CACNA1H Mutations in Autism Spectrum Disorders

Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by impaired social interaction, communication skills, and restricted and repetitive behavior. The genetic causes for autism are largely unknown. Previous studies implicate CACNA1C (L-type CaV1.2) calcium channel mutations in a disorder associated with autism (Timothy syndrome). Here, we identify missense mutations in the calcium channel gene CACNA1H (T-type CaV3.2) in 6 of 461 individuals with ASD. These mutations are located in conserved and functionally relevant domains and are absent in 480 ethnically matched controls (p = 0.014, Fisher's exact test). Non-segregation within the pedigrees between the mutations and the ASD phenotype clearly suggest that the mutations alone are not responsible for the condition. However, functional analysis shows that all these mutations significantly reduce CaV3.2 channel activity and thus could affect neuronal function and potentially brain development. We conclude that the identified mutations could contribute to the development of the ASD phenotype. Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by impaired social interaction, communication skills, and restricted and repetitive behavior. The genetic causes for autism are largely unknown. Previous studies implicate CACNA1C (L-type CaV1.2) calcium channel mutations in a disorder associated with autism (Timothy syndrome). Here, we identify missense mutations in the calcium channel gene CACNA1H (T-type CaV3.2) in 6 of 461 individuals with ASD. These mutations are located in conserved and functionally relevant domains and are absent in 480 ethnically matched controls (p = 0.014, Fisher's exact test). Non-segregation within the pedigrees between the mutations and the ASD phenotype clearly suggest that the mutations alone are not responsible for the condition. However, functional analysis shows that all these mutations significantly reduce CaV3.2 channel activity and thus could affect neuronal function and potentially brain development. We conclude that the identified mutations could contribute to the development of the ASD phenotype. Autism spectrum disorders affect ∼0.5% of children in the general population and cause great morbidity (1Bryson S.E. Rogers S.J. Fombonne E. Can. J. Psychiatry. 2003; 48: 506-516Crossref PubMed Scopus (169) Google Scholar, 2Volkmar F.R. Pauls D. Lancet. 2003; 362: 1133-1141Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Epidemiologic studies estimate that up to 400,000 children are affected in the United States alone (3Fombonne E. J. Autism Dev. Disord. 2003; 33: 365-382Crossref PubMed Scopus (1043) Google Scholar). The primary features of ASD are severe difficulties in social interaction, communication deficits, and unusual behaviors, including repetitive and/or ritualistic actions. There is considerable variation in the severity of phenotypes in autism spectrum disorders, which include autism, Asperger syndrome, childhood disintegrative disorder, Rett syndrome, and pervasive developmental disorder not otherwise specified. Despite the high prevalence and importance of autism spectrum disorders, very little is known about underlying molecular and cellular mechanisms (4Zoghbi H.Y. Science. 2003; 302: 826-830Crossref PubMed Scopus (538) Google Scholar). Timothy syndrome (TS) 3The abbreviations used are: TS, Timothy syndrome; WT, wild type; ANOVA, analysis of variance.3The abbreviations used are: TS, Timothy syndrome; WT, wild type; ANOVA, analysis of variance. is a complex physiological and developmental disorder, which includes autism spectrum disorders. We discovered that TS resulted from a recurrent, de novo CACNA1C calcium (Ca2+) channel mutation, G406R. Our findings that individuals with TS met the criteria for autism, or had severe deficits of language and social development, suggest that abnormal Ca2+ signaling may cause these disorders (5Splawski I. Timothy K.W. Sharpe L.M. Decher N. Kumar P. Bloise R. Napolitano C. Schwartz P.J. Joseph R.M. Condouris K. Tager-Flusberg H. Priori S.G. Sanguinetti M.C. Keating M.T. Cell. 2004; 119: 19-31Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar). Based on these results we hypothesized that mutations in other Ca2+ channel genes might be responsible for non-syndromic forms of ASD. Neuroanatomical studies of autistic patients have found histological abnormalities in the major regions of the limbic system including the hippocampus and amygdala and in the cerebellum and cerebral cortex (6Sadamatsu M. Kanai H. Xu X. Liu Y. Kato N. Congenit. Anom. (Kyoto). 