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TGFbRIIb Mutations Trigger Aortic Aneurysm Pathogenesis by
DOI: 10.1161/CIRCGENETICS.112.964064 TGFbRIIb Mutations Trigger Aortic Aneurysm Pathogenesis by Altering TGFb2 Signal Transduction Running title: Bee et al.; TGFbRIIb mutations in aortic aneurysms Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 Deve vere ve reux re ux,, MD ux MD;; Katharine J. Bee, PhD; David C. Wilkes, PhD; Richard B. De Devereux, Craig T. Basson, MD, PhD; Cathy J. Hatcher, PhD C enter for M olec eccularr C ardiiollogy,, G reen nbeerg gD ivission off C arrdiolog ogyy, og Center Molecular Cardiology, Greenberg Division Cardiology, W il We illl Cornell Corn Co rnel rn elll Medical el Medi Me d ca di call College, Coll Co lleg eg ge, e New e York, ew Yor ork, k, NY NY Weill Corresponding authors: Cathy J. Hatcher, PhD Craig T. Basson, MD, PhD Greenberg Division of Cardiology Novartis Institutes for BioMedical Research Weill Cornell Medical College 220 Massachusetts Avenue 525 E. 68th Street Cambridge, MA 02139 New York, NY 10065 Tel: 212-746-2201 Tel: 617-871-7652 Fax: 212-746-6669 Fax: 617-871-5203 E-mail: cjhatche@med.cornell.edu E-mail: craig.basson@novartis.com Journal Subject Code: [109] Clinical genetics; [89] Genetics of cardiovascular disease; [147] Growth factors/cytokines 1 DOI: 10.1161/CIRCGENETICS.112.964064 Abstract: Background - Thoracic aortic aneurysm (TAA) is a common progressive disorder involving gradual dilation of the ascending and/or descending thoracic aorta that eventually leads to dissection or rupture. Nonsydromic TAA can occur as a genetically triggered, familial disorder that is usually transmitted in a monogenic autosomal dominant fashion and is known as familial TAA (FTAA). Genetic analyses of families affected with TAA have identified several chromosomal loci and further mapping of FTAA genes has highlighted disease-causing mutations in at least four genes: myosin heavy chain 11 (MYH11), a-smooth muscle actin Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 (ACTA2), transforming growth factor beta receptors I and II (TGFERI and TGFERII). ) Methods and Results - We evaluated 100 probands to determine the mu muta mutation tati ta tion ti on frequency freq fr eque eq uenncy in ue n MYH11, ACTA2, TGFbRI and TGFbRII in an unbiased population of indivi individuals idu dual alss wi al with th ggenetically e e en mediated TAA. AA. AA A. In n tthis his study, hi stud udy, 9% of patients had a mu ud m mutation tation in one onne off the the genes analyzed. 33% of patients had dm mutations utations inn ACTA2, ACTA AC TA22, 3% iin TA n MYH11, MYH MY H11, 1% % in 7*)ȕ5II 7*)ȕ 7* )ȕ5 )ȕ 5II II and ndd nno o mu m mutations taati tion onss were on were fo found o in 7*)ȕ5, Additionally, Add dditional ally,, w al wee id identified denttif i ie ied mu mut mutations tat on tati onss inn a 755 ba base ase ppair a r alt ai alternatively ternat attiv i elyy spliced spplicedd TGFbRII TG exon, exon 1a WKDWSURGXFHVWKH7*)ȕ5,,E WKDWSURGXFH FHVV WKKH FH H7* 7* *)ȕ ȕ5, 5 ,E isoform issoffor isof orm m and an nd accounted a co ac coun unnte tedd fo forr 2% ooff patients with O r in vvitro Ou itroo analyses it a al an a ys y es ind diccat atee that t at tthe th hee 7* *)ȕȕ5,,EEac acti t va vati t ngg mut utat atio onss aalter ltter e rec ecep ep p mutations. Our indicate 7*)ȕ5,,Eactivating mutations receptor pon po on TGFb2 TGFb TG Fb22 si Fb sign signaling. gnal alin al ingg. in function upon Conclusions - We propose that TGFbRIIb expression is a regulatory mechanism for TGFb2 signal transduction. Dysregulation of the TGFb2 signaling pathway, as a consequence of TGFbRIIb mutations, results in aortic aneurysm pathogenesis. Key words: aneurysm; aorta; cardiovascular diseases; genetics; TGF-beta pathway aneurysm 2 DOI: 10.1161/CIRCGENETICS.112.964064 Introduction Thoracic aortic aneurysm (TAA) is a common progressive disorder involving gradual dilation of the ascending and/or descending thoracic aorta that eventually leads to dissection or rupture. TAAs are often clinically silent and unsuspected until dissection or rupture occurs. The result is significant morbidity and mortality despite advances in surgical and percutaneous treatments for aortic disease. Although TAA is often a feature of Mendelian complex connective tissue disorders such as Marfan syndrome (MFS), Ehlers-Danlos syndrome type IV (EDS), or LoeysDownloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 Dietz syndrome (LDS), most TAAs occur as isolated nonsyndromic disorders. Nonsydromic isorders. Nonsydrom omi om TAA can also be a familial disorder that is usually transmitted in a monogenic autosomal onogeniic aaut tosomal al dominant fashion. approximately TAA. a hi ashi hion on. These on T es Th e e genetically ge triggered TAAs As account account for appro roximately 20% of TA ro A 1-6 Genetic analyses alysses of familial al famillial TAA TAA AA ((FTAA) FTAA A ) havee iidentified denntifieed sseveral e eral ev al chr chromosomal romos osomaal lo os loci. ocii. F Further urthh ur FTA AA ggenes enees has has hi high ig ligh ghte gh tedd di te ise seas asee-caaus as usin i g muta in m mu uta tation onns in i at at least leas astt four as four ggenes: enes en es:: mapping off FTAA highlighted disease-causing mutations myosin heavy a y chain 11 ((MYH11), avy MYH1 MY H111) 1 , a-smooth h muscl muscle le actin actiin ((ACTA2), AC CTA TA2) 2), tran transforming sforming f g growth growth factor faa -10 10 beta receptors I and d II ((TGF TGFERII and d TGFERII RII). I) 77-10 Recentt studies R t di hhave also l iid identified d tif tifi ifii d mutations t in two novel genes, MYLK and SMAD3, that are linked to syndromic aortic aneurysms and dissections.11, 12 Also, disease genes remain to be determined at additional loci such as AAT1 (also known as FAA1) on chromosome 11q2313 and AAT2 (also known as TAAD1) on chromosome 5q13.14 Because of the identification of TGFERI and TGFERII mutations in aortic aneurysm syndromes such as LDS, considerable attention has been devoted to the role that TGFE may play in FTAA pathogenesis. The TGFE receptor superfamily is comprised of cytokines that control numerous diverse cellular processes including cell proliferation, differentiation, angiogenesis and modification of the extracellular matrix (ECM).13-16 Canonical TGFȕ signaling is initiated when 3 DOI: 10.1161/CIRCGENETICS.112.964064 a TGFE ligand binds to TGFERII, resulting in the recruitment of TGFERI. Upon ligand binding, TGFERII activates TGFERI via trans-phosphorylation of its kinase domain and propagates downstream signaling actions. Receptor-regulated (R-) Smads are substrates of the TGFERI kinase and cytoplasmic phosphorylation of R-Smads allows for translocation of the Smad complexes to the nucleus in order to regulate transcription of target genes.17 Previous studies identified mutations in TGFȕRI, TGFȕRII, ACTA2, and MYH11 in individuals with familial TAA. In most cases, genetic screenings for mutations in these genes Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 ith ther th er with wit ithh an eextensive x en xt