COL4A2 Mutations Impair COL4A1 and COL4A2 Secretion and Cause Hemorrhagic Stroke
2011; Elsevier BV; Volume: 90; Issue: 1 Linguagem: Inglês
10.1016/j.ajhg.2011.11.022
ISSN1537-6605
AutoresMarion Jeanne, Cassandre Labelle‐Dumais, Jeff Jorgensen, W. Berkeley Kauffman, Grazia M.S. Mancini, Jack Favor, Valerie Valant, Steven M. Greenberg, Jonathan Rosand, Douglas B. Gould,
Tópico(s)Cancer-related gene regulation
ResumoCollagen, type IV, alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers and are abundant components of basement membranes, including those of the cerebral vasculature. COL4A1 mutations are an increasingly recognized cause of multisystem disorders, including highly penetrant cerebrovascular disease and intracerebral hemorrhage (ICH). Because COL4A1 and COL4A2 are structurally and functionally associated, we hypothesized that variants in COL4A2 would also cause ICH. We sequence COL4A2 in 96 patients with ICH and identify three rare, nonsynonymous coding variants in four patients that are not present in a cohort of 144 ICH-free individuals. All three variants change evolutionarily conserved amino acids. Using a cellular assay, we show that these putative mutations cause intracellular accumulation of COL4A1 and COL4A2 at the expense of their secretion, which supports their pathogenecity. Furthermore, we show that Col4a2 mutant mice also have completely penetrant ICH and that mutations in mouse and human lead to retention of COL4A1 and COL4A2 within the endoplasmic reticulum (ER). Importantly, two of the three putative mutations found in patients trigger ER stress and activate the unfolded protein response. The identification of putative COL4A2 mutations that might contribute to ICH in human patients provides insight into the pathogenic mechanisms of this disease. Our data suggest that COL4A2 mutations impair COL4A1 and COL4A2 secretion and can also result in cytotoxicity. Finally, our findings suggest that, collectively, mutations in COL4A1 and COL4A2 contribute to sporadic cases of ICH. Collagen, type IV, alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers and are abundant components of basement membranes, including those of the cerebral vasculature. COL4A1 mutations are an increasingly recognized cause of multisystem disorders, including highly penetrant cerebrovascular disease and intracerebral hemorrhage (ICH). Because COL4A1 and COL4A2 are structurally and functionally associated, we hypothesized that variants in COL4A2 would also cause ICH. We sequence COL4A2 in 96 patients with ICH and identify three rare, nonsynonymous coding variants in four patients that are not present in a cohort of 144 ICH-free individuals. All three variants change evolutionarily conserved amino acids. Using a cellular assay, we show that these putative mutations cause intracellular accumulation of COL4A1 and COL4A2 at the expense of their secretion, which supports their pathogenecity. Furthermore, we show that Col4a2 mutant mice also have completely penetrant ICH and that mutations in mouse and human lead to retention of COL4A1 and COL4A2 within the endoplasmic reticulum (ER). Importantly, two of the three putative mutations found in patients trigger ER stress and activate the unfolded protein response. The identification of putative COL4A2 mutations that might contribute to ICH in human patients provides insight into the pathogenic mechanisms of this disease. Our data suggest that COL4A2 mutations impair COL4A1 and COL4A2 secretion and can also result in cytotoxicity. Finally, our findings suggest that, collectively, mutations in COL4A1 and COL4A2 contribute to sporadic cases of ICH. Strokes are common and devastating neurological events with poor clinical outcomes for which effective treatment is limited. This is