Produção Nacional
Revisado por pares
Recessive and Dominant De Novo ITPR1 Mutations Cause Gillespie Syndrome
2016; Elsevier BV; Volume: 98; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2016.03.004
ISSN1537-6605
AutoresS. Gerber, Kamil J. Alzayady, Lydie Bürglen, Dominique Brémond‐Gignac, Valentina Marchesin, Olivier Roche, Marlène Rio, Benoît Funalot, Raphaël Calmon, Alexandra Dürr, Vera Lúcia Gil‐da‐Silva‐Lopes, Maria Fernanda Ribeiro Bittar, Christophe Orssaud, Bénédicte Héron, Edward Ayoub, Patrick Berquin, Nadia Bahi‐Buisson, Christine Bole, Cécile Masson, Arnold Münnich, Matias Simons, Marion Delous, Hélène Dollfus, Nathalie Boddaert, Stanislas Lyonnet, Josseline Kaplan, Patrick Calvas, David I. Yule, Jean‐Michel Rozet, Lucas Fares‐Taie,
Tópico(s)Cellular transport and secretion
ResumoGillespie syndrome (GS) is a rare variant form of aniridia characterized by non-progressive cerebellar ataxia, intellectual disability, and iris hypoplasia. Unlike the more common dominant and sporadic forms of aniridia, there has been no significant association with PAX6 mutations in individuals with GS and the mode of inheritance of the disease had long been regarded as uncertain. Using a combination of trio-based whole-exome sequencing and Sanger sequencing in five simplex GS-affected families, we found homozygous or compound heterozygous truncating mutations (c.4672C>T [p.Gln1558∗], c.2182C>T [p.Arg728∗], c.6366+3A>T [p.Gly2102Valfs5∗], and c.6664+5G>T [p.Ala2221Valfs23∗]) and de novo heterozygous mutations (c.7687_7689del [p.Lys2563del] and c.7659T>G [p.Phe2553Leu]) in the inositol 1,4,5-trisphosphate receptor type 1 gene (ITPR1). ITPR1 encodes one of the three members of the IP3-receptors family that form Ca2+ release channels localized predominantly in membranes of endoplasmic reticulum Ca2+ stores. The truncation mutants, which encompass the IP3-binding domain and varying lengths of the modulatory domain, did not form functional channels when produced in a heterologous cell system. Furthermore, ITPR1 p.Lys2563del mutant did not form IP3-induced Ca2+ channels but exerted a negative effect when co-produced with wild-type ITPR1 channel activity. In total, these results demonstrate biallelic and monoallelic ITPR1 mutations as the underlying genetic defects for Gillespie syndrome, further extending the spectrum of ITPR1-related diseases. Gillespie syndrome (GS) is a rare variant form of aniridia characterized by non-progressive cerebellar ataxia, intellectual disability, and iris hypoplasia. Unlike the more common dominant and sporadic forms of aniridia, there has been no significant association with PAX6 mutations in individuals with GS and the mode of inheritance of the disease had long been regarded as uncertain. Using a combination of trio-based whole-exome sequencing and Sanger sequencing in five simplex GS-affected families, we found homozygous or compound heterozygous truncating mutations (c.4672C>T [p.Gln1558∗], c.2182C>T [p.Arg728∗], c.6366+3A>T [p.Gly2102Valfs5∗], and c.6664+5G>T [p.Ala2221Valfs23∗]) and de novo heterozygous mutations (c.7687_7689del [p.Lys2563del] and c.7659T>G [p.Phe2553Leu]) in the inositol 1,4,5-trisphosphate receptor type 1 gene (ITPR1). ITPR1 encodes one of the three members of the IP3-receptors family that form Ca2+ release channels localized predominantly in membranes of endoplasmic reticulum Ca2+ stores. The truncation mutants, which encompass the IP3-binding domain and varying lengths of the modulatory domain, did not form functional channels when produced in a heterologous cell