Shared functional defect in IP3R-mediated calcium signaling in diverse monogenic autism syndromes.

Autism spectrum disorder (ASD) affects 2% of children, and is characterized by impaired social and communication skills together with repetitive, stereotypic behavior. The pathophysiology of ASD is complex due to genetic and environmental heterogeneity, complicating the development of therapies and making diagnosis challenging. Growing genetic evidence supports a role of disrupted Ca(2+) signaling in ASD. Here, we report that patient-derived fibroblasts from three monogenic models of ASD-fragile X and tuberous sclerosis TSC1 and TSC2 syndromes-display depressed Ca(2+) release through inositol trisphosphate receptors (IP3Rs). This was apparent in Ca(2+) signals evoked by G protein-coupled receptors and by photoreleased IP3 at the levels of both global and local elementary Ca(2+) events, suggesting fundamental defects in IP3R channel activity in ASD. Given the ubiquitous involvement of IP3R-mediated Ca(2+) signaling in neuronal excitability, synaptic plasticity, gene expression and neurodevelopment, we propose dysregulated IP3R signaling as a nexus where genes altered in ASD converge to exert their deleterious effect. These findings highlight potential pharmaceutical targets, and identify Ca(2+) screening in skin fibroblasts as a promising technique for early detection of individuals susceptible to ASD.

Mild phenotype in a male with pyruvate dehydrogenase complex deficiency associated with novel hemizygous in-frame duplication of the E1α subunit gene (PDHA1).

Pyruvate dehydrogenase complex (PDHC) deficiency is an inborn error of metabolism that occurs most commonly due to mutations in the X-linked E1α subunit gene (PDHA1). We report a novel duplication of PDHA1 associated with a mild phenotype in a 15-year-old boy who was diagnosed with PDHC deficiency at 4 years of age following a history of seizures and lactic acidosis. The novel c.1087_1119 mutation in exon 11 resulted in an in-frame duplication of 11 amino acids. Measurements of PDHC activity in cultured skin fibroblasts were low, corresponding to 18.6 and 11.6% of the mean with respect to prior controls, whereas the E1 PDH component was absent. He has borderline intellectual functioning and maintains normal lactate levels on a ketogenic diet in between relapses due to illness. Review of the literature reveals wide variation of clinical phenotype in patients with mutations of the E1α subunit gene (PDHA1). There appears to be a higher incidence of normal or borderline intellectual ability in individuals who have insertions or deletions that are in-frame versus those that are out-of-frame. Furthermore, there is no correlation between mean residual PDH activity and phenotype in these patients.

Rapid identification of mitochondrial DNA (mtDNA) mutations in neuromuscular disorders by using surveyor strategy.

Mutations of mitochondrial genome are responsible for respiratory chain defects in numerous patients. We have used a strategy, based on the use of a mismatch-specific DNA endonuclease named " Surveyor Nuclease", for screening the entire mtDNA in a group of 50 patients with neuromuscular features, suggesting a respiratory chain dysfunction. We identified mtDNA mutations in 20% of patients (10/50). Among the identified mutations, four are not found in any mitochondrial database and have not been reported previously. We also confirm that mtDNA polymorphisms are frequently found in a heteroplasmic state (15 different polymorphisms were identified among which five were novel).

A novel missense ATP1A2 mutation in a Finnish family with familial hemiplegic migraine type 2.

Familial hemiplegic migraine (FHM), a rare autosomal dominant subtype of migraine with aura, has been linked to two chromosomal loci, 19p13 and 1q23. Mutations in the Na+K+-ATPase alpha2 subunit gene, ATP1A2, on 1q23 have recently been shown to cause familial hemiplegic migraine type 2 (FHM2). We sequenced the coding regions of this gene in a Finnish chromosome 1q23-linked FHM family with associated symptoms such as coma and identified a novel A1033G mutation in exon 9. This mutation results in a threonine-to-alanine substitution at codon 345. This residue is located in a highly conserved N-terminal region of the M4-5 loop of the Na+,K+-ATPase. Furthermore, the T345A mutation co-segregated with the disorder in our family and was not present in 132 healthy Finnish control individuals. For these reasons it is most likely the FHM-causing mutation in this family.