ensi sivv si have focused primarily on patients referred to genetic subspecialists either ective ti iss ssue u disorder ue dis i or orde d r and, family history or with obvious features of a complex Mendelian connective tissue therefore, these hesse ppatients hes atien nts hhave ave an increased likelihoodd of harboring ng a m mutation. utation. However, su such u reppresent a sm re mall su ubseet of thos se wit th ge eneetica caall call llyy me ediated TAA AA. T AA he vvast ast m ajj individuals represent small subset those with genetically mediated TAA. The majority p with lim mit i ed or unknown un history and and n are without with thou th o t evidence of a of patients present limited familyy history ynd ynd ndro romi ro micc di mi diso sord so rder rd er. Th er Thes esee pa es pati tien ti ents en ts rrepresent epre ep rese re sent se nt ddiagnostic iagn ia gnos gn osti os ticc di ti dile lemm le mmas mm as ffor or ppracticing ract ra ctic ct icin ic ingg in complex syndromic disorder. These patients dilemmas physicians. This study addresses the potential impact of genetic testing for these four TAA genes on clinical management of TAA patients. We determined the frequency of mutations in these four TAA genes in an unbiased population that is more representative of the population of individuals with genetically mediated TAA seen in cardiovascular clinical practice. Methods Patient cohort collection The cohort of patients enrolled in this study consisted of 100 consecutive adult probands from a clinical population with non-syndromic, potentially genetically triggered aortic aneurysms. FTAA patients were collected from those presenting to cardiologists and cardiothoracic surgeons at Weill Cornell Medical Center. Written informed consent was obtained from all subjects 4 DOI: 10.1161/CIRCGENETICS.112.964064 according to a protocol approved by the institutional review board of Weill Cornell Medical College. To enroll, subjects needed to have been diagnosed with thoracic aortic dilation, aneurysm, or dissection and meet at least one of these criteria: 1. Age at diagnosis of aortic disease less than 50 years 2. Positive family history of aortic aneurysm or dissection in at least one 1st or 2nd degree relative 3. Features of a connective tissue disorder, such as arachnodactyly, pectus Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 carinatum, or pectus excavatum. These inclusion criteria were established to represent patients that might reasonably clinically ght reasonab bly ly bbee cl cli in in suspected too have genetically excluded hav avee a ge av gen neti ticcally mediated disorder. Pa ti Patients were excl lud u ed if they met clinical clinii diagnostic criteria MFS, rare syndromes well criteeria for MF cr FS, LDS, LDS, or o EDS D since DS sin nce eetiologies tiolo logies es for for these theese ra aree sy yndrom o ess are om are w ell known and do not ott generally generrallly ppresent rese re s nt ddiagnostic iagnosti ia ticc di ti ddilemmas lemmass ttoo physicians. lemm phhyssici iccians ians. DNA Isolation and Mutation Analysis t tion Mutatiion A naly lysis ly Blood or saliva patients. ali al li a samples le were ere obtained btaii d ffrom r patients atiient Genomic Ge mii DNA DNA was as isolated isolat l ed d from f lymphoblasts separated from whole blood (QIAamp DNA Blood kit, Qiagen) and saliva (Oragene-DNA kit, DNA Genotek) per manufacturer’s instructions. Exons of ACTA2, MYH11, TGFȕRI and TGFȕRII were PCR amplified with gene-specific primers from genomic DNA isolated from each patient. Primer sequences are available upon request. Additional mutational analyses of TGFȕRII focused on an alternatively spliced exon, exon 1a that substitutes a 26 amino acid peptide for Val51 in the receptor’s extracellular domain. This resultant TGFȕRII is often referred to as TGFȕRIIb, and the specific properties and function of TGFȕRIIb are not well documented.18 PCR products were purified by vacuum filtration using a MultiScreen-PCR filter plate 5 DOI: 10.1161/CIRCGENETICS.112.964064 (Millipore) per manufacturer’s instructions. Purified PCR products were bidirectionally sequenced and analyzed on an automated sequencer (ABI 3130XL) with BigDye Terminator v3.1 (Applied Biosystems). Exons with sequence variants were analyzed in family members when available. In addition, a minimum of 200 control chromosomes from a population of ‘normal’ samples (mixed ethnicity unaffected individuals without known aortic disease) were also analyzed either by restriction fragment length polymorphism (RFLP) analysis or denaturing high-performance liquid chromatography (dHPLC) on a WAVE Nucleic Acid Fragment Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 Analysis System (Transgenomic). Additional sets of ethnically matched used ed controls were us e as noted in the results section below. Sequence were mutations u nce vvariants uen aria ar iaantss w ere considered mutation onns if they (1) caused ed d a non-synonomous amino acid change could potentially protein (2) were absent population chaange or cou uld d pot tenti tiially alter alter pro oteein structure, sttruct ctur ure, ur e (2 e, 2) wer re ab bsent ffrom room a po opull of at least 200 ethnically matched control chromosomes, co-segregated with disease 2 et ethn hnic hn i ally ic ym atch at tch hed d con ontr on trol chr tr h om hr omosom omees, and and (3) (33 co co-se o segr gregat gr ated at ed d wit ithh di it dise seas a e in the family if samples also available analysis. addition, i family y member samp ples were als l o avai ilabl ble for an alys ly is i . IIn n ad ddition,, we examined exam m the National Biotechnology Information all Center C te ffor o Bi Biot Biotechnolog hnoll Inffo atiio (NCBI) (NCB (N CBI) I) single ingll nucleotide n cl cleotide l tid ide polymorphism pol oll morphism phi his (SNP) database of 4356 chromosomes for ACTA2, MYH11, TGFȕRI and TGFȕRII polymorphisms in genome build 37.3 released in October 2011. RNA Analyses Total RNA was isolated from either lymphocytes or homogenized human aortic tissue using TRIzol reagent (Invitrogen) per manufacturer’s instructions. RNA was subjected to reverse transcriptase PCR (RT-PCR; One-Step RT-PCR Kit, Qiagen) to preferentially amplify either the TGFȕRII or TGFȕRIIb isoform with exon-specific primers surrounding, or internal to, the alternatively spliced exon. RT-PCR reactions were performed under the following conditions: 50oC for 30 mins (cDNA synthesis), 95oC for 15 mins (polymerase heat activation) followed by 6 DOI: 10.1161/CIRCGENETICS.112.964064 94oC for 45 secs, 52oC for 80 secs and 72oC for 60 secs for 35 cycles. Plasmid Constructs Full-length cDNAs of both TGFȕRII and TGFȕRIIb were reverse transcribed as described above from RNA extracted from patient fibroblasts using primers immediately flanking the coding region of TGFȕRII. RT-PCR products were cloned into the pCR2.1-TOPO vector using the TOPO TA cloning kit (Invitrogen). Plasmid DNA was isolated (QIAprep Miniprep kit, Qiagen). The entire coding region of TGFȕRII and TGFERIIb in each construct was sequenced biDownloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 