especially true for intracerebral hemorrhages (ICHs), which are associated with the highest rate of mortality despite only accounting for 10–15% of all strokes.1Qureshi A.I. Tuhrim S. Broderick J.P. Batjer H.H. Hondo H. Hanley D.F. Spontaneous intracerebral hemorrhage.N. Engl. J. Med. 2001; 344: 1450-1460Crossref PubMed Scopus (1322) Google Scholar Up to 50% of individuals die within the first year following ICH, and the majority of survivors suffer life-long disability.2Rosand J. Eckman M.H. Knudsen K.A. Singer D.E. Greenberg S.M. The effect of warfarin and intensity of anticoagulation on outcome of intracerebral hemorrhage.Arch. Intern. Med. 2004; 164: 880-884Crossref PubMed Scopus (506) Google Scholar Prevention is therefore of central importance for reducing the personal and societal burden of ICH. Determining the genetic factors leading to cerebrovascular diseases allows identification of individuals who are at greater risk of developing ICH and for whom preventative interventions might be efficacious. Additionally, identifying causative or predisposing genetic risk factors facilitates understanding of the biological mechanisms underlying disease. Although sporadic ICH is often associated with cerebral amyloid angiopathy (CAA) or hypertensive vasculopathy, the precipitating events leading to hemorrhage are poorly defined. Using genetic screens in mice and candidate gene approaches in humans, we showed that mutations in the gene coding for collagen, type IV, alpha 1 (COL4A1 [MIM 120130]) cause porencephaly (MIM 175780) and multisystem small vessel disease (MIM 607595), including nephropathy and ICH.3Gould D.B. Phalan F.C. van Mil S.E. Sundberg J.P. Vahedi K. Massin P. Bousser M.G. Heutink P. Miner J.H. Tournier-Lasserve E. John S.W. Role of COL4A1 in small-vessel disease and hemorrhagic stroke.N. Engl. J. Med. 2006; 354: 1489-1496Crossref PubMed Scopus (415) Google Scholar, 4Gould D.B. Phalan F.C. Breedveld G.J. van Mil S.E. Smith R.S. Schimenti J.C. Aguglia U. van der Knaap M.S. Heutink P. John S.W.M. Mutations in Col4a1 cause perinatal cerebral hemorrhage and porencephaly.Science. 2005; 308: 1167-1171Crossref PubMed Scopus (409) Google Scholar Col4a1 mutant mice had a broad spectrum of cerebrovascular diseases, including pre- and perinatal ICH, porencephalic cavities, progressive, multifocal, and recurrent ICH, and, occasionally, subarachnoid hemorrhages.3Gould D.B. Phalan F.C. van Mil S.E. Sundberg J.P. Vahedi K. Massin P. Bousser M.G. Heutink P. Miner J.H. Tournier-Lasserve E. John S.W. Role of COL4A1 in small-vessel disease and hemorrhagic stroke.N. Engl. J. Med. 2006; 354: 1489-1496Crossref PubMed Scopus (415) Google Scholar, 4Gould D.B. Phalan F.C. Breedveld G.J. van Mil S.E. Smith R.S. Schimenti J.C. Aguglia U. van der Knaap M.S. Heutink P. John S.W.M. Mutations in Col4a1 cause perinatal cerebral hemorrhage and porencephaly.Science. 2005; 308: 1167-1171Crossref PubMed Scopus (409) Google Scholar Dominant COL4A1 mutations are being increasingly recognized as an important cause of highly penetrant cerebrovascular diseases, including porencephaly and ICH.3Gould D.B. Phalan F.C. van Mil S.E. Sundberg J.P. Vahedi K. Massin P. Bousser M.G. Heutink P. Miner J.H. Tournier-Lasserve E. John S.W. Role of COL4A1 in small-vessel disease and hemorrhagic stroke.N. Engl. J. Med. 2006; 354: 1489-1496Crossref PubMed Scopus (415) Google Scholar, 4Gould D.B. Phalan F.C. Breedveld G.J. van Mil S.E. Smith R.S. Schimenti J.C. Aguglia U. van der Knaap M.S. Heutink P. John S.W.M. Mutations in Col4a1 cause perinatal cerebral hemorrhage and porencephaly.Science. 2005; 308: 1167-1171Crossref PubMed Scopus (409) Google Scholar, 5Breedveld G. de Coo I.F. Lequin M.H. Arts W.F.M. Heutink P. Gould D.B. John S.W.M. Oostra B. Mancini G.M.S. Novel mutations in three families confirm a major role of COL4A1 in hereditary porencephaly.J. Med. Genet. 