Novel truncated isoform of SK3 potassium channel is a potent dominant-negative regulator of SK currents: implications in schizophrenia.

The small-conductance calcium-activated K(+) channel SK3 (SKCa3/KCNN3) regulates electrical excitability and neurotransmitter release in monoaminergic neurons, and has been implicated in schizophrenia, ataxia and anorexia nervosa. We have identified a novel SK3 transcript, SK3-1B that utilizes an alternative first exon (exon 1B), but is otherwise identical to SK3. SK3-1B, mRNA is widely distributed in human tissues and is present at 20-60% of SK3 in the brain. The SK3-1B protein lacks the N-terminus and first transmembrane segment, and begins eight residues upstream of the second transmembrane segment. When expressed alone, SK3-1B did not produce functional channels, but selectively suppressed endogenous SK3 currents in the pheochromocytoma cell line, PC12, in a dominant-negative fashion. This dominant inhibitory effect extended to other members of the SK subfamily, but not to voltage-gated K(+) channels, and appears to be due to intracellular trapping of endogenous SK channels. The effect of SK3-1B expression is very similar to that produced by expression of the rare SK3 truncation allele, SK3-Delta, found in a patient with schizophrenia. Regulation of SK3 and SK3-1B levels may provide a potent mechanism to titrate neuronal firing rates and neurotransmitter release in monoaminergic neurons, and alterations in the relative abundance of these proteins could contribute to abnormal neuronal excitability, and to the pathogenesis of schizophrenia.

Respiratory complex II defect in siblings associated with a symptomatic secondary block in fatty acid oxidation.

The mitochondrial oxidative phosphorylation and fatty acid oxidation pathways have traditionally been considered independent major sources of cellular energy production; however, case reports of patients with specific enzymatic defects in either pathway have suggested the potential for a complex interference between the two. This study documents a new site of interference between the two pathways, a site in respiratory complex II capable of producing clinical signs of a block in fatty acid oxidation and reduced in vitro activity of acyl-CoA dehydrogenases. The initial patient, and later her newborn sibling, had mildly dysmorphic features, lactic acidosis and a defect in mitochondrial respiratory complex II associated with many biochemical features of a block in fatty acid oxidation. Results of in vitro probing of intact fibroblasts from both patients with methyl[2H3]palmitate and L-carnitine revealed greatly increased [2H3]butyrylcarnitine; however, the ratio of dehydrogenase activity with butyryl-CoA with anti-MCAD inactivating antibody (used to reveal SCAD-specific activity) to that with octanoyl-CoA was normal, excluding a selective SCAD or MCAD deficiency. Respiratory complex II was defective in both patients, with an absent thenoyltrifluoroacetone-sensitive succinate Q reductase activity that was partially restored by supplementation with duroquinone. Although secondary, the block in fatty acid oxidation was a major management problem since attempts to provide essential fatty acids precipitated acidotic decompensations. This study reinforces the need to pursue broadly the primary genetic defect within these two pathways, making full use of increasingly available functional and molecular diagnostic tools.

Phenotype and genotype variation in primary carnitine deficiency.

PURPOSE: Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective carnitine transport. This disease is caused by mutations in the carnitine transporter gene SLC22A5. The objective of this study was to extend mutational analysis to four additional families with this disorder and determine whether recurrent mutations could be found. METHODS: The SLC22A5 gene encoding the OCTN2 carnitine transporter was sequenced, and the missense mutations identified were expressed in Chinese hamster ovary (CHO) cells. RESULTS: DNA sequencing revealed four novel mutations (Y4X; dup 254-264, 133X; R19P; R399Q). Alleles introducing premature STOP codons reduced the levels of OCTN2 mRNA. Carnitine transport in CHO cells expressing the R19P and R399Q mutations was reduced to < 5% of normal. The 133X mutation was found in two unrelated European families. Two patients within the same family, both homozygous for the same mutation (R399Q) had completely different clinical presentation. CONCLUSIONS: Heterogeneous mutations in the SLC22A5 gene cause primary carnitine deficiency. Different presentations are observed even in children with identical mutations.