directionally in each cDNA construct to confirm the correct full-length h sequence for bot both. th. The (Quik ikCh Changee kit, kit itt, H56N TGFȕRIIb mutation was generated by site-directed mutagenesiss (QuikChange Stratagene) per peer manufacturer’s m nu ma n fa facttur urer’s instructions. The D40N D40 4 N TGFȕRIIb TGFȕ FȕRIIbb mu mutation uta tation was generated by Overlap Extension x ension PCR.1199 Initial xte In nitiial PCR P R reactions PC reacctiionns were re per performed rfo form r ed rm d und under derr the he follo following l wi lo winng con conditions: nd m minute, t , 550 0oC for for 2 mi minutes, inute tes, and 772 2oC for 2 minutes minute i tes for 25 cycles. cyc y les. P PCR CR R products prodductt were 95oC for 1 minute subjected to o gel gel eelectrophoresis lect le ctro ct roph ro phor ores or esis es is aand nd ggel el purified pur urif ifie iedd (Q (QIA (QIAquick IAqu quic qu ickk Gel Gel Extraction Extr Ex trac tr acti ac tion on Kit, Kit it,, Qiagen) Qiag Qi agen ag en)) pe en per manufacturer’s ’ iinstructions. i Subsequent S b PCR reactions i were performed f d using i purified ifi d DNA DN from the initial PCR product using the same cycle conditions. Wildtype TGFȕRII as well as wildtype and mutant isoforms of TGFȕRIIb were subcloned into XhoI and BamHI sites of the pcDNA 3.1(-) expression vector 3’ to a cassette encoding a Kozak sequence in order to generate TGFȕRII- or TGFȕRIIb-cDNA3.1. PCR amplification with XhoI-Kozak-TGFEII-F and BamHI-TGFERII-R primers facilitated cloning into pcDNA3.1 (Invitrogen). The entire coding region of each TGFȕRII and TGFȕRIIb construct was bi-directionally sequenced to confirm the correct full-length sequence. Cell Culture, Transfection and TGFE E stimulation A skin biopsy containing primary dermal fibroblasts from an individual harboring the H56N 7 DOI: 10.1161/CIRCGENETICS.112.964064 TGFȕRII mutation was cultured in Dulbecco’s Modification of Eagle’s Medium (DMEM, Mediatech) supplemented with 10% fetal bovine serum (FBS) and 0.1 mg/mL primocin. Cells were maintained and studied at low passage (passages 2-5). Normal human dermal fibroblasts (NHDF) and L6 rat myoblast cells were obtained from ATCC and grown in DMEM with 10% FBS. Low passage primary dermal fibroblasts (passages 2-5) were serum starved for 24 hours and then stimulated with 5 ng/mL recombinant human (rh) TGFȕ1 or rhTGFȕ2 (R&D Systems) Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 in the presence of 10% serum. Following stimulation, cells were washed PBS ed twice in cold P B BS containing 1 mM Na 3 VO 4 , and lysed at 0, 0.5, 1, 2, 4, and 24 hours post-stimulation ost-sttim i ullattion in R RIPA IP M TrisHCl Tris Tr isHC is HC HCl C pH7.6, pH H7.6, 150 mM NaCl, 1% IIGEPAL, G PAL, 0.5% sodium GE soodi d um deoxycholate, 0.1% 0 buffer (50 mM SDS) supplemented lemented lem em mented with protease protteaase in iinhibitors hibi bittorrs (1X bi 1X X Protease Prooteasse In Inh Inhibitor hibiito or C Cocktail ock ckta tail Tablets, ta Tab ablets ab tss, Roche; R Roche oche 1% otein Ph Phos hos o phat attas asee In IInhibitor hi or C hi hibito Cocktail, ock ktail tail ta il, Mi Millipore) illi llipor ll ipore) e)) ffor or sub subsequent ubse ub sequen sequ ent Western We n blot We blott aana bl n na Ser/Thr Protein Phosphatase analysis as described d below. Cell sti stimulation imulati l ion was pe pperformed rfformedd iin n tripl triplicate. p ic i ate. 3x10 05 L6 cells ell lls were ere plat plated l ed d one da dday bbefore eff transfection tr sff tii and ndd transfected t fected d with ith ithh 500 500 ng of either wildtype (wt) or mutant TGFȕRII-cDNA3.1 constructs (wt-TGFȕRII-, wt-TGFȕRIIb-, H56N-TGFȕRIIb- or D40N-TGFȕRIIb-cDNA3.1) in low serum media using LipofectAMINE (Invitrogen). Cells either remained unstimulated or were stimulated with 50 pM TGFȕ1 or 50 pM TGFȕ2 for 0, 0.5, 1 or 2 hours and lysed as described above in RIPA buffer supplemented with protease inhibitors for Western blot analysis. All cell transfections and stimulations were performed in triplicate. SDS-PAGE and Western Blotting Lysate protein concentrations were determined (Coomassie Plus Bradford Assay kit, Pierce). 20 mg of cell lysate were electrophoresed on 10% polyacrylamide gels (Pierce) and transferred to 8 DOI: 10.1161/CIRCGENETICS.112.964064 PVDF membrane (GE Healthcare). Protein expression was determined by Western blotting with primary antibodies to anti-phosphorylated Smad2 (pSMAD2, Cell Signaling) or anti-b-actin. (Sigma). Bound antibodies were detected by incubation with goat anti-rabbit secondary antibody (Cell Signaling) followed by chemiluminescence (ECL Plus, GE Healthcare). Densitometry was performed on a BioRad Gel Doc MultiAnalyst system. Statistical Analysis All values are expressed as mean + SEM or % and 95% confidence intervals for categorical Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 variables. Statistical analyses were performed using ANOVA and Student’s udent’s t-test. P<0.05 P<0. 0.05 0. 0 was considered significant. A Sharp-Wilk normality test of our Western blot lot data datta reve revealed aled l tthat hat it ha followed normal o ma orma mall di dist distributions stri st ribu ri b tiions with a P>0.01 and ha bu had ad eequal qual variance. Bi B Binomial nomial power calculationss indicate inndicate that oour ur ppower ower e to de er ddetect teectt mut mutations utatio ons with witth a prevalence prrevalen en ncee aass lo low ow ass 1% % iiss 555% in cont n rol ch nt hro romo moso s mees aand nd 95 995% % in in 5588 888 con onntrol ol chr hrom hr omosoomes omos es. A es lso so, bi bino nom m a sample off 2000 co control chromosomes control chromosomes. Also, binomial power calculations u ulations indicate th that hat our ppower ower to ddetect etect NC NCB NCBI BI SN S SNPs P withh a prevalence Ps prevalence of 5/4356 5/4355 chromosomes mes is is 21% 21% iin 200 200 chromosomes ch h and d 49 49% % iin n 588 588 chromosomes, chromosomes hr whereas hhereas our o r ddet detection et power is 37% in 200 chromosomes and 74% in 588 chromosomes with a SNP having a prevalence of 10/4356 chromosomes. Results Patient Cohort The cohort consisted of predominantly male patients (77 men and 23 women) (Table 1). This distribution is consistent with previous studies in which men predominate in a series of clinically apparent genetically meditated TAAs.1 The majority of patients were Caucasian (80/100) and the remainder were of Hispanic, African-American, Native American or Asian descent. Patients 9 DOI: 10.1161/CIRCGENETICS.112.964064 ranged in age from 21-93 years old with an average age of 53. The average weight of patients was 85.1 kg and average height was 177.3 cm with an average body surface area of 2m2. Of the 100 patients, 64 were diagnosed at less than 50 years of age. Sixty-seven had a family history of TAA or thoracic aortic dissection. Only 14 exhibited connective tissue abnormalities, including joint hypermobility, pectus excavatum and pectus carinatum, but none met or nearly met diagnostic criteria for Marfan or other syndromes.20 At the time of enrollment, 42 patients had previously undergone aortic surgery. Aortic dissections had been reported in 27 of the patients. Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 Aneurysm Gene Mutational Analyses All patients in this cohort underwent sequencing-based mutational analyses ACTA2, alyses off tthe h AC he ACTA TA22, TA MYH11, 7*)ȕ5,and *)ȕ *)ȕ )ȕ5, 5,an 5, andd 7*)ȕ5,, an 7*)ȕ 7* ) 5,, genes. Sequencing ACTA2 missense mutations, T108M, R118Q, uen ncing of thee AC A CTA TA A2 ggene ene re rrevealed eveealed d tthree hreee mis isse is seens n e mu m tattioonss, T10 08M 8M, R 118 18Q Q and G270E (Fig. g 1) g. 1). ). Tw Two in intronic ntr t on onic i ppolymorphisms ic o ym ol ymor orphisms or ms w were eree se see seen, en, n hhowever, ow wever err, no eexonic xoni nicc poly ni polymorphisms ymo morp rphi his hi were detected. ted. The R118Q 8Q Q mutatio mutation i n in i ffamily amil ilyy JNW il JNW ha hhass been ppreviously reviiouslly repo reported p rted in two oth other TAA families. iies 21 T1 T108M T108 08M M andd G270E G270 G2 70E E were ere not ot ffo found ndd in i 200 200 Caucasian/Northern C Caa casian/Northern ian/N /Northhe E European ropeann control chromosomes. All three affected probands had TAAs, two of which led to acute dissections, and all required surgery. Both the R118Q (Family JNW) and G270E (Family ANS) mutations co-segregated with disease in families. Family members of SY92, who carried the T108M mutation, were unavailable for genetic analysis. Patient SY92 also had an atrial septal defect (ASD) and patent ductus arteriosus (PDA). None of the ACTA2 mutations were found in the NCBI SNP database. Three mutations and 13 polymorphisms were identified in the MYH11 gene (Fig. 2), including two missense mutations (R1590Q and E1899D), and one splice site alteration: a seven nucleotide substitution located five base pairs 3’ to exon 27. None of the variants were detected 10 DOI: 10.1161/CIRCGENETICS.112.964064 in 200 Caucasian/Northern European control chromosomes. The mutations co-segregated with aortic disease in families JNE and ANHH. Family members of patient ANO II-2 (carrying a seven nucleotide substitution located five base pairs 3’ to exon 27) were unavailable for genetic analysis. Individual ANHH II-2 exhibited TAAs and also a bicuspid aortic valve, however, ANO II-2 had a tricuspid aortic valve. Individual JNE II-1 exhibited TAA resulting in two dissections. None of these mutations were identified in the NCBI SNP database except for E1899D MYH11 that was found in 10/4356 chromosomes corresponding to a frequency of Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 0.23%. The significance of this polymorphism is unknown given that the NCBI database se includes patients with cardiovascular disease whose aortic aneurysm status tatus iiss unknown. unkn k own. In our study, we screened chromosomes who weree uunaffected individuals c en creen ened ed d 20 2000 ch hromosomes from controll patients pa naffected individua a without known own aortic disease. ow diseease.. Members were screened mutations TGFȕR type m s of tthe mbers he ccohort ohor oh hortt we w re aalso lso ls s scre reeened re ened e ffor or m utat ut atio at io ons iin n both both h TG GFȕR ty ype I aand ndd type II genes, TGFȕRI FȕȕRII and TGFȕRII. F TGFȕ FȕRI RIII. In In TGFȕRI, T FȕRI TG RI, three th hree exonic exoniic ppolymorphisms olym y orph phhis i ms and andd no mutations were w found. Two polymorphisms mutation TGFȕRII o eexonic onic nii pol l morphisms hi andd one m tation tatii were ere detected detected d iinn TG TGFȕ TGF FȕRI RIII (F (Fig (Fi ig 3). 33)) The Thh T TGFȕRII missense mutation (A414T) in patient KNA II-2 is located in the protein’s kinase domain, co-segregates with disease in the family, and was absent in 206 ethnically matched (Ashkenazi Jewish) control chromosomes. All four family members carrying the mutation were diagnosed with aortic aneurysm. Three of the four also have pectus excavatum, one of whom was also diagnosed with an ostium secundum ASD. There was no evidence of other LDS features such as vascular tortuosity or bifid uvula in probands or family members. This 7*)ȕ5,, mutation was not found in the NCBI SNP database. TGFȕRII has an additional alternatively spliced isoform containing a 75 base pair exon (exon 1a) in its extracellular domain that produces TGFȕRIIb. TGFȕRII exon 1a was also 11 DOI: 10.1161/CIRCGENETICS.112.964064 sequenced in all 100 individuals. No polymorphisms were found in exon 1a, but two missense mutations were identified. The D40N mutation (family KNK) was not detected in 224 Caucasian/Northern European control chromosomes. The H56N mutation (family ANV) cosegregated with disease in the family, and was absent from 588 control chromosomes including 384 ethnically matched (Ashkenazi Jewish) chromosomes. KNK II-1 had a TAA and also a bicuspid aortic valve. ANV II-1 had an aortic aneurysm and pectus carinatum. H56N was not found in the NCBI SNP database, but D40N 7*)ȕ5,,E, identified in patient KNKII-1 for which Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 we were unable to determine cosegregation with disease, was found in chromosomes n 5/4356 chromoso ome m corresponding to a frequency of 0.11%. The significance of this polymorphism unknown morph phhi m is phis i unkno now no wn given that the h NC he NCBI BI database BI dataaba da base includes patients withh cardiovascular cardiovascular disease dis issea e se whose aortic aneurysm status patients tatus ta atu us is unknown. unknoown. Inn our ouur study, stud udy, we ud we sc sscreened creeneed 2224 24 cchromosomes hrom omosom mes from m co control ontrol ppat at who were unaff individuals uunaffected ffec ff eccted teed indi divi vidu idu d al als ls without wiith thou out known ou know ownn aortic ow ao orti rttic ic disease. dis isea ease ease se. In total, o , 9 of 100 probands otal, proba b nds d were ffound oundd to hhave ave a mutation in i one off the th he four ggenes enes analyzed. No genotype-phenotype were observed N genot ot pe phenot he t pe correlations latii ere obser bs ed d amongstt the th h probands’ obb ds’’ available a ail i family members, and mutations within the same gene did not necessarily correspond to any specific phenotypic variation. Families ANV, KNK, and KNA each have TGFȕRII mutations yet exhibit considerable interfamilial variation. Four of the seven individuals with a TGFȕRII mutation (including those with a mutation in the alternatively spliced exon) exhibited noncardiovascular connective tissue abnormality. Two were diagnosed with a congenital heart defect. Although MYH11 mutations have been previously associated with PDA9, none of the individuals with MYH11 mutations detected in the current study were known to have PDA. Livedo reticularis or iris flocculi were not observed in individuals in this study with ACTA2 mutations. 