2006; 43: 490-495Crossref PubMed Scopus (129) Google Scholar, 6Vahedi K. Kubis N. Boukobza M. Arnoult M. Massin P. Tournier-Lasserve E. Bousser M.-G. COL4A1 mutation in a patient with sporadic, recurrent intracerebral hemorrhage.Stroke. 2007; 38: 1461-1464Crossref PubMed Scopus (100) Google Scholar, 7Sibon I. Coupry I. Menegon P. Bouchet J.-P. Gorry P. Burgelin I. Calvas P. Orignac I. Dousset V. Lacombe D. et al.COL4A1 mutation in Axenfeld-Rieger anomaly with leukoencephalopathy and stroke.Ann. Neurol. 2007; 62: 177-184Crossref PubMed Scopus (109) Google Scholar, 8Plaisier E. Gribouval O. Alamowitch S. Mougenot B. Prost C. Verpont M.C. Marro B. Desmettre T. Cohen S.Y. Roullet E. et al.COL4A1 mutations and hereditary angiopathy, nephropathy, aneurysms, and muscle cramps.N. Engl. J. Med. 2007; 357: 2687-2695Crossref PubMed Scopus (256) Google Scholar, 9de Vries L.S. Koopman C. Groenendaal F. Van Schooneveld M. Verheijen F.W. Verbeek E. Witkamp T.D. van der Worp H.B. Mancini G. COL4A1 mutation in two preterm siblings with antenatal onset of parenchymal hemorrhage.Ann. Neurol. 2009; 65: 12-18Crossref PubMed Scopus (96) Google Scholar, 10Shah S. Kumar Y. McLean B. Churchill A. Stoodley N. Rankin J. Rizzu P. van der Knaap M. Jardine P. A dominantly inherited mutation in collagen IV A1 (COL4A1) causing childhood onset stroke without porencephaly.Eur. J. Paediatr. Neurol. 2010; 14: 182-187Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 11Rouaud T. Labauge P. Tournier Lasserve E. Mine M. Coustans M. Deburghgraeve V. Edan G. Acute urinary retention due to a novel collagen COL4A1 mutation.Neurology. 2010; 75: 747-749Crossref PubMed Scopus (16) Google Scholar, 12Plaisier E. Chen Z. Gekeler F. Benhassine S. Dahan K. Marro B. Alamowitch S. Paques M. Ronco P. Novel COL4A1 mutations associated with HANAC syndrome: a role for the triple helical CB3[IV] domain.Am. J. Med. Genet. A. 2010; 152A: 2550-2555Crossref PubMed Scopus (78) Google Scholar, 13Bilguvar K. DiLuna M.L. Bizzarro M.J. Bayri Y. Schneider K.C. Lifton R.P. Gunel M. Ment L.R. Pacifier and Breastfeeding Trial GroupCOL4A1 mutation in preterm intraventricular hemorrhage.J. Pediatr. 2009; 155: 743-745Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 14Meuwissen M.E.C. de Vries L.S. Verbeek H.A. Lequin M.H. Govaert P.P. Schot R. Cowan F.M. Hennekam R. Rizzu P. Verheijen F.W. et al.Sporadic COL4A1 mutations with extensive prenatal porencephaly resembling hydranencephaly.Neurology. 2011; 76: 844-846Crossref PubMed Scopus (44) Google Scholar, 15Vermeulen R.J. Peeters-Scholte C. Van Vugt J.J.M. Barkhof F. Rizzu P. van der Schoor S.R. van der Knaap M.S. Fetal origin of brain damage in 2 infants with a COL4A1 mutation: fetal and neonatal MRI.Neuropediatrics. 2011; 42: 1-3Crossref PubMed Scopus (34) Google Scholar COL4A1 mutations in mice and humans are pleiotropic and can affect multiple organ systems with different levels of severity. Using controlled genetic approaches in mice, we have shown that at least some of the variability in the penetrance and severity of Col4a1-induced pathology can be attributed to environmental influences and to genetic context.3Gould D.B. Phalan F.C. van Mil S.E. Sundberg J.P. Vahedi K. Massin P. Bousser M.G. Heutink P. Miner J.H. Tournier-Lasserve E. John S.W. Role of COL4A1 in small-vessel disease and hemorrhagic stroke.N. Engl. J. Med. 2006; 354: 1489-1496Crossref PubMed Scopus (415) Google Scholar, 16Gould D.B. Marchant J.K. Savinova O.V. Smith R.S. John S.W.M. Col4a1 mutation causes endoplasmic reticulum stress and genetically modifiable ocular dysgenesis.Hum. Mol. Genet. 