system. Furthermore, ITPR1 p.Lys2563del mutant did not form IP3-induced Ca2+ channels but exerted a negative effect when co-produced with wild-type ITPR1 channel activity. In total, these results demonstrate biallelic and monoallelic ITPR1 mutations as the underlying genetic defects for Gillespie syndrome, further extending the spectrum of ITPR1-related diseases. Aniridia (MIM: 106210) refers to a rare inborn error of eye development whose most prominent sign is the partial or complete absence of iris. Associated ocular malformations include macular and optic nerve hypoplasia, cataract, and corneal opacification. Most aniridia cases are accounted for by dominant mutations affecting PAX6 (MIM: 607108) at the AN locus (MIM: 106210).1Hingorani M. Hanson I. van Heyningen V. Aniridia.Eur. J. Hum. Genet. 2012; 20: 1011-1017Crossref PubMed Scopus (154) Google Scholar Wilms tumor, genitourinary abnormalities, and intellectual disability are not uncommon in individuals with aniridia. This complex form of the disease known as WAGR syndrome (MIM: 194072) has been ascribed to 11q13 deletions involving the PAX6 locus and the adjacent Wilms tumor 1 (WT1) locus (MIM: 607102).1Hingorani M. Hanson I. van Heyningen V. Aniridia.Eur. J. Hum. Genet. 2012; 20: 1011-1017Crossref PubMed Scopus (154) Google Scholar Occasionally, aniridia can be associated with cerebellar ataxia and intellectual disability, defining a clinical entity known as Gillespie syndrome (GS [MIM: 206700]).2Gillespie F.D. Aniridia, cerebellar ataxia, and oligophrenia in siblings.Arch. Ophthalmol. 1965; 73: 338-341Crossref PubMed Scopus (74) Google Scholar GS is usually diagnosed in the first year of life by the presence of fixed dilated pupils in a hypotonic infant. Ophthalmologic examination reveals partial aniridia with the pupil border of the iris containing festooned edge and iris strands extending onto the anterior lens surface at regular intervals.3Nelson J. Flaherty M. Grattan-Smith P. Gillespie syndrome: a report of two further cases.Am. J. Med. Genet. 1997; 71: 134-138Crossref PubMed Scopus (25) Google Scholar These eye findings are mandatory to make the diagnosis of GS because they demonstrate the developmental nature of the iris anomaly, thereby excluding neurogenic congenital mydriasis in an infant with neuropsychomotor symptoms. Since Gillespie's initial description in 1965, 22 affected families have been reported.2Gillespie F.D. Aniridia, cerebellar ataxia, and oligophrenia in siblings.Arch. Ophthalmol. 1965; 73: 338-341Crossref PubMed Scopus (74) Google Scholar, 3Nelson J. Flaherty M. Grattan-Smith P. Gillespie syndrome: a report of two further cases.Am. J. Med. 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Essoussi A.S. [Gillespie syndrome: 2 familial cases].Arch. Pediatr. 2006; 13: 1323-1325Crossref PubMed Scopus (6) Google Scholar, 13Ticho B.H. Hilchie-Schmidt C. Egel R.T. Traboulsi E.I. Howarth R.J. Robinson D. Ocular findings in Gillespie-like syndrome: association with a new PAX6 mutation.Ophthalmic Genet. 2006; 27: 145-149Crossref PubMed Scopus (35) Google Scholar, 14Luquetti D.V. Oliveira-Sobrinho R.P. Gil-da-Silva-Lopes V.L. Gillespie syndrome: additional findings and parental consanguinity.Ophthalmic Genet. 2007; 28: 89-93Crossref PubMed Scopus (9) Google Scholar, 15Graziano C. D'Elia A.V. Mazzanti L. Moscano F. Guidelli Guidi S. Scarano E. Turchetti D. Franzoni E. Romeo G. Damante G. Seri M. A de novo nonsense mutation of PAX6 gene in a patient with aniridia, ataxia, and mental retardation.Am. J. Med. Genet. A. 2007; 143A: 1802-1805Crossref PubMed Scopus (27) Google Scholar, 16Mariën P. Brouns R. Engelborghs S. Wackenier P. Verhoeven J. Ceulemans B. De Deyn P.P. Cerebellar cognitive affective syndrome without global mental retardation in two relatives with Gillespie syndrome.Cortex. 