Coucke, Paul

De Paepe, Anne

Demuynck, L

Guillemyn, Brecht

Kayserili, H

Malfait, Fransiska

Symoens, Sofie

Syx, Delfien

English

Ghent University Academic Bibliography

A homozygous CREB3L1 missense mutation expands the mutational spectrum of CREB3L1-related osteogenesis imperfectaGuillemyn B.1, Syx D.1, Demuynck L.1, Kayserili H.2, De Paepe A.1, Coucke P.1, Malfait F.1, Symoens S.1BackgroundOsteogenesis imperfecta (OI) comprises a heterogeneous group of disorders characterized by bonedeformities, low bone mass, brittle bones, and connective tissue manifestations. Dominant mutations inthe COL1A1 or COL1A2 genes account for more than 80% of the cases, whereas recessive defects canbe found in a plethora of genes. In 2013, our group identified CREB3L1 (encoding the endoplasmicreticulum (ER)-stress transducer old astrocyte specifically induced substance (OASIS)) as a novelautosomal recessive (AR) lethal/severe OI gene in a fetus in whom this gene was homozygouslydeleted.2 Hitherto, only two additional homozygous mutations have been reported, an in-framedeletion c.934_936delAAG (p.(Lys312del)) and a frameshift mutation c.1365del (p.(Pro458Argfs*25)) in afamily with severe AR OI.3,4OASIS and its role in ER stressIn the absence of ER stress, OASIS is expressed at the ER membrane, with cytosolic N-terminal DNA-binding and transcription activation domains. In regulated intramembrane proteolysis (RIP), OASIS istransported from the ER to the golgi membrane, where it is sequentially cleaved by theendopeptidases S1P and S2P. Finally, the N-terminal domain of OASIS (OASIS-N) is translocated to thenucleus where transcriptional activation of the COL1A1 promoter can occur.In addition, Keller et al demonstrated that OASIS regulates the expression of the COPII componentSEC24D.3 Together with SEC23A, this protein forms the inner coat of COPII vesicles, complexes whichare involved in the trafficking of secreted proteins (such as type I collagen) from the ER to the golgi.Figure 1: Working mechanism and proteinfunction of the tissue-specific transcription factorOASIS.1ConclusionWe successfully used an overexpression model to study the pathogenicnature of the homozygous missense variant p.(Ala304Val).Quantitative reverse transcription polymerase chain reaction shows aneffect on the expression of both COPII components SEC23A andSEC24D, whereas Western blot levels only link SEC24D to the CREB3L1-related disease mechanism.In addition to these findings, which are in line with an earlier report 3,luciferase assays could show that the variant might also alter COL1A1transcription, thereby influencing the formation of the secretory coatprotein II complex. This is in turn postulated to disturb the OASIS-mediated secretory pathways, necessary for normal bone development.Figure 3: qPCR and Western blot results of transient overexpressed HEK293 cells.(a) and (b): qPCR shows that both variants A304V (p.(Ala304Val), reported here) andK312del (p.(Lys312del), included as a positive control 3) have an effect on the expression ofthe COPII components SEC23A and SEC24D, when compared to WT overexpressedCREB3L1 (Tukey test, * P < 0.05, ** P < 0.05; values shown are the mean of threeindependent experiments (n = 3); NTC, non transfected cells; Empty, empty vector–transfected control; WT, wild type-transfected OASIS).(c) and (d): Western blot shows that the newly identified missense variant A304V has aneffect on the protein level of SEC24D, but not on SEC23A (Tukey test, * P < 0.05; valuesshown are the mean of four independent experiments (n = 4)).RationaleWe report the first homozygous CREB3L1 missense mutation associated with lethal AR OI, and investigated its pathogenicity in relation toSEC24D/SEC23A expression and COL1A1 transcription.qPCRFigure 4: Luciferase assays of transient overexpressed HEK293 cells.Luciferase assays, in which both variants A304V (p.(Ala304Val), reported here)and K312del (p.(Lys312del), included as a positive control 3) were overexpressedtogether with ms COL1A1 promoter (a) or ms COL1A1 promoter mUPRE (upstreamregulatory elements) (b) constructs, indicate a possible negative effect of thetranscriptional capacity of both variants. Statistics were not yet possible as thesegraphs display results of only 2 independent experiments (n = 2) (NTC, nontransfected cells; Empty, empty vector–transfected control; WT, wild type-transfected OASIS).Western blotmsCOL1A1prommsCOL1A1prom +EmptymsCOL1A1prom +WTmsCOL1A1prom +A304VmsCOL1A1prom +K312delNTCArbitrary units(a)msCOL1A1prom mUPREmsCOL1A1prom +EmptymsCOL1A1prom +WTmsCOL1A1prom +A304VmsCOL1A1prom +K312delNTCArbitrary units(b)Clinical phenotypeProtein changeDEOGEN2 (mutation effect prediction tool for proteins) 5,6Homozygous p.(Ala304Val)cDNA change c.911C>TProtein domain Highly conserved basic leucine zipper domain, positioned four amino acids upstream of the DNA binding domainAmino acid similarityOverall scoreAbsentPopulation frequency (gnomad)Fetus, aborted at 19 weeks of gestation because of multiple fractures/short bowed extremitiesEthnicity TurkishFigure 2: Clinical phenotype and molecular findings.Contact informationBrecht.Guillemyn@UGent.beSofie.Symoens@UGent.beAuthor affiliations1 Center for MedicalGenetics, Ghent UniversityHospital, Ghent, Belgium2 University of Istanbul,Medical Genetics, MilletCad. Capa, 34390, Istanbul,TurkeyReferences1 Kang H et al, Translational Research, 2017: Osteogenesis imperfecta: newgenes reveal novel mechanisms in bone dysplasia2 Symoens S et al, Orphanet J Rare Dis, 2013: Deficiency for the ER-stresstransducer OASIS causes severe recessive osteogenesis imperfecta inhumans.3 Keller R et al, Genetics in Medicine, 2017: Monoallelic and biallelicCREB3L1 variant causes mild and severe osteogenesis imperfecta,respectively4 Micha D, 13th International Conference on OI, 2017: Severe OsteogenesisImperfecta presentation in a family with a novel CREB3L1 mutation.5 Raimondi et al, Nucleic Acids Research, 2017: DEOGEN2: prediction andinteractive visualization of single amino acid variant deleteriousness inhuman proteins.6 Tanyalcin I et al, Computing in Science & Engineering, 2018: LexiconVisualization Library and JavaScript for Scientific Data Visualization.Arbitrary unitsArbitrary unitsRelative band intensity / beta tubulinRelative band intensity / beta tubulinEmpty WTA304VK312delNTCEmpty WTA304VK312delNTCOASIS80kDa60kDaBTUB55kDaSEC23A86kDaBTUB55kDaSEC24D113kDaBTUB55kDaSEC23A SEC23AEmpty WTA304VK312delNTCEmpty WTA304VK312delNTCEmpty WTA304VK312delNTCEmpty WTA304VK312delNTCSEC24D SEC24DEmpty WTA304VK312delNTC****** ****(a)(b)(c)(d)*** *PolyPhen Probably damaging (0.992)Sift Tolerated (0.09)

A homozygous CREB3L1 missense mutation expands the mutational spectrum of CREB3L1-related osteogenesis imperfecta

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A homozygous CREB3L1 missense mutation expands the mutational spectrum of CREB3L1-related osteogenesis imperfecta

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