2007; 16: 798-807Crossref PubMed Scopus (97) Google Scholar, 17Labelle-Dumais C. Dilworth D.J. Harrington E.P. de Leau M. Lyons D. Kabaeva Z. Manzini M.C. Dobyns W.B. Walsh C.A. Michele D.E. Gould D.B. COL4A1 mutations cause ocular dysgenesis, neuronal localization defects, and myopathy in mice and Walker-Warburg syndrome in humans.PLoS Genet. 2011; 7: e1002062Crossref PubMed Scopus (109) Google Scholar Accumulating evidence suggests that allelic heterogeneity might also contribute to variable expressivity of COL4A1 mutations. A subset of individuals with COL4A1 mutations are reported to have a distinct clinical phenotype referred to as hereditary angiopathy, nephropathy, aneurysms, and cramps syndrome (HANAC [MIM 611773]), and the mutations in these individuals cluster within a 31 amino acid region of the COL4A1 protein that encompasses integrin binding sites.8Plaisier E. Gribouval O. Alamowitch S. Mougenot B. Prost C. Verpont M.C. Marro B. Desmettre T. Cohen S.Y. Roullet E. et al.COL4A1 mutations and hereditary angiopathy, nephropathy, aneurysms, and muscle cramps.N. Engl. J. Med. 2007; 357: 2687-2695Crossref PubMed Scopus (256) Google Scholar, 12Plaisier E. Chen Z. Gekeler F. Benhassine S. Dahan K. Marro B. Alamowitch S. Paques M. Ronco P. Novel COL4A1 mutations associated with HANAC syndrome: a role for the triple helical CB3[IV] domain.Am. J. Med. Genet. A. 2010; 152A: 2550-2555Crossref PubMed Scopus (78) Google Scholar, 18Plaisier E. Alamowitch S. Gribouval O. Mougenot B. Gaudric A. Antignac C. Roullet E. Ronco P. Autosomal-dominant familial hematuria with retinal arteriolar tortuosity and contractures: a novel syndrome.Kidney Int. 2005; 67: 2354-2360Crossref PubMed Scopus (30) Google Scholar, 19Alamowitch S. Plaisier E. Favrole P. Prost C. Chen Z. Van Agtmael T. Marro B. Ronco P. Cerebrovascular disease related to COL4A1 mutations in HANAC syndrome.Neurology. 2009; 73: 1873-1882Crossref PubMed Scopus (108) Google Scholar These data suggest that alterations of specific functional domains could contribute to the phenotypic variability associated with COL4A1 mutations. COL4A1 forms heterotrimers with COL4A2 (MIM 120090) and, together, they are the most abundant and prevalent proteins in basement membranes, including those of the cerebral vasculature. Heterotrimers composed of one COL4A2 and two COL4A1 peptides are assembled and modified within the endoplasmic reticulum (ER) before trafficking to the Golgi where they are packaged into vesicles for secretion into the vascular basement membranes.20Mayne R. Wiedemann H. Irwin M.H. Sanderson R.D. Fitch J.M. Linsenmayer T.F. Kühn K. Monoclonal antibodies against chicken type IV and V collagens: electron microscopic mapping of the epitopes after rotary shadowing.J. Cell Biol. 1984; 98: 1637-1644Crossref PubMed Scopus (32) Google Scholar, 21Trüeb B. Gröbli B. Spiess M. Odermatt B.F. Winterhalter K.H. Basement membrane (type IV) collagen is a heteropolymer.J. Biol. Chem. 1982; 257: 5239-5245Abstract Full Text PDF PubMed Google Scholar, 22Fatemi S.H. The role of secretory granules in the transport of basement membrane components: radioautographic studies of rat parietal yolk sac employing 3H-proline as a precursor of type IV collagen.Connect. Tissue Res. 1987; 16: 1-14Crossref PubMed Scopus (10) Google Scholar In the extracellular space, heterotrimers polymerize into flexible sheets, which not only provide strength to basement membranes but also participate in dynamic biological processes through interactions with growth factors and cell surface receptors, including integrins.23Paralkar V.M. Vukicevic S. Reddi A.H. Transforming growth factor beta type 1 binds to collagen IV of basement membrane matrix: implications for development.Dev. Biol. 1991; 143: 303-308Crossref PubMed Scopus (300) Google Scholar, 24Parkin J.D. San Antonio J.D. Pedchenko V. Hudson B. Jensen S.T. Savige J. Mapping structural landmarks, ligand binding sites, and missense mutations to the collagen IV heterotrimers predicts major functional domains, novel interactions, and variation in phenotypes in inherited diseases affecting basement membranes.Hum. Mutat. 