2008; 44: 54-67Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 17Edén U. Beijar C. Riise R. Tornqvist K. Aniridia among children and teenagers in Sweden and Norway.Acta Ophthalmol. 2008; 86: 730-734Crossref PubMed Scopus (25) Google Scholar, 18Agarwal P.K. Awan M.A. Strang N. Dutton G.N. Gillespie syndrome with impaired accommodation.J. Pediatr. Ophthalmol. Strabismus. 2009; 46: 317Crossref PubMed Scopus (1) Google Scholar, 19Dell'acqua Cassão B. de Rezende D.T. Silva L.C. Herbella F.A. Esophageal dysmotility in gillespie syndrome.J. Neurogastroenterol. Motil. 2013; 19: 538-539Crossref PubMed Scopus (3) Google Scholar Current GS-affected families include three sibpairs describing an autosomal-recessive inheritance, two families with disease transmission from an affected mother to the progeny, and 18 simplex families in which recessive and dominant de novo mutations might underlie the disease. De novo PAX6 mutations have been identified in two simplex families but otherwise molecular studies have failed to identify the disease-causing mutation, supporting genetic heterogeneity of GS.9Glaser T. Ton C.C. Mueller R. Petzl-Erler M.L. Oliver C. Nevin N.C. Housman D.E. Maas R.L. Absence of PAX6 gene mutations in Gillespie syndrome (partial aniridia, cerebellar ataxia, and mental retardation).Genomics. 1994; 19: 145-148Crossref PubMed Scopus (51) Google Scholar, 13Ticho B.H. Hilchie-Schmidt C. Egel R.T. Traboulsi E.I. Howarth R.J. Robinson D. Ocular findings in Gillespie-like syndrome: association with a new PAX6 mutation.Ophthalmic Genet. 2006; 27: 145-149Crossref PubMed Scopus (35) Google Scholar, 15Graziano C. D'Elia A.V. Mazzanti L. Moscano F. Guidelli Guidi S. Scarano E. Turchetti D. Franzoni E. Romeo G. Damante G. Seri M. A de novo nonsense mutation of PAX6 gene in a patient with aniridia, ataxia, and mental retardation.Am. J. Med. Genet. A. 2007; 143A: 1802-1805Crossref PubMed Scopus (27) Google Scholar We collected five trios with index case subjects presenting with partial aniridia, cerebellar ataxia, and intellectual disability with or without additional systemic symptoms (Table 1 and Figures 1A and 2). Informed consents were obtained from all the research participants. The study received approval from the Comité de Protection des Personnes "Ile-de-France II." Three index case subjects (F1:II1, F2:II1, F5:II1), one of which was reported previously (F2:II1),14Luquetti D.V. Oliveira-Sobrinho R.P. Gil-da-Silva-Lopes V.L. Gillespie syndrome: additional findings and parental consanguinity.Ophthalmic Genet. 2007; 28: 89-93Crossref PubMed Scopus (9) Google Scholar were born to consanguineous parents (Figure 1A). F1:II1, F3:II1, F4:II1, and F5:II1 had neither point mutations nor micro-rearrangements affecting PAX6 as determined by Sanger sequencing of the 5′ regulatory elements,20Bhatia S. Bengani H. Fish M. Brown A. Divizia M.T. de Marco R. Damante G. Grainger R. van Heyningen V. Kleinjan D.A. Disruption of autoregulatory feedback by a mutation in a remote, ultraconserved PAX6 enhancer causes aniridia.Am. J. Hum. Genet. 