2006; 46: 1-9Crossref PubMed Scopus (54) Google Scholar). CaV3.2, a T-type calcium channel encoded by the CACNA1H gene, is abundantly expressed in these and other regions. T-type Ca2+ channels activate with relatively small depolarization of the neuron membrane triggering low threshold spikes that contribute to rebound burst firing and oscillatory behavior in central neurons (7Perez-Reyes E. Physiol. Rev. 2003; 83: 117-161Crossref PubMed Scopus (1325) Google Scholar). In the thalamus, this behavior maintains normal transitions in sensory gating, sleep, and arousal (8McCormick D.A. Bal T. Annu. Rev. Neurosci. 1997; 20: 185-215Crossref PubMed Scopus (997) Google Scholar, 9Joksovic P. Nelson M. Jevtovic-Todorovic V. Patel M. Perez-Reyes E. Campbell K. Chen C.C. Todorovic S. J. Physiol. 2006; (in press)PubMed Google Scholar). Abnormal CaV3.2 activity, however, has been implicated in childhood absence epilepsy (10Khosravani H. Altier C. Simms B. Hamming K.S. Snutch T.P. Mezeyova J. McRory J.E. Zamponi G.W. J. Biol. Chem. 2004; 279: 9681-9684Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 11Khosravani H. Bladen C. Parker D.B. Snutch T.P. McRory J.E. Zamponi G.W. Ann. Neurol. 2005; 57: 745-749Crossref PubMed Scopus (97) Google Scholar, 12Vitko I. Chen Y. Arias J.M. Shen Y. Wu X.R. Perez-Reyes E. J. Neurosci. 2005; 25: 4844-4855Crossref PubMed Scopus (156) Google Scholar). Here we identify missense mutations in the calcium channel gene CACNA1H (T-type CaV3.2) of six families affected by ASD. 461 individuals with ASD were screened for both mutations in the CACNA1H gene and the specific G406R mutation in the CACNA1C gene we had previously found associated with TS. Missense mutations were only found in the CACNA1H gene and were absent in 480 ethnically matched individuals unaffected by ASD. We show that heterologous expression of the mutated T-type channels functionally alters several biophysical properties of human CaV3.2 including current density and voltage-dependent gating properties. Within the complex circuitry of the neuronal system these mutated CaV3.2 channels may contribute to the development of the ASD phenotype. Subjects—Our samples included 461 unrelated probands from families with ASD from the publicly available data base supported by the National Institute of Mental Health, which consists of DNA materials and genotypic and phenotypic data. Each family was ascertained on the condition that at least two individuals were diagnosed with ASD (13Geschwind D.H. Sowinski J. Lord C. Iversen P. Shestack J. Jones P. Ducat L. Spence S.J. Am. J. Hum. Genet. 2001; 69: 463-466Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Diagnostic tests included autism diagnostic interview-revised (14Lord C. Rutter M. Le Couteur A. J. Autism Dev. Disord. 1994; 24: 659-685Crossref PubMed Scopus (6724) Google Scholar) and the autism diagnostic observational schedule (15Lord C. Risi S. Lambrecht L. Cook Jr., E.H. Leventhal B.L. DiLavore P.C. Pickles A. Rutter M. J. Autism Dev. Disord. 2000; 30: 205-223Crossref PubMed Scopus (5523) Google Scholar). Our control group consisted of 480 ethnically matched individuals. Informed consent or assent was obtained from all individuals or their guardians according to standards established by local institutional review boards. Genotypic and DNA Sequence Analyses—Oligonucleotides (see supplemental Table 1) to all known exons of the CACNA1H gene were designed to genomic sequences found in the Celera data base using Oligo 6.6 (Molecular Biology Insights). PCR amplification of DNA samples and mutational analyses were carried out as described previously (16Splawski I. Tristani-Firouzi M. Lehmann M.H. Sanguinetti M.C. Keating M.T. Nat. Genet. 