2011; 32: 127-143Crossref PubMed Scopus (80) Google Scholar, 25Wang, X., Harris, R., Bayston, L., and Ashe, H. (2008). Type IV collagens regulate BMP signalling in Drosophila Nature 425, 72–77.Google Scholar, 26Bunt S. Hooley C. Hu N. Scahill C. Weavers H. Skaer H. Hemocyte-secreted type IV collagen enhances BMP signaling to guide renal tubule morphogenesis in Drosophila.Dev. Cell. 2010; 19: 296-306Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar At the carboxy terminus of COL4A1 and COL4A2 is a globular noncollagenous (NC1) domain responsible for initiating heterotrimer formation within the ER.27Khoshnoodi J. Cartailler J.-P. Alvares K. Veis A. Hudson B.G. Molecular recognition in the assembly of collagens: terminal noncollagenous domains are key recognition modules in the formation of triple helical protomers.J. Biol. Chem. 2006; 281: 38117-38121Crossref PubMed Scopus (153) Google Scholar A 7S domain that is involved in lateral intertrimer associations in the extracellular matrix is present at the amino terminus of both proteins. Flanked by these terminal domains are the large triple-helix-forming domains that constitute more than 80% of both the COL4A1 and COL4A2 proteins. The triple-helix-forming domain is characteristic of collagens and is composed of repeating Gly-Xaa-Yaa amino acid residues where there is a requirement for a glycine every third amino acid and where the X and Y amino acids are often proline or lysine residues that undergo posttranslational processing in order to crosslink the constituent peptides of a heterotrimer.28Gorres K.L. Raines R.T. Prolyl 4-hydroxylase.Crit. Rev. Biochem. Mol. Biol. 2010; 45: 106-124Crossref PubMed Scopus (406) Google Scholar, 29Vranka J.A. Sakai L.Y. Bächinger H.P. Prolyl 3-hydroxylase 1, enzyme characterization and identification of a novel family of enzymes.J. Biol. Chem. 2004; 279: 23615-23621Crossref PubMed Scopus (151) Google Scholar The triple-helix-forming domains of type IV collagens also contain frequent and positionally conserved interruptions within the Gly-Xaa-Yaa repeats, which are thought to confer molecular flexibility to type IV collagens. Pathogenic mutations are reported in the NC1 domain, however, the vast majority of disease-causing mutations reported to date for various types of collagens, including type IV collagens, have been shown to interfere with triple-helix formation or stability.30Engel J. Prockop D.J. The zipper-like folding of collagen triple helices and the effects of mutations that disrupt the zipper.Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 137-152Crossref PubMed Scopus (267) Google Scholar Because of the intimate structural and functional association between COL4A1 and COL4A2 and because of the presumed mechanisms by which COL4A1 mutations lead to disease, we expect that COL4A2 mutations will cause pathologies similar to those resulting from COL4A1 mutations. In support of this hypothesis, mutations in COL4A2 orthologs phenocopy mutations in COL4A1 orthologs in mice and in Caenorhabditis elegans.31Favor J. Gloeckner C.J. Janik D. Klempt M. Neuhäuser-Klaus A. Pretsch W. Schmahl W. Quintanilla-Fend L. Type IV procollagen missense mutations associated with defects of the eye, vascular stability, the brain, kidney function and embryonic or postnatal viability in the mouse, Mus musculus: an extension of the Col4a1 allelic series and the identification of the first two Col4a2 mutant alleles.Genetics. 