. DNA analysis revealed a missense mutation (R1193Q) in the SCN5A gene, previously linked with familial sudden unexpected nocturnal death syndrome, also known as Brugada syndrome.

Red cell dimorphism in a young man with a constitutional chromosomal translocation t(11;22)(p15.5;q11.21).

A constitutional, balanced chromosomal translocation t(11;22)(p15.5;q11.21) was discovered in a tall young man during investigation of a red cell dimorphism. The red cells are predominantly normochromic and normocytic with a small population of hypochromic, microcytic cells. Contained within the regions involved in the translocation are determinants of height (IGF2:11p15.5), red cell haemoglobinization (non-alpha globin gene complex: 11p15.5) and oncogenesis (cHa-Ras-1, Beckwith-Wiedemann syndrome: 11p15.5; BCR, Burkitts lymphoma, Ewings sarcoma: 22q11.21). To map these regions in the patient, somatic cell hybrids were generated and cell lines that segregated the chromosomes 11, 22 and 22q- were obtained. All 11p15.5 sequences investigated, in particular the whole of the non-alpha globin gene complex including its 5' and 3' regulatory sequences, were found to be translocated to 22q-. All chromosome 22 sequences studied were missing from the 22q- cell lines, including the proximal anonymous marker D22S24, and therefore assumed to be translocated to 11p+. These results suggest that the non-alpha globin gene complex has been moved close to the centromeric region of chromosome 22q-. It is postulated that such a positioning subjects the complex to a variegated position-effect bringing about a clonal exclusion of the complex and thus producing a beta-thalassaemia trait mosaic.

Evidence for recessive as well as dominant forms of startle disease (hyperekplexia) caused by mutations in the alpha 1 subunit of the inhibitory glycine receptor.

Startle disease, or hyperekplexia, is characterized by an exaggerated startle reflex and neonatal hypertonia. An autosomal dominant form of the disorder is associated with mutations in the same codon of the alpha 1 subunit of the inhibitory glycine receptor (GLRA 1) resulting in the substitution of an uncharged amino acid for Arg271 in the mature protein. However, recessive transmission is seen in the mouse mutant spasmodic which resembles startle disease phenotypically and is also associated with mutations in Glra 1. We have confirmed the finding of Arg271 mutations in individuals with startle disease in a UK family showing autosomal dominant transmission. In addition we describe an apparently sporadic case, the offspring of a consanguineous mating, who is homozygous for a novel mutation (T1112A) in GLRA 1, which results in the substitution of asparagine for isoleucine at position 244 of the mature protein. This suggests that human startle disease can display recessive as well as dominant inheritance resulting from different mutations in GLRA 1.

Further evidence of autosomal dominant congenital zonular pulverulent cataracts linked to 13q11 (CZP3) and a novel mutation in connexin 46 (GJA3).

We describe a four-generation family with fully penetrant, autosomal dominant, congenital cataracts (ADCC), presenting with morphologically homogeneous "zonular pulverulent" cataracts (CZP) and typical early-onset phenotype. Linkage analysis was performed with a panel of polymorphic markers mapped to all genomic regions of ADCC susceptibility. Contiguous significant two-point lod scores were generated at autosomal region 13q11-q13 and further linkage and haplotype studies confined the disease locus to 13q11, supporting a previous linkage of CZP (specifically CZP3) to 13q11. Mutations in a gap-junction protein, connexin 46 (alphaa3 subunit or GJA3), have recently been reported as being linked to the 13q11 region. Mutational analysis of connexin 46 in our family revealed a C-->T at position 560 (P187L) of the cDNA sequence creating a novel MnlI restriction site that segregated with affected members of the pedigree. This family represents a second report of CZP3 linkage to 13q and is associated with a novel mutation in the connexin 46 (GJA3) gene.