2013; 93: 1126-1134Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar the exons and the intron-exon boundaries, nor deletions in, or around, PAX6 as determined by array comparative genomic hybridization and multiplex ligation-dependent probe amplification (MLPA; SALSA MLPA kit P219 PAX6, In vitrotek), respectively.Table 1Clinical Features of the Individuals Affected with Gillespie SyndromeF1:II1F2:II114Luquetti D.V. Oliveira-Sobrinho R.P. Gil-da-Silva-Lopes V.L. Gillespie syndrome: additional findings and parental consanguinity.Ophthalmic Genet. 2007; 28: 89-93Crossref PubMed Scopus (9) Google ScholarF3:II1F4:II1F5:II1Age (years)4.5167.5181.5GenderFFFFFFamily historynononononoConsanguinityyesyesnonoyesPrenatal historypersistent truncus arteriosusnonononoOcular FeaturesIris presentationbilateral partial aniridiabilateral partial aniridiabilateral partial aniridiabilateral partial aniridiabilateral partial aniridiaFundus aspectunremarkableunremarkableunremarkableunremarkableunremarkableOCTunremarkableunknownunknownunknownunknownNeurologic FeaturesAge at onsetbirth3 monthsbirthbirthbirthInitial symptomhypotonia with iris hypoplasiahypotonia with iris hypoplasiahypotonia with iris hypoplasiahypotonia with iris hypoplasiahypotonia with iris hypoplasiaCerebellar SignsNystagmusYesyesyesyesyesAtaxiayesyesyesyesyesPostural tremoryesunknownyesnounknownSlurred speechnot evaluableyesyesyesunknownGeneral hypotoniayesyesyesyesyesPyramidal signnounknownnononoExtrapyramidal signnounknownnonoyesPeripheral neuropathynounknownareflexia of lower limbsnonoEpileptic seizuresnonononotransitory myoclonus in the neonatal periodIntellectual disabilitysevere (uses two/three words)mild (phrases at 4 years)moderatenounevaluableMotor DevelopmentHead controldelayed12 months18 months18 months10 monthsSit without support3.5 years3 years18 months (with support)3 yearsnoStand with support4.5 yearsyesyes3 yearsnoWalk unassistednonono16 years (a few steps only)noMRI findingsunremarkable at 1.5 months; marked cerebellar atrophy, thin corpus callosum, discrete ventricular dilatation at 4.5 yearsreported unremarkable at ages 6 months and 8 yearsmoderate cerebellar atrophy at 2.5 years; marked cerebellar atrophy, at 8 yearsunremarkable at 6 months; cerebellar atrophy at 4 years (vermis > hemispheres)unremarkable at 3.5 months; marked cerebellar atrophy at 1.5 yearsOther findingshymeneal imperforationfacial dysmorphy–short 4th metatarsalmarked kyphosis, left pectoral agenesis Open table in a new tab Figure 2Brain MRI and Eye Findings in Individuals with Mutations in ITPR1Show full caption(A–J) MRIs were performed at 1.5 months and 4.5 years, 2.5 and 8 years, 3.5 months and 1.5 years, and 8 years of age in individuals F1:II1 (A–C), F3:II1 (D–F), F5:II1 (G–I), and F4:II1 (J) by means of the following modalities: sagittal T1 (A, B, D, E, G, H) or T2 (J) and axial T2 (C, F, I). In the first months of life, sagittal T1 images are unremarkable (A, G). The cerebellar atrophy appears with age as shown on sagittal T1 or T2 images by the moderate atrophy at age 1.5 years and 2.5 years (∗∗) (D, H) and the marked cerebellar atrophy at 4.5 and 8 years (∗∗) (B, D, J). In addition to cerebellar atrophy, individual F1:II1 presents with thin corpus callosum (∗) (B) and discrete ventricular dilatation (∗∗∗) (C).(K) Photograph of an eye of individual F5:II1 showing typical partial iris hypoplasia with iris strands extending onto the anterior lens surface.