2007; 175: 725-736Crossref PubMed Scopus (91) Google Scholar, 32Gupta M.C. Graham P.L. Kramer J.M. Characterization of alpha1(IV) collagen mutations in Caenorhabditis elegans and the effects of alpha1 and alpha2(IV) mutations on type IV collagen distribution.J. Cell Biol. 1997; 137: 1185-1196Crossref PubMed Scopus (101) Google Scholar, 33Guo X.D. Johnson J.J. Kramer J.M. Embryonic lethality caused by mutations in basement membrane collagen of C. elegans.Nature. 1991; 349: 707-709Crossref PubMed Scopus (88) Google Scholar, 34Sibley M.H. Graham P.L. von Mende N. Kramer J.M. Mutations in the alpha 2(IV) basement membrane collagen gene of Caenorhabditis elegans produce phenotypes of differing severities.EMBO J. 1994; 13: 3278-3285Crossref PubMed Scopus (50) Google Scholar However, to date, pathogenic COL4A2 mutations have not been reported in humans. To test the hypothesis that COL4A2 mutations cause ICH, we sequenced COL4A2 in a large cohort of unrelated individuals diagnosed with sporadic hemorrhages not related to arteriovenous malformations, tumors, or impaired coagulation. We identified putative pathogenic variants in four out of 96 people, showed that COL4A2 proteins are sequestered within the ER, and, in some cases, caused activation of the unfolded protein response (UPR). For the affected cohort, we selected 96 individuals from among 800 consecutive participants with ICH presenting to Massachusetts General Hospital.35Biffi A. Halpin A. Towfighi A. Gilson A. Busl K. Rost N. Smith E.E. Greenberg M.S. Rosand J. Viswanathan A. Aspirin and recurrent intracerebral hemorrhage in cerebral amyloid angiopathy.Neurology. 2010; 75: 693-698Crossref PubMed Scopus (247) Google Scholar Cranial computed tomography scans and/or magnetic resonance images of the head were completed for all individuals, and hemorrhages were classified according to the Boston Criteria.36Knudsen K.A. Rosand J. Karluk D. Greenberg S.M. Clinical diagnosis of cerebral amyloid angiopathy: validation of the Boston criteria.Neurology. 2001; 56: 537-539Crossref PubMed Scopus (816) Google Scholar For the present study, 48 individuals with probable CAA-related ICH and 48 individuals with presumed hypertension-related deep ICH were chosen according to the inclusion and exclusion criteria published previously36Knudsen K.A. Rosand J. Karluk D. Greenberg S.M. Clinical diagnosis of cerebral amyloid angiopathy: validation of the Boston criteria.Neurology. 2001; 56: 537-539Crossref PubMed Scopus (816) Google Scholar (summarized in Table 1). Simultaneously, we collected DNA from ethnically and age-matched individuals who were free of a history of hemorrhagic stroke and were drawn from the primary care practices at Massachusetts General Hospital. All participants or their surrogates provided informed consent for study participation and the Massachusetts General Hospital institutional review board approved all study procedures.Table 1Inclusion and Exclusion CriteriaPatientControlInclusion Criteriaeither gendereither genderability and willingness to consentability and willingness to consentsymptomatic intracerebral hemorrhageExclusion Criteriaantecedent head traumahistory of intracerebral hemorrhageischemic stroke during 2 weeks prior to intracerebral hemorrhageintracerebral tumorintracerebral tumorarteriovenous malformation, aneurysm, or central nervous system vasculitisarteriovenous malformation, aneurysm, or central nervous system vasculitisprimary coagulopathy, blood dyscrasia, or active liver disorderprimary coagulopathy, blood dyscrasia, or active liver disorderuse of cocaine or sympathomimetic druguse of cocaine or sympathomimetic drugalcohol abusealcohol abuse Open table in a new tab We performed direct sequence analysis (ABI BigDye v3.1) of the entire coding sequence of COL4A2 including the flanking intronic