12 DOI: 10.1161/CIRCGENETICS.112.964064 TGFȕRII Expression and Activity Previous studies22 established that TGFER kinase domain mutations inactivate the receptor although downstream TGFE signaling is paradoxically increased. Similarly, we identified the A414T TGFERII mutation in a patient and examined its kinase activity through in vitro expression of A414T-TGFERII in a luciferase vector. Mutant TGFERII was inactive (data not shown). In this study, we sought to understand how the novel TGFERIIb mutations we observed Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 outside of the TGFERII kinase domain might alter TGFE signaling. Tissue expression patterns of TGFȕRII alternative splicing have not been established. Using RT-PCR, -PC PCR, PC R, we we de dete determined term te rmin rm ined in ed that aort rtic rt ic w alll an al aand d both spliced isoforms, TGFȕRII and TGFȕRIIb, are expressed in the humann ao aortic wall lymphocytes e (Fig. es (F 4) aand n aalso nd lso iinn cultured c ltur cu ured dermal fibroblasts fibrooblastts and d ao aaortic rtic smoot smooth othh muscle ot musccle cellss (not shown). d ffec ff eccts ooff mu uta tant ntt TGFȕRIIb TGF GFȕ ȕRI RIIb I oonn TGFȕ Ib TG GFȕ si ign gnal alin al in ng, w To determine the eeffects mutant signaling, wee compared rel relative GFȕ1 and TG TGF Fȕ2 si sign gnal gn alin al ing (indicated ing (ind (i (ind ndic dic icat atted ated ed bby y pSMA pS SMAD2 MA AD2 D2 llevels) e el ev els) s) iin s) n norm no orm rmal al ddermal ermal levels of TGFȕ1 TGFȕ2 signaling pSMAD2 normal fibroblasts with dermal fibroblasts isolated from individual ANV I-1 who is heterozygous for the H56N TGFȕRIIb mutation (Fig 5). Although the specific contributions of canonical TGFE signaling via Smads vs. noncanonical TGFE signaling via MEK/ERK pathways to the pathogenesis of specific aneurysm and aneurysm related phenotypes remain under active investigation,23-25 pSMAD2 provides a valuable biomarker of TGFE activity.26 Upon TGFȕ1 stimulation of normal dermal fibroblasts, we observed an increase in pSMAD2 levels peaking at 0.5 hours post-stimulation before declining by 4 hours. By contrast, TGFȕ1 stimulation of ANV I-1 dermal fibroblasts resulted in delayed SMAD2 phosphorylation that peaked at 2 hours. Upon stimulation with TGFȕ2, normal dermal fibroblasts exhibited similar kinetics to TGFȕ1 stimulation; pSMAD2 levels peaked at 0.5 hours although high levels of pSMAD2 persisted 13 DOI: 10.1161/CIRCGENETICS.112.964064 even 4 hours post–stimulation. TGFȕ2 stimulation of ANV I-1 dermal fibroblasts exhibited distinct kinetics of SMAD2 phosphorylation. Although pSMAD2 levels peaked at 0.5 hours, these levels rapidly declined by 1 hour and were markedly reduced at all time points compared to normal dermal fibroblasts. Since dermal fibroblasts from individual ANV I-1 express both wt- and mutant TGFȕRIIb isoforms, one cannot distinguish the activities of each isoform in these cells. Therefore, we utilized L6 rat myoblast cells lacking both endogenous TGFȕRIIb and TGFȕRIII Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 to compare 7*)ȕVLJQDOLQJDFWLYLWLHVin wt- and mutant TGFȕRIIb. L6 6 myoblasts were transfected with constructs encoding wt-TGFȕRII, wt-TGFȕRIIb, H56N-TGFȕRIIb, 6N-TG TGF TG FȕRI RIIb Ib,, or D40NIb D40 TGFȕRIIb and stimulated preliminary examination a d tthen heen stim st ti ul ulated with 50 pM TGFȕ1 or or TGFȕ2. Our pr prel e iminary examinatio o of pSMAD2 levels post-stimulation evels measured during ev durrinng the the h first firrst 2 hhours ouurs pos ost-st stim imul im ulatio ul ion showed sho howeed that ho att 7*)ȕ5,, 7 )ȕ5, 7* 5,, activity peaked hours a d att 0. aked 00.5 5 ho our urss LQ7*)ȕ-stimulated L 7*) LQ * ȕ ȕ-stimul ulat ul ated at ted e L6 L6 cells, cellls, whereas ce wherea wh h ea eass it i peaked peake kedd aatt 1 hhour ke ourr iin ou n 7*)ȕ-stimulated cells. Therefore, m mulated Thereffore,, in subsequent subbsequ q ent studies, studdie i s,, we assessed assessed d peak p ak pe k 7*)ȕreceptor 7*)ȕ ȕrecept po activity in genetically geneticall tii ll engineered gii ed d L6 cells ell lls following ffollo oll ll iing n stimulation stim sti im llation atiio with iith th h either ithhe TGFȕ1 TGF TG Fȕ1 for fo 00.5 5 hours (Fig. 6) or TGFȕ2 for 1 hour (Fig. 7). L6 cells transfected with either wt-TGFȕRII or wtTGFȕRIIb exhibited comparable responsiveness to TGFȕ1 (Fig 6A, n=3). Similarly, introduction of neither H56N-TGFȕRIIb nor D40N-TGFȕRIIb significantly modified TGFȕ1 responsiveness (Fig 6B, n=3). However, when cells were stimulated with TGFȕ2 (Fig. 7), pSmad2 levels were reduced by 74% in cells transfected with wt-TGFȕRIIb compared to those transfected with wt-TGFȕRII (Fig 7A; n=3, p=0.02). Introduction of either H56N-TGFȕRIIb or D40N-TGFȕRIIb, then, ablated this reduction in receptor activity, and both resulted in a nearly three-fold increase in pSmad2 levels (Fig 7B; n=3, p=0.009 and p=0.02, respectively). 14 DOI: 10.1161/CIRCGENETICS.112.964064 Discussion FTAA is a clinically heterogeneous disorder exhibiting variation in both age of onset and degree of aortic dilatation prior to dissection. FTAA can be part of a complex syndrome, such as LDS, or an isolated finding. The four genes analyzed in this study (ACTA2, 0<+7*)ȕ5,, and 7*)ȕ5,,) were initially identified as associated with syndromic FTAA, and the cause of FTAA in many families remains unknown. The utility of mutational analyses in clinical strategies for isolated FTAA diagnostic workup is unclear. Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 The principal goal of our study was to address the potential value genetic lue of clinical gene neettiic testing of ACTA2, MYH11, TGFȕRI, and TGFȕRII in nonsyndromic FTAA TAA A in in order ordder ttoo improve impr patient caree an and diagnosis. FTAA nd di diag agno ag nosiis. Although these four FT no TAA causative ge ggenes ness are known to be ne prevalent in molecular n cohorts coohorts ascertained ascerrtaained ed d ffor orr m ollecuulaar ggenetic en netic ic studies, stu udi diees, their th heir contribution co ontriibutionn to disease diseeasse in a population relevant clinical studied. study, relev van nt to cli lini nica icall practice practiicee hhas pr as nnot ott ppreviously revi viou iousl slly be been en stu tudi tu died di ed d. In th this is stu udy, dy wee determined the frequency mutations in these cohort frequ q ency y off muta tions i thhese four four TA TAA A ge ggenes nes in i a coho h rt routinely y seen iin cardiology clinical Individuals known MFS, LDS, linii l practice. practice tii IIndi ndi di id als ls ddiagnosed ia ed d with ith ithh kno kn n MFS MF S LDS LDS or EDS EDS were ere excluded. In this study, 9% of patients had a mutation in one of the genes analyzed. 