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A–J) MRIs were performed at 1.5 months and 4.5 years, 2.5 and 8 years, 3.5 months and 1.5 years, and 8 years of age in individuals F1:II1 (A–C), F3:II1 (D–F), F5:II1 (G–I), and F4:II1 (J) by means of the following modalities: sagittal T1 (A, B, D, E, G, H) or T2 (J) and axial T2 (C, F, I). In the first months of life, sagittal T1 images are unremarkable (A, G). The cerebellar atrophy appears with age as shown on sagittal T1 or T2 images by the moderate atrophy at age 1.5 years and 2.5 years (∗∗) (D, H) and the marked cerebellar atrophy at 4.5 and 8 years (∗∗) (B, D, J). In addition to cerebellar atrophy, individual F1:II1 presents with thin corpus callosum (∗) (B) and discrete ventricular dilatation (∗∗∗) (C). (K) Photograph of an eye of individual F5:II1 showing typical partial iris hypoplasia with iris strands extending onto the anterior lens surface. To identify the molecular defect underlying GS, we subjected the genomic DNA (1 μg) from an index case subject (F1:II1) and her consanguineous parents of Tunisian origin (F1:I1 and F1:I2) to exome resequencing, using the SureSelect Human All Exon kit version 5 (Agilent). Each genomic DNA fragment was analyzed on a sequencer using the paired-end strategy and an average read length of 75 bases (Illumina HISEQ, Illumina). Image analysis and base calling were performed with Real Time Analysis (RTA) Pipeline v.1.9 with default parameters (Illumina). Sequences were aligned to the human genome reference sequence (hg19 assembly) and SNPs were called based on allele calls and read depth using the CASAVA pipeline (Consensus Assessment of Sequence and Variation 1.8, Illumina). Genetic variation annotation was performed by an in-house pipeline. We first focused our analysis on consensus splice-site changes, non-synonymous variants, and insertion and/or deletion in coding regions. Considering the rarity of the disease and the uncertain mode of inheritance, we assumed that the affected individual was either single heterozygous for a variant absent in parental DNA (dominant de novo model) and in the Exome Aggregation Consortium (ExAC), 1000 Genomes, dbSNP132, and Imagine déjà-vu databases, or homozygous for a variant found in the heterozygote state in parental DNA and absent or with a minor allele frequency < 0.001 in databases (recessive model). This filtering strategy pointed to one de novo and three homozygous variants (Tables S1 and S2). The de novo change occurred in KLK9 (c.88C>T [p.Arg30Cys]; GenBank: NM_012315.1), which encodes the kallikrein-related peptidase 9 (MIM: 605504). This variant was predicted to be deleterious both by PolyPhen-2 and SIFT softwares (Table S2), but its frequency in the ExAC database (7.5:10,000) was significantly higher than that of Gillespie syndrome, making unlikely its causality in the disease. Two out of the three homozygote variants were predicted to be benign according to PolyPhen-2 and SIFT and were filtered out (Table S2). The unique remaining candidate variant consisted of a nonsense mutation (c.4672C>T [p.Gln1558∗]) in ITPR1 (GenBank: NM_001099952.2), which encodes the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), type 1 also known as IP3R1 (UniProt: Q14643; MIM: 147265) (Tables 2 and S2). Sanger sequencing confirmed homozygosity and single heterozygosity of the change in the affected child and her parents, respectively (Figure 1A).Table 2ITPR1 Mutations Identified in the Individuals Affected with Gillespie SyndromePedigreeParental OriginAllele 1Allele 2Mutation (origin)Predicted EffectMutation (origin)Predicted EffectFamily 1S, CMoroccoc.4672C>T (paternal)p.Gln1558∗c.4672C>T (maternal)p.Gln1558∗Family 2S, CBrazilc.2182C>T (paternal)p.Arg728∗c.2182C>T (maternal)p.Arg728∗Family 3S, NCFrancec.6366+3A>T (paternal)p.Gly2102Valfs5∗1Predicted from mRNA analysis. Nucleotide positions according to the reference sequence