regions (more than 50 nucleotides for most introns and never less than 20 nucleotides) by using 42 pairs of primers (Table S1, available online). Using Sequencher software (Gene Codes Corporation), we identified 168 sequence variants of which 137 were intronic (Table S2). Because we expected that pathogenic mutations having a strong effect will be rare, nonsynonymous variants, we focused our attention on these. It is possible that intronic or synonymous variants are pathogenic, in which case our estimation of the role of COL4A2 in ICH would be understated. Of the 31 coding variants, ten were nonsynonymous (Table 2). Four nonsynonymous variants, c.1550G>A, c.2048G>C, c.2152C>T, and c.4195G>A, were found at high frequency and considered unlikely to be causative mutations (p.Arg517Lys, p.Gly683Ala, p.Pro718Ser, and p.Val1399Ile, hereafter referred to as COL4A2R517K, COL4A2G683A, COL4A2P718S and COL4A2V1399I, respectively). Notably, one of these variants changes a glycine residue, and glycine missense mutations within the triple-helix domain constitute the prototypic pathogenic mutation for several types of collagens. However, this variant, COL4A2G683A, is highly polymorphic (17 out of 92 affected individuals were heterozygous and 4 out of 92 were homozygous for the minor allele) and impacts a glycine that is part of a repeat interruption and not part of the Gly-Xaa-Yaa tripeptide repeat pattern necessary for triple-helix formation. Thus, out of a cohort of 96 individuals, we identified six nonsynonymous rare variants that we deemed to be potentially pathogenic (Figure 1A ).Table 2Coding SNPs IdentifiedVariantPatientsControlsExonDNAProteinHomozygous MajorHeterozygousHomozygous MinorTotalHomozygous MajorHeterozygousHomozygous MinorTotalNonsynonymous9c.574G>Tp.Val192PheV192FG/G (91)G/T (1)T/T (0)92G/G (144)G/T (0)T/T (0)14422c.1550G>Ap.Arg517LysR517KA/A (33)A/G (45)G/G (15)9327c.2048G>Cp.Gly683AlaG683AG/G (71)G/C (17)C/C (4)9228c.2102A>Gp.Lys701ArgK701RA/A (94)A/G (1)G/G (0)95A/A (137)A/G (0)G/G (0)13728c.2152C>Tp.Pro718SerP718SC/C (57)C/T (27)T/T (11)95C/C (92)C/T (41)T/T (4)13736c.3326G>Ap.Arg1109GlnR1109QG/G (92)G/A (1)A/A (1)94G/G (132)G/A (1)A/A (0)13337c.3368A>Gp.Glu1123GlyE1123GA/A (91)A/G (2)G/G (0)93A/A (142)A/G (0)G/G (0)14237c.3448C>Ap.Gln1150LysQ1150KC/C (92)C/A (1)A/A (0)93C/C (142)C/A (0)A/A (0)14244c.4195G>Ap.Val1399IleV1399IG/G (86)G/A (8)A/A (0)9448c.5068G>Ap.Ala1690ThrA1690TG/G (92)G/A (1)A/A (0)93G/G (143)G/A (0)A/A (0)143Synonymous3c.49C>Tp.Leu17LeuL17LC/C (91)C/T (1)T/T (0)925c.297A>Gp.Thr99ThrT99TA/A (91)A/G (2)G/G (0)9310c.594C>Tp.Pro198ProP198PC/C (92)C/T (1)T/T (0)9317c.1008C>Tp.Pro336ProP336PC/C (26)C/T (48)T/T (17)91C/C (30)C/T (68)T/T (38)13617c.1011G>Ap.Lys337LysK337KG/G (92)G/A (1)A/A (0)93G/G (135)G/A (1)A/A (0)13619c.1095G>Ap.Pro365ProP365PG/G (86)G/A (0)A/A (8)9419c.1179C>Tp.Ile393IleI393IC/C (82)C/T (2)T/T (10)9420c.1308C>Tp.Pro436ProP436PC/C (92)C/T (1)T/T (0)9322c.1488G>Ap.Pro496ProP496PA/A (33)A/G (44)G/G (16)9334c.3204C>Tp.Ser1068SerS1068SC/C (91)C/T (1)T/T (0)9241c.3804A>Tp.Pro1268ProP1268PA/A (88)A/T (4)T/T (0)9241c.3807T>Cp.Gly1269GlyG1269GT/T (88)T/C (4)C/C (0)9241c.3876C>Tp.Leu1292LeuL1292LC/C (91)C/T (1)T/T (0)9243c.4083T>Cp.Thr1361ThrT1361TT/T (89)T/C (3)C/C (0)9243c.4089G>Ap.Ala1363AlaA1363AG/G (61)G/A (25)A/A (6)9245c.4292T>Cp.Phe1430PheF1430FT/T (90)T/C (4)C/C (0)9446c.4428G>Ap.Pro1476ProP1476PG/G (92)G/A (1)A/A (0)9346c.4515A>Gp.Pro1505ProP1505PG/G (63)G/A (28)A/A (2)9347c.4617G>Ap.Ala1539AlaA1539AG/G (78)G/A (15)A/A (0)9347c.4737C>Tp.Ala1579AlaA1579AC/C (92)C/T (1)T/T (0)9348c.4929G>Ap.Pro1643ProP1643PG/G (92)G/A (1)A/A (0)93G/G (141)G/A (2)A/A (0)143 Open table in a new tab We resequenced at least 264 control chromosomes for each exon in which one of the six nonsynonymous