3% of patients had mutations in ACTA2, 3% in MYH11, and 3% in TGFȕRII. No mutations were found in TGFȕRI, consistent with the reported rarity of TGFȕRI mutations outside of LDS.27-29 Previous studies reported higher rates of mutation (14% in ACTA2 and 5-10% in TGFȕRII) than observed here upon screening the same genes.9, 10, 30 Our study differs from those studies whose cohorts may have been ascertained through family-based programs and/or medical genetic clinics to which patients are largely referred if they are believed to have signs or symptoms of known disorders, such as LDS, MFS, and EDS. Patients in those studies are more likely to harbor a mutation in one of these genes, and our cohort may be more representative of 15 DOI: 10.1161/CIRCGENETICS.112.964064 the patient population routinely presenting to cardiovascular clinical practices. Our study provides an estimate of the potential value of genetic testing for mutations in known aortic aneurysm disease genes as part of the diagnostic workup of these patients who are often seen by the general cardiologist or cardiothoracic surgeon. The 95% confidence interval for the point estimate of 9% in our population is consistent with finding a potentially causative mutation in 5-16% of such patients in cardiovascular clinical practices. Genetic testing can be a valuable adjunct for diagnostic management of aortic aneurysm since this disorder often goes Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 undiagnosed until a dissection or rupture occurs. Individuals identifiedd by genetic testingg as a at risk for aortic aneurysm development can undergo interval imaging earlier monitor arlier tto o moni ito t r th thee progression dissection. n off aaortic orti or tiic di ddilation laati tion o and to facilitate intervention interve vent ve n ion prior to rupture ruppture tu and/or dissection tu This study provides foundation future that prro rovi v des a foun unndattioon ffor o fut or turee sstudies tuddiees th hat will wil illl likely lik like kely ly provide proovide dee insight iins nsig ghhtt into into nt how h w ho enhanced diagnos algorithms improve ddiagnostic ossti ticc algo gori rith ithms thms iincorporating ncor orpo or porating po ng routine rou o tiine TAA ou TAA A ggenetic enetticc ttesting en esti es tinng ccan ti a imp an mpro rove ve ppatient at at outcomes and survival. Rega FTAA Regardless, g rddless,, our stud study dy critically crit i icallly ly hhighlights ig ghl h ig i hts the th he need d ffor or further FTA gene identification, triggered aneurysm study ffication icatiio since in mostt genetically geneticall tii ll tri ig d aortic rtiic ane r sm patients tii ts iin our o r st t d hhad no evidence of mutation in any of the genes analyzed. With clinical deployment of exonic and genome wide sequencing that do not rely on family-based analyses, cohorts such as the one followed here will provide a rich source for such gene identification. Although no genotype-phenotype correlation was found in this study, the statistical power to detect correlations may have been inadequate due to the small number of individuals with a mutation. Nonetheless, the study already highlights certain clinical diagnostic hazards. For instance, PDA has been strongly associated with MYH11 mutations. In fact, though, we observed PDA in the setting of ACTA2 mutations as well. Thus, the presence of PDA should not provoke presumption of MYH11 mutations. 16 DOI: 10.1161/CIRCGENETICS.112.964064 TGFȕ signaling has become an emerging target for novel therapies for aortic aneurysms. Previous studies have established that dysregulated TGFE signaling contributes to aortic aneurysms.22, 31 However, a paradox in the mode of pathogenesis obfuscates a clear functional role for TGFE in aneurysm development.16 Identification of TGFȕR mutations in LDS and characterization of novel 7*)ȕ5,,b mutations in an alternatively spliced gene segment in our study highlight the contribution of enhanced TGFE signaling to FTAA. Although previously identified TGFȕR mutations modified the receptors’ kinase domain, this study identifies novel Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 mutations in an alternatively spliced segment of TGFȕRII that is not involved nvvol olve vedd in kinase ve kin inas a e ac as acti activity. ti Functional analyses of several kinase domain mutations have revealed d conse consequent equ quen e t lo en loss loss-ofss-oofss function that a ne at nev nevertheless verthe heelesss ddisplayed isplayed a paradoxical enh enhancement nhancement ooff TG TGFȕ GFȕ signaling in patient patie e By e. By contrast, contrastt, mu utaationns in the aalternatively lterrnative vely sspliced plic pl i ed se ic egm mennt off TG GFȕRI RIIb de ddescribed escc aortic tissue. mutations segment TGFȕRIIb here are unique i e since th ique they y aaugment uggment nt rec rreceptor ecep ptor activity activi activity, ty,, and th ty these hes e e findings findings prompted prompted ro ted us to evaluate eva m ca mica mi call si sign gnif gn ific if ican ic ance an ce of of th thes esee mu es muta tati ta tion ti onss. on the biochemical significance these mutations. Prior mutational analyses of TGFERII have rarely included the alternatively spliced segment.9, 27 Little is known about the function of this alternative receptor isoform.32, 33 A previous study asserted that TGFERII requires an accessory receptor, TGFERIII, for efficient binding of TGFE2 and subsequent signaling.34 Rotzer et al.33 proposed that TGFERIIb alone is capable of binding TGFE1, TGFE2 and TGFE3 whereas del Re et al.32 suggested that TGFERIIb alone is capable of binding only TGFE1 and TGFE3. In contrast to the binding data, del Re et al. further proposed that TGFERIIb mediates in vitro TGFE2 signaling in a dose-dependent manner.32 We demonstrate that the segment encoded by exon 1a does not alter receptor function upon stimulation with TGFE1, but does alter TGFE2 signaling. TGFERIIb has a lower TGFE2– stimulated activity than TGFERII, and mutations in this alternatively spliced segment reverse 17 DOI: 10.1161/CIRCGENETICS.112.964064 this effect, increasing receptor activity levels similar to that of prototypical TGFERII. We then propose that TGFERIIb expression is a regulatory mechanism for TGFE2 signal transduction, and dysregulation of the TGFE2 signaling pathway resulting from TGFERIIb mutations can contribute to aneurysm pathogenesis. Regulation of the TGFE signaling pathway is important in determining cellular outcome and the underlying mechanisms are complex. This pathway depends upon several factors Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 LQFOXGLQJWKHVWRLFKLRPHWULFEDODQFHRI7*)ȕOLJDQGVDQGUHFHSWRUVH[SUHVVHGZLWKLQWKHFHOO n TG TGF TGFb1 Fb1 si sign signaling gnal gn a in al ingg in Although TGFERIIb binds TGFE1,32, 33 we did not observe a change in response to mutant TGFERIIb expression. Upon TGFE2 stimulation of cells ls eexpressing xpre xp ress re ssin ss ingg either in et ei wt-TGFERII II orr wt-TG II wt-TGFERIIb, GFE ERI R Ib,, wee obs observed bsser bser erveed sign significantly g if gn i ican ntlyy le less s TGF ss TGFERIIb G ERIIIb GF I activity act ctiv ct ivit iv i y re relative ela l ti tive v too TGFERII activity. cti cti tivi vity vi t . However, How owev ow ev ver, mutant mutaant n TGFERIIb TG GFERIIb b iisoforms sofo form ms ab ablated blaatedd thiss rreduction ed duction ucc n in in receptor reecept ptorr activity by increasing TGFE2-stimulated TGF FE22-st stim st im muullatted TGFERIIb TGF GFE ER RIIIb activity act ctiv ivityy too levels iv lev e el elss equivalent equi eq uiva ui vale va l nt to that of wtw TGFERII. T The he incre increase easse in nT TGFE2 G E2 ssi GF signaling