Genomic organization and promoter analysis of human KCNN3 gene.

KCNN3 is a member of the gene family, KCNN1-4, encoding the small and intermediate conductance calcium-activated potassium channels. Long CAG-repeat alleles of this gene have been found to be over-represented in patients with schizophrenia in a number of population-based association studies, and this gene maps to human chromosome 1q21, a region recently implicated in schizophrenia by linkage. To set the stage for a further functional evaluation of KCNN3, we defined the nature of the genomic locus in the size, structure, and sequence of its introns and exons and the function of potential upstream regulatory regions. We isolated P1-derived artificial chromosome (PAC) clones from a genomic library and identified an overlapping available bacterial artificial chromosome (BAC) clone. Cosmids subcloned from the PAC and BAC clones were then sequenced and merged with the sequence in the public database. The KCNN3 gene spans over 163.1 kb and is composed of eight exons and seven introns. All of the exon-intron junctions conform closely to consensus splice sites. The proximal 2.5 kb of the 5'-flanking sequence was obtained and analyzed for potential transcription factor binding sites. In the proximal 2.5 kb upstream region, potential sites for the Ikaros factor (IK2), homeodomain factor Nkx-2.5/Csx (NKX25), nuclear factor of activated T-cells (NFAT), upstream stimulating factor (USF), c-AMP responsive element binding protein (CREB), POU factor Brn2 (BRN-2), myeloid zinc finger protein (MZF1), vitellogenin binding protein (VBP), HNF3 forkhead homologue 2 (HFH2), and transcription initiation were identified, as well as several potential AP-1 and AP-4 sites. Finally, a 2261-bp fragment of this upstream region was cloned into a promoterless pGL3-luciferase vector, where it produced orientation-dependent expression of the reporter gene in transiently transfected PC12 cells, cells which natively express functional KCNN3 channels, suggesting that this cloned fragment includes competent promoter elements of this gene.

Design and characterization of a highly selective peptide inhibitor of the small conductance calcium-activated K+ channel, SkCa2.

Apamin-sensitive small conductance calcium-activated potassium channels (SKCa1-3) mediate the slow afterhyperpolarization in neurons, but the molecular identity of the channel has not been defined because of the lack of specific inhibitors. Here we describe the structure-based design of a selective inhibitor of SKCa2. Leiurotoxin I (Lei) and PO5, peptide toxins that share the RXCQ motif, potently blocked human SKCa2 and SKCa3 but not SKCa1, whereas maurotoxin, Pi1, Tskappa, and PO1 were ineffective. Lei blocked these channels more potently than PO5 because of the presence of Ala(1), Phe(2), and Met(7). By replacing Met(7) in the RXCQ motif of Lei with the shorter, unnatural, positively charged diaminobutanoic acid (Dab), we generated Lei-Dab(7), a selective SKCa2 inhibitor (K(d) = 3.8 nm) that interacts with residues in the external vestibule of the channel. SKCa3 was rendered sensitive to Lei-Dab(7) by replacing His(521) with the corresponding SKCa2 residue (Asn(367)). Intracerebroventricular injection of Lei-Dab(7) into mice resulted in no gross central nervous system toxicity at concentrations that specifically blocked SKCa2 homotetramers. Lei-Dab(7) will be a useful tool to investigate the functional role of SKCa2 in mammalian tissues.

Nuclear localization and dominant-negative suppression by a mutant SKCa3 N-terminal channel fragment identified in a patient with schizophrenia.