1997; 17: 338-340Crossref PubMed Scopus (671) Google Scholar). PCR fragments were purified using a QIAquick PCR purification kit (Qiagen), and sequencing was performed with an ABI 3700 automated DNA sequencer. Oligonucleotide sequences and PCR conditions are included in the supplemental information. Statistical Analysis—Fisher's exact test was used to determine the p value for the association of ASD and CACNA1H mutations. Northern Blot—Blot analyses were performed using human brain II and V Northern blots (Clontech). An ∼1060 base pair (AvrII/PshA1) fragment from the C-terminal end of CaV3.2 was used as a probe. The fragment was labeled with the Prime-It II labeling kit (Stratagene). Hybridization and washing conditions followed the manufacturer's suggestions. The blots were exposed to film for 3 days. Cell Culture and Transfection—Transformed human embryonic kidney-293 (HEK-293T) cells were grown in Dulbecco's modified Eagle's medium + F-12 supplemented with 10% fetal calf serum and 1% penicillin/streptomycin at 5% CO2 and 37 °C. Cells grown to 90% confluence in 35-mm Petri dishes were transiently transfected with plasmid DNAs encoding each Cav3.2 construct (4 μg) and green fluorescent protein (0.1 μg) using Lipofectamine 2000 (Invitrogen). The WT human CaV3.2 cDNA (Genbank™ accession number AF051946) was a kind gift from E. Perez-Reyes (University of Virginia, Charlottesville, VA). 24-36 h after transfection green fluorescent protein-positive cells expressed sufficient levels of the Ca2+ channels to proceed with electrophysiological recordings. Cells were split via trypsin EDTA and plated on glass coverslips at 5-10% confluence and given 2-3 h to settle and attach. Site-directed Mutagenesis—Mutations were introduced to the WT human CaV3.2 cDNA using the QuikChange site-directed mutagenesis kit (Stratagene). Oligonucleotide sequences are detailed in the supplemental materials. The presence of the desired mutations and absence of all other changes were verified by sequencing the entire CaV3.2 cDNA (Molecular Genetics Core, Children's Hospital, Boston and Harvard Medical School). To ensure against modifications that may have occurred in the pcDNA3 vector (Invitrogen) during mutagenesis unique restriction sites were used to place fragments containing the CaV3.2 mutations into an identically digested WT construct that had not been subject to PCR. Electrophysiology—Electrophysiological experiments were performed using the whole-cell configuration of the patch-clamp technique. Recordings were obtained using an Axo-patch 200B amplifier, Digidata 1322A analog-to-digital converter, and pClamp 8.01 software (Molecular Devices, Union City, CA). Data were filtered at 2 kHz and digitized at 5 kHz. Modified Ringer's solution with low chloride contained (in mm): 140 sodium gluconate, 5 KCl, 1 MgCl2, 2 CaCl2, 20 HEPES, and 10 glucose (pH 7.4; 310 mosm). The internal pipette solution contained (in mm): 120 cesium methanesulfonate, 10 EGTA, 8 NaCl, 2 Mg-ATP, and 20 HEPES (pH 7.4; 290 mosm). Borosilicate glass pipettes (World Precision Instruments, Sarasota, FL) were pulled with a DMZ-Universal puller (Dagan Corp., Minneapolis, MN) to a typical pipette resistance of 3 mΩ after fire polishing. The pipettes were parafilm-wrapped prior to patching. Cell capacitance was measured for each cell and access resistance compensated to 80%. Data Acquisition and Analysis—The current-voltage protocol stepped the cell membrane potential from −100 mV to test potentials starting at −90 and increasing to +20 mV in 10-mV increments. Test potentials were 150 ms in duration, and the membrane potential was returned to −100 mV for 10 s between acquisitions to allow complete recovery from inactivation. Peak Ca2+ were as a function of the test potential to current-voltage The were by the cell capacitance for the of current and were with a modified of the = + and is the is the is the and is the that is to the gating of the and of the Ca2+ obtained during the protocol the and were The was from of obtained during the current a to mV was given to the channels the membrane potential of the cell to test between and To the of the cell membrane was stepped from a potential of −100 mV to potentials 1 s in between and mV in 10-mV to a test potential of mV for 150 from which the Ca2+ were The of was from of = + the protocol the membrane