ign nal a in ng that th hat a we we ob observe bseerv rvee ma may be rrelated elat el ated at ed to to complex comp co m lex stoichiometric interactions at the cell surface between TGFE ligands and various TGFERII isoforms as suggested by del Re et al.32. The precise mechanism whereby TGFE ligand binding may induce receptor activation is conflicting. Some models propose that TGFE ligands bind TGFERII dimers that recruit TGFERI dimers to form a heterotetrameric signaling complex.35 Other models, which propose the existence of inactive preformed complexes of TGFERI and TGFERII dimers36, 37, are supported by potential cooperative TGFb2 ligand binding to a TGFERI-TGFERIIb complex in which the receptors make physical contact32. Krishnaveni et al. suggest that TGFERIIb favors heterodimerization with TGFERII because this interaction is more robust.38 Overall, these data suggest a complex TGFE signaling process further depending upon 18 DOI: 10.1161/CIRCGENETICS.112.964064 the stoichiometric interactions between TGFE ligands and various receptor isoforms. Further investigation in vivo of these interactions will add to our understanding of aortic aneurysm pathogenesis. Aberrant TGFȕsignaling that result from type I and II receptor mutations has been implicated in the pathogenesis of cardiovascular disorders involving TAAs. We showed that TGFȕ2 signaling is decreased in cells expressing TGFȕRIIb and mutations in this receptor result in increased TGFȕ2 signaling. Identification of TGFȕRIIb activating mutations in two TAA Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 patients supports the hypothesis that an increase in TGFȕ signaling contributes ntributes to aortic pathogenesis. Furthermore, this evidence highlights the scientific and clinic clinical i all iimport mport off expanding diag alternative ddiagnostic ag gno nost stic st ic strategies strat ateegies to include the altern at nat ative segmentt of TGFȕRIIb TGFȕ TG FȕRIIb in genetic screening of individuals with Taken findings suggest that TGFȕRIIb of in ndividuals w ith TAA. TAA A. Take k n together, to ogeeth her,, tthese hesee fi findin ngs sug ugggeestt tha at TG TGF FȕRI RIIb RI expression is like likely important regulatory k ly an impo p rtant regu g latory mechanism mechhani nism off TGFȕ2 TGF TG Fȕ2 signaling signal i al aling in in the aorta, and ann that there may be contributions TGFE1 TGFE2 signaling be ddifferential iffe if fere rent re ntia nt iall co cont ntri nt ribu buti tion onss of T on GFE GF E1 and and TG TGF FE2 si sign gnal gn alin ingg to aneurysm ane neur urys ur ysm ys m pathogenesis. Acknowledgments: We are grateful to the family members, cardiologists and cardiothoracic surgeons at Weill Cornell Medical Center for their participation in this study. We thank Dr. Harry Ostrer for contributing the Ashkenazi Jewish control samples. Funding Sources: This work was supported by grants from NIH [R01 HL61785 (C.T.B., C.J.H.)], the Snart Cardiovascular Fund [C.J.H.] and Raymond and Beverly Sackler [C.J.H.]. Conflict of Interest Disclosures: None. References: 1. Coady MA, Davies RR, Roberts M, Goldstein LJ, Rogalski MJ, Rizzo JA, et al. Familial patterns of thoracic aortic aneurysms. Arch Surg. 1999;134:361-367. 2. Palmieri V, Bella JN, Arnett DK, Roman MJ, Oberman A, Kitzman DW, et al. Aortic root 19 DOI: 10.1161/CIRCGENETICS.112.964064 dilatation at sinuses of valsalva and aortic regurgitation in hypertensive and normotensive subjects: The Hypertension Genetic Epidemiology Network Study. Hypertension. 2001;37:12291235. 3. Bella JN, MacCluer JW, Roman MJ, Almasy L, North KE, Welty TK, et al. Genetic influences on aortic root size in American Indians: the Strong Heart Study. Arterioscler Thromb Vasc Biol. 2002;22:1008-1011. 4. Albornoz G, Coady MA, Roberts M, Davies RR, Tranquilli M, Rizzo JA, et al. Familial thoracic aortic aneurysms and dissections--incidence, modes of inheritance, and phenotypic patterns. Ann Thorac Surg. 2006;82:1400-1405. Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 5. Devereux RB, de Simone G, Arnett DK, Best LG, Boerwinkle E, Howard BH, et al. TwoDimensional Echocardiographic Aortic Root Dimensions in Adolescents and Adults: Normal Limits in Relation to Age, Body Size and Gender. J Am Coll Cardiol. 2010;55:A87.E824. 2010;55:A87.E824 24.. 24 6. Devereux RB, Cooper RN, Weder A, Seto TB, Hanis C, Mosley TH, and H, et al. all. 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J Med Genet. 2009;46:607-613. 28. Singh KK, Rommel K, Mishra A, Karck M, Haverich A, Schmidtke J, et al. TGFBR1 and TGFBR2 mutations in patients with features of Marfan syndrome and Loeys-Dietz syndrome. Hum Mutat. 2006;27:770-777. 29. Stheneur C, Collod-Beroud G, Faivre L, Gouya L, Sultan G, Le Parc JM, et al. Identification of 23 TGFBR2 and 6 TGFBR1 gene mutations and genotype-phenotype investigations in 457 patients with Marfan syndrome type I and II, Loeys-Dietz syndrome and related disorders. Hum Mutat. 2008;29:E284-295. Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 30. Akutsu K, Morisaki H, Okajima T, Yoshimuta T, Tsutsumi Y, Takeshita S, et al. Genetic analysis of young adult patients with aortic disease not fulfilling the diagnostic criteria for Marfan syndrome. Circ J. 2010;74:990-997. 31. Mizuguchi T, Collod-Beroud G, Akiyama T, Abifadel M, Harada N, Morisaki N M oris or isak is akii T, ak T eett al al. Heterozygous TGFBR2 mutations in Marfan syndrome. Nat Genet. 2004;36:855-860. 004;3 36: 6 85 8555 86 8600. 32. del Re E, Ba receptor, the Babi Babitt biitt JL, JL, L Pirani Pir irani A, Schneyer AL, Linn HY. H . In the absence HY absen nce of type III receptor transforming II-B receptor requires receptor bind ng growth ng growth factor facttor ((TGF)-beta fa TGF) TG F))-be beeta ttype yp pe II I-B B rec ceptorr re requ q ir ires es tthe he ttype ypee I re ece c ptor pttor or tto o bin n TGF-beta2.. J B Biol iol Chem. 22004;279:22765-22772. 004;;2799:2 227765--222772 72. 33. Rotzer D, M,, Lu Lutz M,, Li Lindemann D,, S Sebald D Roth Roth M Ro L tz M tz L nddem emann m D ebal eb bald ld W, W Knaus Kna nauus P. P. Type T pe III Ty II TGF-beta TGF-be b ta be ta receptorrec ece independent alternatively n signalling off TGF-beta2 nt T FTG F-be beta be t 2 via ta via TbetaRII-B, Tbet Tb etaR et aRII aR III-B,, an an alte tern te rnnat ativ iv vel elyy sp sspliced lice li cedd TGF-beta type ce typp II receptor. Embo 2001;20:480-490. m J. 2001;2 mbo ; 0:48 4800-49 48 4990. 34. Lopez-Casillas JL, Massag Massague Betaglycan Casillas C Ca sill ill F, F Wrana Wr JL M a e JJ. Betagl B etagll can presents nt ligand li ndd to t the th h TGF TGF beta beta signaling receptor. Cell. 1993;73:1435-1444. 35. ten Dijke P, Yamashita H, Ichijo H, Franzen P, Laiho M, Miyazono K, et al. Characterization of type I receptors for transforming growth factor-beta and activin. Science. 1994;264:101-104. 36. Chen RH, Moses HL, Maruoka EM, Derynck R, Kawabata M. Phosphorylation-dependent interaction of the cytoplasmic domains of the type I and type II transforming growth factor-beta receptors. J Biol Chem. 1995;270:12235-12241. 