The small conductance calcium-activated K+ channel gene SKCa3/KCNN3 maps to 1q21, a region strongly linked to schizophrenia. Recently, a 4-base pair deletion in SKCa3 was reported in a patient with schizophrenia, which truncates the protein at the end of the N-terminal cytoplasmic region (SKCa3Delta). We generated a green fluorescent protein-SKCa3 N-terminal construct (SKCa3-1/285) that is identical to SKCa3Delta except for the last two residues. Using confocal microscopy we demonstrate that SKCa3-1/285 localizes rapidly and exclusively to the nucleus of mammalian cells like several other pathogenic polyglutamine-containing proteins. This nuclear targeting is mediated in part by two polybasic sequences present at the C-terminal end of SKCa3-1/285. In contrast, full-length SKCa3, SKCa2, and IKCa1 polypeptides are all excluded from the nucleus and express as functional channels. When overexpressed in human Jurkat T cells, SKCa3-1/285 can suppress endogenous SKCa2 currents but not voltage-gated K+ currents. This dominant-negative suppression is most likely mediated through the co-assembly of SKCa3-1/285 with native subunits and the formation of non-functional tetramers. The nuclear localization of SKCa3-1/285 may alter neuronal architecture, and its ability to dominantly suppress endogenous small conductance K(Ca) currents may affect patterns of neuronal firing. Together, these two effects may play a part in the pathogenesis of schizophrenia and other neuropsychiatric disorders.

hKCa3/KCNN3 potassium channel gene: association of longer CAG repeats with schizophrenia in Israeli Ashkenazi Jews, expression in human tissues and localization to chromosome 1q21.

We demonstrate a significant association between longer CAG repeats in the hKCa3/KCNN3 calcium-activated potassium channel gene and schizophrenia in Israeli Ashkenazi Jews. We genotyped alleles from 84 Israeli Jewish patients with schizophrenia and from 102 matched controls. The overall allele frequency distribution is significantly different in patients vs controls (P = 0.00017, Wilcoxon Rank Sum test), with patients showing greater lengths of the CAG repeat. Northern blots reveal substantial levels of approximately 9 kb and approximately 13 kb hKCa3/KCNN3transcripts in brain, striated muscle, spleen and lymph nodes. Within the brain, hKCa3/KCNN3transcripts are most abundantly expressed in the substantia nigra, lesser amounts are detected in the basal ganglia, amygdala, hippocampus and subthalamic nuclei, while little is seen in the cerebral cortex, cerebellum and thalamus. In situ hybridization reveals abundant hKCa3/KCNN3 message localized within the substantia nigra and ventral tegmental area, and along the distributions of dopaminergic neurons from these regions into the nigrostriatal and mesolimbic pathways. FISH analysis shows that hKCa3/KCNN3 is located on chromosome 1q21.

Lack of linkage or association between schizophrenia and the polymorphic trinucleotide repeat within the KCNN3 gene on chromosome 1q21.

To determine the importance of a candidate gene KCNN3 (formerly named hSKCa3) in the susceptibility to schizophrenia, we have studied the genotypes of a (CAG)n polymorphism within this gene in the DNAs of the members of 54 multiplex families with this disease. Parametric and nonparametric linkage analysis did not provide evidence for linkage between KCNN3 (that we mapped to chromosome 1q21) and schizophrenia. Furthermore, we observed no difference in the distribution of the (CAG)n alleles between affected and normal individuals. These results do not support the hypothesis that larger KCNN3 alleles are preferentially associated with schizophrenia [Chandy et al. 1998 Mol Psychiatr 3:32-37] in individuals from multiply affected families.

A piece in the puzzle: an ion channel candidate gene for schizophrenia.