GenBank: NM_001099952.2. The p.Phe2553Leu changes was predicted to be deleterious according to PolyPhen-2, SIFT, and Mutation Taster available through the Alamut Interpretation Software 2.0.c.6664+5G>T (maternal)p.Ala2221Valfs23∗1Predicted from mRNA analysis. Nucleotide positions according to the reference sequence GenBank: NM_001099952.2. The p.Phe2553Leu changes was predicted to be deleterious according to PolyPhen-2, SIFT, and Mutation Taster available through the Alamut Interpretation Software 2.0.Family 4S, NCFrancec.7687_7689del (de novo)p.Lys2563del––Family 5S, NCFrance2French Caribbean Island La Guadeloupe.c.7659T>G (de novo)p.Phe2553Leu––Abbreviations are as follows: S, simplex family; C, consanguineous; NC, nonconsanguineous.1 Predicted from mRNA analysis. Nucleotide positions according to the reference sequence GenBank: NM_001099952.2. The p.Phe2553Leu changes was predicted to be deleterious according to PolyPhen-2, SIFT, and Mutation Taster available through the Alamut Interpretation Software 2.0.2 French Caribbean Island La Guadeloupe. Open table in a new tab Abbreviations are as follows: S, simplex family; C, consanguineous; NC, nonconsanguineous. Three major ITPR1 mRNA isoforms are reported which in total represents 62 unique exons.21Nucifora Jr., F.C. Li S.H. Danoff S. Ullrich A. Ross C.A. Molecular cloning of a cDNA for the human inositol 1,4,5-trisphosphate receptor type 1, and the identification of a third alternatively spliced variant.Brain Res. Mol. Brain Res. 1995; 32: 291-296Crossref PubMed Scopus (46) Google Scholar Variant 1 (GenBank: NM_001099952.2) includes 59 exons, 57 of which are coding. Compared to variant 1, variant 2 (GenBank: NM_0022225) lacks exon 12 whereas isoform 3 (GenBank: NM_001168272.1) lacks exon 12, uses an alternate in-frame splice site in intron 23, and includes three additional exons between variant 1 exons 39 and 40. Sanger sequencing of the 62 ITPR1 exons and intron-exon boundaries (Table S3) detected five additional unique ITPR1 mutations in the other four index case subjects (Table 2 and Figure 1A). F2:II1, born to consanguineous parents from Brazil,14Luquetti D.V. Oliveira-Sobrinho R.P. Gil-da-Silva-Lopes V.L. Gillespie syndrome: additional findings and parental consanguinity.Ophthalmic Genet. 2007; 28: 89-93Crossref PubMed Scopus (9) Google Scholar carried another homozygous nonsense mutation (c.2182C>T [p.Arg728∗]) and F3:II1, born to non-consanguineous parents from mainland France, had two splice-site mutations (c.6366+3A>T and c.6664+5G>T). Segregation analysis confirmed the biparental transmission in the two families (Table 2 and Figure 1A). F4:II1, born to non-consanguineous parents from mainland France, carried a 3-base pair (bp) deletion (c.7687_7689del [p.Lys2563del]) absent from parental DNA. (F4:II1 is the same individual as 1388_1388 who is described, with the same ITPR1 mutation, in the accompanying report by McEntagert et al.22McEntagart M. Williamson K.A. Rainger J.K. Wheeler A. Seawright A. De Baere E. Verdin H. Bergendahl L.T. Quigley A. Rainger J. et al.A restricted repertoire of de novo mutations in ITPR1 cause Gillespie syndrome with evidence for dominant-negative effect.Am. J. Hum. Genet. 