37. Zhu HJ, Sizeland AM. Extracellular domain of the transforming growth factor-beta receptor negatively regulates ligand-independent receptor activation. J Biol Chem. 1999;274:2922029227. 38. Krishnaveni MS, Hansen JL, Seeger W, Morty RE, Sheikh SP, Eickelberg O. Constitutive homo- and hetero-oligomerization of TbetaRII-B, an alternatively spliced variant of the mouse TGF-beta type II receptor. Biochem Biophys Res Commun. 2006;351:651-657. 22 DOI: 10.1161/CIRCGENETICS.112.964064 Table 1. Description of Cohort Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 Gender (n) Male Female 77 23 Ethnicity (n) Caucasian Caucasian/Hispanic Caucasian/Native American African American African American/Hispanic Asian Other 80 6 2 2 2 2 6 Age at Enrollment (years) Mean Median Range 53 52 21-93 Body Surface ace ac ce Area (BSA (BSA, A, m2) Mean (n=65) n=6 n=6 65) Median 1.9 1.99 99 22.00 2. 00 Aortic Surgery gery (n) 4 42 Dissection (n) (n) 27 Inclusion Criteria (n) Diagnoses <50 years old Connective Tissue Abnormality Family History 64 14 67 Figure Legends: Figure 1. Mutational analysis of ACTA2 in families ANS, JNW and SY92. A. Automated sequence analyses of ACTA2 from affected individuals in families ANS, JNW and SY92 are shown. A 1-bp deletion in families ANS, JNW and SY92 (arrow) generates a missense mutation indicated by the amino acid code. B. Pedigrees of families ANS, JNW and SY92 with an ACTA2 23 DOI: 10.1161/CIRCGENETICS.112.964064 mutation indicate that mutations co-segregate with disease. Mutations are present only in family members affected with TAA. Arrow indicates family proband. Figure 2. Mutational analysis of MYH11 in families JNE, ANHH and ANO. A. Automated sequence analyses of MYH11 from affected individuals in families JNE, ANHH and ANO are shown. A 1-bp deletion in families JNE and ANHH (arrow) generates a missense mutation indicated by the amino acid code. A 7-bp intronic substitution in family ANO (arrow) produces Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 a splice site alteration located at the nucleotide position relative to the preceding exon. B. Pedigrees of families JNE, ANHH and ANO are shown and indicate that MYH11 hat M YH11 YH 11 mutations muttati tioons coti segregate with w h ddisease. issea ease se.. Mu se Mutations are present only iin n family memberss aaffected ffected with TAA. Arrow indicates proband. catees family pr ca roban nd. Figure 3. TGFbRII and TG TGFbIIb mutations T GFbIIIb mutati ions in in families famil i ie il i s KNA, KNA, A, ANV ANV V and d KNK. KNK N . A. Automated Autom m sequence analyses KNA, ANV nall ses off T TGFbRII GFbR GF bRII II and ndd TGFbRIIb TGF GFbbRI RIIb Ib from f aff affected ffected d indi iindividuals ndi di id d als ls iin families famili ili KNA KNA A and KNK are shown. A 1-bp deletion in families KNA, ANV and KNK (arrow) generates a missense mutation indicated by the amino acid code. B. Pedigree of family KNA with a TGFERII mutation and families ANV and KNK with TGFbRIIb mutations indicate that mutations co-segregate with disease. Mutations are present only in family members affected with TAA. Arrow indicates family proband. Figure 4. mRNA expression of TGFERII isoforms in aortic tissue and lymphocytes. RT-PCR performed using various pairs of TGFbRII and TGFbRIIb isoform specific primers to amplify 24 DOI: 10.1161/CIRCGENETICS.112.964064 exons 1, 1a, 2 and 3 (a), exons 1, 2 and 3 (b) and exons 1a, 2 and 3 (c) from isolated human aortic tissue (lanes 1-2) and lymphocyte (lanes 3-4) RNA. Figure 5. TGFE1 and TGFE2 signaling in dermal fibroblasts. A-B. Cultured fibroblasts were isolated from normal patient with wt-TGFbRII and ANV I-1 patient with H56N-TGFbRIIb mutation. Representative Western blot analyses shown for pSMAD2 protein expression in wtTGFbRII or H56N-TGFbRIIb fibroblasts stimulated with either TGFb1 (A) or TGFb2 (B) for Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 indicated time points. Corresponding b-actin blots shown. (A) H56N-TGFbRIIb -TGFbRIIb dermal dermaal fibroblasts exhibit delayed TGFE1 signaling compared to normal dermal fibroblasts. H56Nmal fibr b oblasts.. (B) (B) H TGFbRIIb der ddermal e ma er m l fi ffibroblasts brroblaasts exhibit decreased TG TGFE2 GFE2 signaling g comp compared mp pared to normal dermal derm m fibroblasts. Figure 6. ,QYLWUR7*)ȕ-VWLPXODWLRQGRHVQRWDOWHU7*)ȕ5,,DQG7*)ȕ5,,EDFWLYLW\. Q YLW LWUR UR 7*) *)ȕ ȕ-VW ȕ VWLP VW LPXO LP XODW XO DWLR DW LRQQGR LR GRHV GR HV QRW DOW OWHU HU 7*) *)ȕ5 ȕ ,, DQG 7*) ȕ5 *)ȕ5 ȕ5,, ȕ5 ,,EEDF ,, DFWL DF WLYL WL YLW\ YL W\. A. W\ A. Relative quantification of pSmad2 expression after in vitro 7*)ȕVWLPXOation. Bar graph displays densitometric quantification of pSmad2 protein expression relative to b-actin in untransfected control, wt-7*)ȕ5,,DQGZW-7*)ȕ5,,E/FHOOVQ 1RVLJQLILFDQWFKDQJHLQ 7*)ȕ5,,RU7*)ȕ5,,E activity compared to control cells. B. Relative quantification of pSmad2 expression after in vitro 7*)ȕVWLPXODWLRQ%DUJUDSKGLVSOD\VGHQVLWRPHWULFTXDQWLILFDWLRQRI pSmad2 relative to b-actin in control wt-7*)ȕ5,,E+1-7*)ȕ5,,EDQG'1-7*)ȕ5,,E/ cells (n=3). No significant change in aFWLYLW\LQPXWDQW7*)ȕ5,,EFHOOVFRPSDUHGWRZW7*)ȕ5,,EFHOOVLQresponse to 0.5 hour S07*)ȕVWLPXODWLRQ. Values normalized to control L6 cells. All data presented as mean+SEM. P values shown. NS= not significant. 25 DOI: 10.1161/CIRCGENETICS.112.964064 Figure 7. In vitro 7*)ȕ-VWLPXODWLRQDOWHUV7*)ȕ5,,DQG7*)ȕ5,,EDFWLYLW\ A. Relative quantification of pSmad2 expression after in vitro 7*)ȕVWLPXODWLRQ%DUJUDSKGLVSOD\V densitometric quantification of pSmad2 protein expression relative to b-actin in untransfected control, wt-7*)ȕ5,,DQGZW-7*)ȕ5,,E/FHOOVQ wt-7*)ȕ5,,EFHOOVH[KLELWGHFUHDVHG 7*)ȕVLJQDOLQJFRPSDUHGWRZW-7*)ȕ5,,FHOOV B. Relative quantification of pSmad2 expression after in vitro 7*)ȕVWLPXODWLRQ%DUJUDSKGLVSOD\VGHQVLWRPHWULFTXDQWLILFDWLRQRI pSmad2 relative to b-actin in control wt-7*)ȕ5,,E+1-7*)ȕ5,,EDQG'1-7*)ȕ5,,E/ Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 cells (n=3). Mutant 7*)ȕ5,,E cells exhibit increased response after 1 hour S07*)ȕ S07*)ȕ )ȕ )ȕ stimulation compared to wt-7*)ȕ5,,EFHOOV Values normalized to control L6 cells. ntroll L 6 cell lls. All ll Alll data dat presented aass m mean+SEM. eann+SEM ea SE EM. P values shown. 26 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 TGFβRIIb Mutations Trigger Aortic Aneurysm Pathogenesis by Altering TGFβ2 Signal Transduction Katharine J. Bee, David C. Wilkes, Richard B. Devereux, Craig T. Basson and Cathy J. Hatcher Downloaded from http://circgenetics.ahajournals.org/ by guest on November 17, 2016 Circ Cardiovasc Genet. published online October 24, 2012; Circulation: Cardiovascular Genetics is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2012 American Heart Association, Inc. All rights reserved. Print ISSN: 1942-325X. Online ISSN: 1942-3268 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circgenetics.ahajournals.org/content/early/2012/10/24/CIRCGENETICS.112.964064 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation: Cardiovascular Genetics can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. 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