Mutations in ion channels have been found to cause a variety of mendelian genetic diseases, and polyglutamine repeat expansion is a newly recognized pathogenic mechanism that causes several rare, genetic, late-onset neurological syndromes. Polymorphic polyglutamine tracts are present in a recently described human, calcium-activated potassium channel, KCNN3 (also known as hKCa3), and alleles of this gene that contain longer repeats have been associated with schizophrenia. The physiological function of the channel is consistent with an etiological role in this disease; drugs designed to target this channel might therefore provide novel psychotherapeutics.

Transmission disequilibrium analysis of a triplet repeat within the hKCa3 gene using family trios with schizophrenia.

hKCa3 is a neuronal small conductance calcium-activated potassium channel which contains a polyglutamine tract, encoded by a polymorphic CAG repeat in the gene. Since an association between longer alleles of the CAG repeat and schizophrenia has been reported, we performed haplotype-based haplotype relative risk (HHRR) and transmission disequilibrium (TDT) in 97 family trios with schizophrenia from SW China. We found no evidence for an excess of longer CAG repeats in the patients, and the ETDT test was not significant for either allele-wise (P = 0.31) or genotype-wise analysis (P = 0.18). However, there was a deficit of transmission of the (CAG)20 repeat allele to affected offspring when this allele was considered individually by TDT (P = 0.012; not corrected for multiple testing). These data do not support a role for larger alleles at the hKCa3 locus in psychosis in Chinese subjects.

CCG1/TAF(II)250 regulates epidermal growth factor receptor gene transcription in cell cycle mutant ts13.

The epidermal growth factor receptor (EGFR) plays a critical role in normal growth and its overexpression is associated with several types of cancer. To learn more about regulation of the expression of this important receptor, we investigated the role of the TAF(II)250 subunit of transcription factor IID in the transcription of the EGFR gene. The EGFR gene has a TATA-less promoter and TAF(II)250 has previously been shown to have an important regulatory role in such promoters. The study was performed in the ts13 hamster cell line which has a temperature-sensitive mutation in the CCG1 gene that encodes TAF(II)250. At the nonpermissive temperature, the transcription of a few cell cycle-dependent genes is depressed in ts13 cells while global RNA synthesis is unaffected. Using this model system, we found that EGFR promoter-driven luciferase expression in transiently transfected ts13 cells decreased 8, 25, and 50-fold after 12, 24, and 48 hours, respectively, at the nonpermissive temperature. The decrease was partially rescued by cotransfection with the wild-type CCG1 gene. The expression of endogenous EGFR also appeared to be regulated by TAF(II)250--the maximum binding capacity of ts13 cells for 125I-labeled EGF decreased approximately twofold when incubated for 2 days at the nonpermissive temperature. Placing these studies in the context of the current understanding of the TFIID transcription complex, we speculate that selective stimulation of EGFR gene transcription may be mediated by TAF(II)250 interaction with enhancer-bound activators and the basal transcription machinery.

Further support for an association between a polymorphic CAG repeat in the hKCa3 gene and schizophrenia.

A recent study has suggested that a polymorphism in the hKCa3 potassium channel may be associated with raised susceptibility to schizophrenia. Despite its modest statistical significance, the study is intriguing for two reasons. First, hKCa3 contains a polymorphic CAG repeat in its coding sequence, with large repeats more common in schizophrenics compared with controls. This is interesting in view of several repeat expansion detection (RED) studies that have reported an excess of large CAG repeats in psychotic probands. Second, the hKCa3 gene is a functional candidate gene because studies of antipsychotic and psychotogenic compounds suggest that glutamatergic systems modulated by SKCa channels may be important in schizophrenia pathogenesis. In the light of the above, we have tested the hypothesis of an association between schizophrenia and the hKCa3 CAG repeat polymorphism using a case control study design. Under the same model of analysis as the earlier study, schizophrenic probands had a higher frequency of alleles with greater than 19 repeats than controls (chi 2 = 2.820, P = 0.047, 1-tail). Our data therefore provide modest support for the hypothesis that polymorphism in the hKCa3 gene may contribute to susceptibility to schizophrenia.