2016; 98 (Published online April 21, 2016)https://doi.org/10.1016/j.ajhg.2016.03.018Abstract Full Text Full Text PDF Scopus (62) Google Scholar) Likewise, F5:II1, born to consanguineous parents from the French Caribbean island La Guadeloupe, carried a missense change (c.7659T>G [p.Phe2553Leu]) absent in parental DNA. All variant numbers refer to ITPR1 isoform 1 (GenBank: NM_001099952.2). The six unique ITPR1 mutations were absent from all databases and were predicted to be deleterious according to the Alamut Mutation Interpretation Software, a decision support system for mutation interpretation based on Align DGVD, PolyPhen-2, SIFT, SpliceSiteFinder-like, MaxEntScan, NNSPLICE, and Human Splicing Finder (Table 2). ITPR1, ITPR2 (MIM: 600144), and ITPR3 (MIM: 147267) encode the three subtypes of the IP3-receptor family (ITPR1, ITPR2, and ITPR3 are also known as IP3R1, IP3R2, and IP3R3, respectively) that form large (>1 MDa), homo- and heterotetrameric Ca2+ release channels localized predominantly in membranes of endoplasmic reticulum (ER) Ca2+ stores.23Foskett J.K. White C. Cheung K.H. Mak D.O. Inositol trisphosphate receptor Ca2+ release channels.Physiol. Rev. 2007; 87: 593-658Crossref PubMed Scopus (905) Google Scholar, 24Yule D.I. Betzenhauser M.J. Joseph S.K. Linking structure to function: Recent lessons from inositol 1,4,5-trisphosphate receptor mutagenesis.Cell Calcium. 2010; 47: 469-479Crossref PubMed Scopus (66) Google Scholar ITPR subtypes are ubiquitously expressed throughout tissues and all cell types express at least one subtype. Notably, the majority of cell types outside the central nervous system express multiple isoforms.25Fujino I. Yamada N. Miyawaki A. Hasegawa M. Furuichi T. Mikoshiba K. Differential expression of type 2 and type 3 inositol 1,4,5-trisphosphate receptor mRNAs in various mouse tissues: in situ hybridization study.Cell Tissue Res. 1995; 280: 201-210PubMed Google Scholar, 26Furuichi T. Simon-Chazottes D. Fujino I. Yamada N. Hasegawa M. Miyawaki A. Yoshikawa S. Guénet J.L. Mikoshiba K. Widespread expression of inositol 1,4,5-trisphosphate receptor type 1 gene (Insp3r1) in the mouse central nervous system.Receptors Channels. 1993; 1: 11-24PubMed Google Scholar, 27Wojcikiewicz R.J. Type I, II, and III inositol 1,4,5-trisphosphate receptors are unequally susceptible to down-regulation and are expressed in markedly different proportions in different cell types.J. Biol. Chem. 1995; 270: 11678-11683Crossref PubMed Scopus (373) Google Scholar Although the expression of various ITPR subtypes is overlapping, the expression of certain subtypes is more enriched in a particular tissue to meet the developmental and physiological needs of that tissue. For example, ITPR1 is specifically enriched in the nervous system whereas ITPR2 and ITPR3 are highly expressed in the digestive system. The structure of ITPR is generally divided into an N-terminal ligand-binding domain, a central transducing/modulatory domain, and a C-terminal channel domain23Foskett J.K. White C. Cheung K.H. Mak D.O. Inositol trisphosphate receptor Ca2+ release channels.Physiol. Rev. 2007; 87: 593-658Crossref PubMed Scopus (905) Google Scholar (Figure 3A). IP3, generated after phospholipase C-mediated hydrolysis of phosphatidyl-inositol 4,5-bisphosphate, engages IP3 receptors and results in Ca2+ release from ER stores.28Berridge M.J. Inositol trisphosphate and calcium signalling mechanisms.Biochim. Biophys. Acta. 2009; 1793: 933-940Crossref PubMed Scopus (577) Google Scholar Here we utilized biological samples obtained from GS-affected individuals and healthy control subjects to characterize ITPR1 mRNA levels and integrity. Further, we generated ITPR1 constructs corresponding to mutants identified in GS-affected individuals and assessed their activity in heterologous cell systems. The effect of the c.4672C>T nonsense mutation and the c.6366+3A>T and c.6664+5G>T splice-site