variants was found (Table 2). One variant, c.3326G>A ([p.Arg1109Gln] hereafter referred to as COL4A2R1109Q), was identified in two affected individuals (including one homozygote) and in a single control individual, suggesting that this variant might not be pathogenic and other variants were present in dbSNP. However, because reduced penetrance is documented in COL4A1 mutations and because genetic context dependency has been demonstrated for Col4a1 mutations in mice, we chose to include these variants for further analysis. We performed multispecies alignments to determine whether the affected amino acids were conserved and whether there was precedence for any of the variants in other species (Figure 1B). Five of the six variants were highly conserved across species and perfectly conserved among mammals. COL4A2R1109Q was less conserved and, in fact, Rhesus monkey (Macaca mulatta) has a glutamine (Q) at this position of its COL4A2 ortholog, further suggesting that this variant might not be pathogenic. We then used commonly referenced algorithms to help predict the potential impact of each variant. The automated tools SIFT (Sorting Intolerant from Tolerant, version 2.0) and PolyPhen (Polymorphism Phenotyping-2) lead to different and even contradictory results. The evolutionary-based approach, SIFT, predicted only one damaging variant (c.574G>T [p.Val192Phe], hereafter referred to as COL4A2V192F), whereas the structure- and evolutionary-based approach, PolyPhen, predicted two different variants to be possibly damaging (c.3448C>A [p.Gln1150Lys] and c.5068G>A [ p.Ala1690Thr]; hereafter referred to as COL4A2Q1150K and COL4A2A1690T) and one different variant to be probably damaging (c.3368A>G [p.Glu1123Gly]; hereafter referred to as COL4A2E1123G). However, PolyPhen also predicted the COL4A2P718S variant to be possibly damaging despite this variant being highly polymorphic and found in both cohorts at high frequencies and therefore highly unlikely to be pathogenic. Based on these results, we contend that in silico approaches are currently unlikely to accurately predict functional consequences of COL4A2 variants. Therefore, to evaluate the functional consequences of putative COL4A2 mutations, we used a cell-culture-based assay that we have previously validated for COL4A1 mutations.17Labelle-Dumais C. Dilworth D.J. Harrington E.P. de Leau M. Lyons D. Kabaeva Z. Manzini M.C. Dobyns W.B. Walsh C.A. Michele D.E. Gould D.B. COL4A1 mutations cause ocular dysgenesis, neuronal localization defects, and myopathy in mice and Walker-Warburg syndrome in humans.PLoS Genet. 2011; 7: e1002062Crossref PubMed Scopus (109) Google Scholar Pathogenic mutations in genes coding for several types of collagen often disrupt normal heterotrimer formation leading to intracellular accumulation of misfolded proteins and decreased heterotrimer secretion. Thus, we used an in vitro secretion assay to test whether rare COL4A2 variants changed the ratio of extracellular to intracellular COL4A2 and COL4A1 proteins in cultured cells. As described previously,17Labelle-Dumais C. Dilworth D.J. Harrington E.P. de Leau M. Lyons D. Kabaeva Z. Manzini M.C. Dobyns W.B. Walsh C.A. Michele D.E. Gould D.B. COL4A1 mutations cause ocular dysgenesis, neuronal localization defects, and myopathy in mice and Walker-Warburg syndrome in humans.PLoS Genet. 2011; 7: e1002062Crossref PubMed Scopus (109) Google Scholar we transfected HT1080 cells (which endogenously express COL4A1 and COL4A2) with a control COL4A2 cDNA (NM_001846.2) or with COL4A2 cDNA that contained one of the six nonsynonymous rare sequence variants. At 80%–90% confluence, cells were serum deprived and treated with ascorbic acid (50 μg/ml), and after 24 hr the cond
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