of the cell was stepped to a of mV for 1 s from mV followed by a test depolarization of mV for 150 Peak obtained the depolarization were to obtained in the absence of the to determine the was used to all data obtained in (Molecular of the and were carried out in Data are as the S.E. Statistical tests included both test and analysis of were in and p are to the number of samples between the the data to tests for the analysis a of Here, we mutations in a Ca2+ channel gene expressed in brain in samples from individuals with ASD. We screened all exons of CACNA1H in 461 individuals with ASD. missense mutations were identified in conserved domains of 5 a in DNA samples in an affected from to the of by at is conserved in from to and is located at the end of the of In an affected from had a which resulted in an to in the of II and is conserved of to in the of a conserved to in an affected of is located in the of II unrelated individuals with autism from and carried an to at mutation a conserved with located the of In all the mutation was on the as an to at The for the autistic samples was was the was individuals with autism the was not (p = The of the and were all The low of these mutations within the affected population may that the changes However, the mutations were not found in 480 control ethnically matched and the Fisher's exact test an association between the CACNA1H mutations and ASD (p = identified in individuals with ASD the properties of Cav3.2 of CACNA1H the of each in current the the current for WT was S.E. = for S.E. = for S.E. = and for S.E. = not not significantly from WT S.E. = The 20 The current In of the six an affected not a CACNA1H mutation was of the pedigrees shows that and several were not carried the described mutations. these of the mutations could be in of other genes in the affected both and of the CACNA1H gene mutation, could be to the phenotype. phenotypic of the normal and was not in the National Institute of Mental of these individuals may be is that the identified mutations are not major to the ASD phenotype in these families only the phenotypic To determine the molecular of the CACNA1H mutations on channel we expressed WT and forms of the CaV3.2 channel in The biophysical properties of the channels were characterized by whole-cell Each of the and the channels that current WT channels analysis the was not for Table 1 Previous of the G406R mutation associated with TS in current density with WT (5Splawski I. Timothy K.W. Sharpe L.M. Decher N. Kumar P. Bloise R. Napolitano C. Schwartz P.J. Joseph R.M. Condouris K. Tager-Flusberg H. Priori S.G. Sanguinetti M.C. Keating M.T. Cell. 2004; 119: 19-31Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar, I. Timothy K.W. Decher N. Kumar P. Sanguinetti M.C. Keating M.T. S. A. 2005; PubMed Scopus Google Scholar). To functional and 1) were To determine the of the mutations on channel we the of all of the mutations were were to activate these the the at which of channel population was was by mV and WT, with in at several and These data that the ASD mutations reduce available Ca2+ current and thus reduce neuronal mutations associated with ASD activate at significantly potentials and/or have a were with a modified and the results Data are as S.E. 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In of the with the are (p = were by test are with a (p are were by (p were by (p (p = were by test are with a (p are (p = were by test are with a (p are were by test are with a (p are were by (p in a Ca2+ channels the membrane potential is during these channels WT channels Despite the in the in for and and Ca2+ that in WT of the including the from WT in their of recovery from or properties Table The had biophysical properties to the for all Our was to determine mutations in Ca2+ channel genes could contribute to the of non-syndromic ASD in individuals. CACNA1H the T-type Ca2+ channel CaV3.2, which Ca2+ and neuronal firing (7Perez-Reyes E. Physiol. Rev. 2003; 83: 117-161Crossref PubMed Scopus (1325) Google Scholar, J. A. C. S. Perez-Reyes E. J. P. 2000; PubMed Scopus Google Scholar). CACNA1H is expressed in regions of the brain with expression in and A. Perez-Reyes E. D.A. J. Neurosci. PubMed Google