mutations was assessed on mRNA from GS-affected individuals and control leucocytes or immortalized lymphocytes (biological samples from family 2 individuals were unavailable for analysis). Considering the c.4672C>T nonsense mutation, we measured the abundance of ITPR1 mRNAs by quantitative reverse-transcription PCR (RT-qPCR) in immortalized lymphocytes of the homozygous index case subject of family F1 and two unrelated control subjects. Compared to control subjects, GS-affected individuals' lymphoblasts had significantly reduced levels of ITPR1 mRNAs (mean relative expression levels of 0.74 ± 0.05 and 0.21 ± 0.01 in control and GS-affected individual cell lines, respectively; Figure S1), suggesting degradation through nonsense-mediated mRNA decay (NMD). According to the SpliceSiteFinder-like, NNSPLICE, and Human Splicing Finder prediction softwares, the c.6366+3A>T and c.6664+5G>T mutations identified in family F3 reduced exons 50 and 52 splice-donor splicing scores below the scores of neighboring exonic and intronic cryptic splice-donor sites, respectively (Figure S2). To assess the activation of these cryptic splice-donor sites, reverse-transcribed leucocyte mRNAs from F3:II1 and her parents F3:I1 and F3:I2 were amplified using primers designed in exons 49 and 53, respectively. Three PCR products were obtained from F3:II1 reverse-transcribed mRNA which Sanger sequencing revealed use of both the wild-type splice-donor sites and neighboring exonic and intronic cryptic splice-donor sites, respectively (Figures 1B and S2). Sequencing analysis showed that both the c.6366+3A>T and c.6664+5G>T mutant alleles encoded wild-type and mutant mRNAs. The mutant mRNA arising from the c.6366+3A>T allele lacked the last 62 bp of exon 50 (r.[ =, 6305_6366del; 6366+3a>u]; Figure 1B) and was predicted to encode a truncated ITPR1 (p.Gly2102Valfs5∗; Figure 3B). The mutant transcript arising from the c.6664+5G>T allele retained the first 70 bp of intron 52 (r.[ =, 6664_6665ins6664+1_6664+70; 6664+5g>u]; Figure 1B) and was predicted to encode another truncated ITPR1 (p.Ala2221Valfs23∗; Figure 3B). Consistent with parental genotypes, the shortened and enlarged mutant mRNA products were specifically detected in the father and mother leucocytes, respectively (Figure 1B). None of the aberrant splice products were detected in control individuals (Figure 1B). The truncation constructs encompass the ligand-binding domain and most of the central regulatory domain but lack the channel domain. To evaluate the impact of these mutants on Ca2+ signaling, mammalian expression vectors co-expressing hcRed and human ITPR1 (ITPR1 WT), ITPR1 p.Gln1558∗, ITPR1 p.Gly2102Valfs5∗, or ITPR1 p.Ala2221Valfs23∗ were created. These truncation mutants (Figure 3B) were transiently transfected into human embryonic kidney cell line (HEK293). At 24 hr after transfection, immunoblot analysis was performed using antibodies that recognize an epitope located on the N-terminal region of ITPR1. The antibody detected a band corresponding to intact endogenous ITPR1 in all lysates (Figure S3A). The antibody also detected bands of appropriate molecular mobility in lysates from cells transfected with ITPR1 p.Gln1558∗, ITPR1 p.Gly2102Valfs5∗, and ITPR1 p.Ala2221Valfs23∗ but not in cells transfected with empty vector (Figure S3A). Furthermore, when expressed in DT40-3KO lymphoma cell line devoid of all three endogenous ITPRs,29Sugawara H. Kurosaki M. Takata M. Kurosaki T. Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-
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