Isolation of a novel potassium channel gene hSKCa3 containing a polymorphic CAG repeat: a candidate for schizophrenia and bipolar disorder?

Many human hereditary neurodegenerative diseases are caused by expanded CAG repeats, and anonymous CAG expansions have also been described in schizophrenia and bipolar disorder. We have isolated and sequenced a novel human cDNA encoding a neuronal, small conductance calcium-activated potassium channel (hSKCa3) that contains two arrays of CAG trinucleotide repeats. The second CAG repeat in hSKCa3 is highly polymorphic in control individuals, with alleles ranging in size from 12 to 28 repeats. The overall allele frequency distribution is significantly different in patients with schizophrenia compared to ethnically matched controls (Wilcoxon Rank Sum test, P=0.024), with CAG repeats longer than the modal value being over-represented in patients (Fisher Exact test, P=0.0035). A similar, non-significant, trend is seen for patients with bipolar disorder. These results provide evidence for a possible association between longer alleles in the hSKCa3 gene and both of these neuropsychiatric diseases, and emphasize the need for more extensive studies of this new gene. Small conductance calcium-activated K+ channels play a critical role in determining the firing pattern of neurons. These polyglutamine repeats may modulate hSKCa3 channel function and neuronal excitability, and thereby increase disease risk when combined with other genetic and environmental effects.

Mutations causing achondroplasia and thanatophoric dysplasia alter bFGF-induced calcium signals in human diploid fibroblasts.

Mutations in the fibroblast growth factor receptor (FGFR) gene family recently have been shown to underlie several hereditary disorders of bone development, with specific FGFR3 mutations causing achondroplasia (Ach) and thanatophoric dysplasia (TD). However, for none of these mutations has the defect in receptor function been demonstrated directly and, therefore, for none has the pathophysiological mechanism of the disease been defined. Using our established techniques for single-cell ratiometric real-time calcium image analysis, we defined the nature of the basic fibroblast growth factor (bFGF)-induced calcium signal in human diploid fibroblasts, and, in blinded studies, have analyzed the bFGF-induced signals from 18 independent fibroblast cell lines, including multiple lines from patients with known mutant alleles of FGFR3 and syndromes of Ach or TD. Control cells responded with transient increases in intracellular calcium, with many cells showing oscillatory calcium waves. Homozygous Ach cell lines failed to signal, whereas heterozygous Ach lines responded nearly normally. We observed heterogeneous signals in TD heterozygotes: the unresponsive lines all turned out to carry TD1 alleles, whereas all responsive lines had TD2 alleles. Since FGFR1, 2 and 3 receptors are known to be expressed in fibroblasts, our results suggest that specific mutant FGFR3 alleles can function in a dosage-dependent dominant-negative fashion to inactivate FGFR signaling.

Reduced epidermal growth factor receptor expression in hypohidrotic ectodermal dysplasia and Tabby mice.

Patients with hypohidrotic ectodermal dysplasia (HED) and Tabby (Ta) mice lack sweat glands and there is compelling evidence that these phenotypes are caused by mutations in the same highly conserved but unidentified X-linked gene. Previous studies showed that exogenous epidermal growth factor (EGF) reversed the Ta phenotype but the EGF status in HED patients has not been studied at all. Studies reported herein investigated the hypothesis that the EGF signaling pathway is involved in HED/Ta. Fibroblasts from HED patients had a two- to eightfold decrease in binding capacity for (125)I-labeled EGF, a decreased expression of the immunoreactive 170-kD EGF receptor (EGFR) protein, and a corresponding reduction in EGFR mRNA. Reduced expression of the EGFR also was observed in Ta fibroblasts and liver membranes. Other aspects of the EGF signaling pathway, including EGF concentration in urine and plasma, were normal in both HED patients and Ta mice. We propose that a decreased expression of the EGFR plays a causal role in the HED/Ta phenotype.