Abbate, Rosanna

Attanasio, Monica

Chiarini, F

Evangelisti, L

Gensini, Gian Franco

Lapini, I

Lucarini, L

Pepe, Guglielmina

Porciani, Maria Cristina

English

Florence Research

May TGFBR1 act also as low penetrance allele in Marfan syndrome?Laura Lucarini a,⁎, Lucia Evangelisti a, Monica Attanasio a, Ilaria Lapini a, Francesca Chiarini a,Maria Cristina Porciani a, Rosanna Abbate a, GianFranco Gensini a,b, Guglielmina Pepe aa Department of Medical and Surgical Critical Care, University of Florence; Marfan Centre, Azienda Ospedaliero-Universitaria Careggi, Florence;Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies,University of Florence, Florence, Italyb Fondazione Don Carlo Gnocchi ONLUS, Centro S.Maria degli Ulivi-IRCCS, Florence, ItalyReceived 31 May 2007; accepted 1 July 2007Available online 22 October 2007AbstractMarfan syndrome, a human disease involving cardiovascular and skeletal apparatuses and ocular and central nervous systems, isassociated to mutations in FBN1 gene; heterozygous mutations in TGFBR2 and TGFBR1 genes were found associated to MFS type 2,characterized by the presence of skeletal and cardiovascular major criteria and absence of eye major criterion.We screened the TGFBR1 gene in 46 Marfan patients in whom mutations in FBN1 and TGFBR2 genes were excluded and the analysis ofEx1 was extended to additional 114 Marfan patients and 237 controls.We detected two potentially pathological sequence variants: the TGFBR1 6Ala allele whose frequency was higher in the group of Marfanpatients (0.13) than in the controls (0.08) (p=0.013; OR=1.69) and an insertion of 20 nucleotides in the 5′UTR that turned out to be afamilial silent rare polymorphism. We hypothesize that TGFBR1 sequence variants may act not only as major, but also as low penetrancealleles of the clinical phenotype in Marfan syndrome.© 2007 Elsevier Ireland Ltd. All rights reserved.Keywords: TGFBR1; Marfan syndrome; MFS type 2; TGFBR1 6Ala alleleMarfan syndrome (MFS;MIM#154700) is a multisystemicdominantly inherited connective tissue disorder with aprevalence of 1 in 5000–10000 [1]; MFS is mostly causedby mutations in fibrillin-1 (FBN1;MIM#134797;chr.15q21.1)[2]. MFS type 2 (MFS2;MIM#154705) is characterized byabsence of major ocular involvement; it has been recentlyassociated to heterozygous dominant mutations in bothTGFBR2 (MIM#190182;chr.3p22) [3–5], and TGFBR1(MIM#190181;chr.9q33-34) [4,5], the two genes coding forthe receptors of transforming growth factor beta (TGFβ).TGFβ induces extracellular matrix (ECM) formation bybinding to its two receptors and by activating the intracellularsignalling through SMAD proteins [6]. Mutations in FBN1,TGFBR1 and TGFBR2 lead to altered TGFβ signallingresulting in degenerative changes of the ECM and causinganeurysms and dissections of the vessel wall in MFS2 [3,7],Thoracic Aortic Aneurysms and Dissections (TAAD;MIM#608967) [4,7], and Loeys–Dietz syndrome (LDS;MIM#609192) [5,8,9]. Noteworthy, in a mouse model ofMFS, excessive TGFβ signalling was demonstrated tocontribute to the