previously associated with ASD P. A. J. 2003; PubMed Scopus Google Scholar). We discovered in conserved of CaV3.2 in six of 461 individuals with ASD. These mutations the functional properties of the with Ca2+ channel mutations in a of autism I. Timothy K.W. Decher N. Kumar P. Sanguinetti M.C. Keating M.T. S. A. 2005; PubMed Scopus Google data suggest that Ca2+ channel be as a potential in the development of ASD. The mutations associated with ASD are all in regions of the protein that be to T-type calcium channel of and the that were to voltage-dependent current may that these CACNA1H mutations of the channels the number of functional channels on the the CaV3.2 mutations may reduce the channel or the not be for a mutation to the or of a protein to and thus is a to in in the CaV3.2 channel to for be using current of voltage-dependent channel gating in which in the of the channel in to a in the B. of Scholar). to the gating Campbell R. Science. 2005; PubMed Scopus Google Scholar). in the domains of by or thus the channel are to the gating The of both mutations to the membrane may the of was not in the obtained in the in the potential of was for the mutation and to a in The was to of which in WT CaV3.2 channels with the neuronal membrane the and may changes to and at a are to calcium sufficient depolarization inactivation. The mutation, not affect by this channel protein were that may be expressed in in Table the biophysical of the mutated CaV3.2 channels and the on neuronal calcium is to the these changes might have on development and neuronal the expression of CaV3.2 in the brain might the these mutations may to the phenotype of ASD. a of these CACNA1H mutations we may an of the these channel in a neuronal an association between brain and autism has been (6Sadamatsu M. Kanai H. Xu X. Liu Y. Kato N. Congenit. Anom. (Kyoto). 2006; 46: 1-9Crossref PubMed Scopus (54) Google may be that within the system identified ASD may cell or associated with ASD are characterized by properties to in a in neuronal on Ca2+ current Ca2+ in Ca2+ of Ca2+ of Ca2+ current Ca2+ in Ca2+ in Ca2+ of Ca2+ of Ca2+ of Ca2+ current Ca2+ in Ca2+ in Ca2+ of Ca2+ of Ca2+ in a of functional analysis was within the of a of the CaV3.2 sites were identified within the CACNA1H gene, several of which the and voltage-dependent gating of the channel X. Liu D.A. Hum. Genet. 2006; PubMed Scopus Google Scholar). of the described in However, be to determine the phenotype of the identified CaV3.2 mutations is or within as control is to a complex in the development of the on the expression and of the changes by mutated CaV3.2 channels may in regions of the is that the identified CACNA1H mutations are not major to the ASD phenotype the phenotypic of the affected individuals we CACNA1H and of the six families contained an affected not a CACNA1H is that is as and several were not carried the described mutations. However, phenotypic was not or was not available in the normal and not be the of a genetic were as studies have that ASD is of R. I. 2004; PubMed Scopus Google Scholar). is that other genetic and contribute to ASD. in other regions of the CACNA1H gene have been identified in childhood absence epilepsy (10Khosravani H. Altier C. Simms B. Hamming K.S. Snutch T.P. Mezeyova J. McRory J.E. Zamponi G.W. J. Biol. Chem. 2004; 279: 9681-9684Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 11Khosravani H. Bladen C. Parker D.B. Snutch T.P. McRory J.E. Zamponi G.W. Ann. Neurol. 2005; 57: 745-749Crossref PubMed Scopus (97) Google Scholar, 12Vitko I. Chen Y. Arias J.M. Shen Y. Wu X.R. Perez-Reyes E. J. Neurosci. 2005; 25: 4844-4855Crossref PubMed Scopus (156) Google Scholar, Y. J. H. Y. Wu H. Xu K. Liu X. Y. X. K. B. P. Shen Y. Wu X. Ann. Neurol. 2003; PubMed Scopus Google Scholar). and ASD have been in a of individuals with ASD R. I. 2004; PubMed Scopus Google Scholar). mutation, which was previously in an with was identified in a family with autism A. M. M. J. M.H. Psychiatry. 2003; PubMed Scopus Google Scholar). studies determine mutations and in Ca2+ channels contribute to the of ASD. We the families have in and to these studies (see supplemental with

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