formation of aortic aneurysms; furthermore,treatment with TGFβ antagonists was shown to block andreverse the demolition of ECM and consequentially thedilatation and dissection of aorta [10].We investigated 46 MF patients (34 men and 12 women,median age 32.5 years, range 7–54 years) diagnosed accordingto Ghent criteria, referred to the Center for Marfan Syndromeand Related Disorders of the Azienda Ospedaliero-Universi-taria, Careggi, University of Florence.DNA mutation analysis of FBN1 [11], TGFBR2 [3] andTGFBR1 genes (Table 1) was performed [11,12]. Clinicalcharacteristics and molecular data of these patients (8 ofthem with MFS2) are shown in Table 2. No mutations inboth FBN1 and TGFBR2 were found. We detected twopotentially pathological mutations both in exon 1 ofTGFBR1. In order to verify their pathogenicity, we ex-tended the analysis to 114 Marfan patients (69 men and 45women, median age 35.5 years, range 7–70 years; 18MFS2, 103 carrying a mutation in FBN1) and 237 healthyunrelated controls (111 men and 126 women, median age51.2 years, range 15–91 years) with primers Ex1ins andEx1-9⁎ (Table 1). The different alleles were separated on a4% agarose gel. Patients and controls, all Italian, gave their⁎ Corresponding author. Department of Medical and Surgical CriticalCare, University of Florence, Viale Morgagni 85, 50134 Florence, Italy. Tel.:+39 055 7949420; fax: +39 055 7949418.E-mail address: lauralucarini@gmail.com (L. Lucarini).281Letters to the EditorTable 1Primers of TGFBR1 geneTGFBR1 gene Forward Reverse TM Size (bp)EX1 5′-agc agt tac aaa ggg ccg g-3′ 5′-gcg cca tgt ttg aga aag ag-3′ 60 °C 313EX1ins 5′-gga cgc gcg tcc tcc gag c-3′ 5′-gac cgc cgc ctc cat ggt c-3′ 62 °C 157EX1-9⁎ 5′-gac cat gga ggc ggc ggt c-3′ 5′-gtc gcc ccc ggg agc ag-3′ 62 °C 99EX2 5′-gag caa caa aca gtg cat aga aa-3′ 5′-tcc ctt cca gtt cta aaa tca ca-3′ 58 °C 448EX3 5′-ggc tct ttg gct aag tgg tg-3′ 5′-ttc tag caa gtt ggc tta tta g-3′ 58 °C 449EX4a 5′-tca gtt ttc tgg gtc act cat t-3′ 5′-gtt ctt ctc tag agg aga ata tc-3′ 58 °C 237EX4b 5′-gtt tgg aga gga aag tgg c-3′ 5′-gtc tca tct act ttg atg atg g-3′ 56 °C 228EX5 5′-ttg cag tgt gtg act cag ga-3′ 5′-agg ccc agc ctc cac ctt-3′ 60 °C 351EX6 5′-ttt ggg ttg gga gaa gag ac-3′ 5′-ttt ggg ttg gga gaa gag ac-3′ 58 °C 399EX7 5′-tgt ctg aaa gga ggt tca tcc-3′ 5′-aat tct tga aca act tct gct c-3′ 60 °C 256EX8 5′-gcc ttg gca tta gct gaa taa-3′ 5′-cat tgg ttt gac tgc tat gaa aa-3′ 58 °C 316EX9a 5′-cgt aaa aat tct tat cca gac c-3′ 5′-cct tct gtt ccc tct cag tg-3′ 60 °C 300EX9b 5′-ctt cag atc tgc tcc tgg g-3′ 5′-cat tca aag tct gaa tca agg-3′ 60 °C 429EX9c 5′-gta cat tct cag agg att ctg-3′ 5′-gaa tac caa atg aca tac cac-3′ 60 °C 403⁎ = primers modified from [13].Accession number NC_000009.10 for TGFBR1 gDNA.informed consent and the study was approved by the localethics committee of the Faculty of Medicine, University ofFlorence.The 6Ala allele polymorphism in Ex1 (Fig. 1; Table 3a)[5,13] was a trinucleotide GCG, corresponding to alanine(Ala), repeated 6 or 9 times. The 6Ala allele was the rarevariant (Fig. 2).Genotype distribution and allele frequency were inHardy–Weinberg equilibrium for the 6Ala allele polymor-phism both in patients and controls. A 9Ala/7Ala genotypewas detected in one control.The 6Ala allele frequency significantly differed between thegroup of Marfan patients and controls (p=0.013; 0.13vs.0.08).The genotype distribution of the 6Ala allele polymorphismwas higher in the group of Marfan patients (9Ala/6Ala 21.9%and 6Ala/6Ala 2.5%) than in the controls (9Ala/6Ala 16%).Furthermore, the homozygous genotype 6Ala/6Ala wasdetected only in 4 MF patients (Table 3b). The odds ratiowas 1.69 (95%CI:1.023–2.785), suggesting that the 6Alaallele may contribute to Marfan syndrome phenotype.The 6Ala allele frequency was 0.13 in both classic MFSand MFS2, although the second group is still too small.No significant different distribution of the 6Ala allele wasfound between genres and between single systems or organs'involvement.The heterozygous insertion of 20nts in Ex1-5′UTR(Fig. 1; Table 3a) was identified in the proband, hishealthy son, and an affected niece of one Marfan family. Itwas a duplication of 20nts (nts 3–22), inserted betweennts 22 and 23 of exon 1, 55nts before the ATG start ofprotein translation. It is located in a sequence that does notseem to contain any site for transcription binding proteinsFig. 1. Sequence variants found in Exon 1 of TGFBR1: A: insertion of the 20 duplicated nucleotides in the Ex1-5′UTR; B: the TGFBR1 6Ala allele is indicatedand the 9nts (3Ala) deleted in the polymorphism are highlighted. Nts = nucleotides; AA = aminoacids; Ala = alanine.Fig. 2. PCR fragments of homozygous and heterozygous genotypes of TGFBR16Ala allele polymorphism and homozygous genotype of normal TGFBR1 9Alaallele on agarose gel (4%). Lanes 1 and 6 = molecular markers; lanes 2 and 5 =9Ala/9Ala genotype; lane 3 = 9Ala/6Ala genotype; lane 4 = 6Ala/6Alagenotype. 9Ala = 319 bp; 6Ala = 310 bp. Ala = alanine; bp = base pairs.282 Letters to the EditorTable 2Clinical manifestations and mutations found in 46 Marfan patientsAge(yrs)Sex Major criteria Minor criteriaFamilial history CV Ocular Skel Skin Pneum TGFBR1 6Ala genotype1 36 F + − + + + − 9Ala/9Ala2 35 M + + − − + − 9Ala/9Ala3 38 M − § + − + − − 9Ala/9Ala4 42 M − ⁎ + − − + − 9Ala/9Ala5 46 M − ⁎ + − − + − 9Ala/9Ala6 46 F − + + + + − 9Ala/9Ala7 40 M − § + − + + − 9Ala/9Ala8 11 M − + + − − − 9Ala/9Ala9 34 F + § + − + + − 9Ala/6Ala10 24 M − § + − + + + 9Ala/9Ala11 8 M − + + − − − 9Ala/9Ala12 23 F + § + − + + − 9Ala/9Ala13 44 M + + − − + − 9Ala/9Ala14 13 M + + + + − − 9Ala/9Ala15 30 M + + − − + − 9Ala/9Ala16 42 M + + − − + − 9Ala/9Ala17 40 M + + + − + − 9Ala/9Ala18 33 F + + + − − − 9Ala/9Ala19 36 M + + − − + − 9Ala/6Ala20 42 M − § + − + + − 9Ala/6Ala21 25 F + − − + + − 9Ala/9Ala22 22 M − + + − + − 9Ala/9Ala23 34 F − + + − − − 9Ala/9Ala24 30 M − ⁎ + − − + − 9Ala/9Ala25 34 F − + + − − − 9Ala/9Ala26 42 M − + + − − − 9Ala/9Ala27 30 M − ⁎ + − − + − 9Ala/9Ala28 44 M + + − − + − 9Ala/9Alains 20nts hetero29 45 M + + − − + − 9Ala/9Ala30 32 M − ⁎ + − − + − 9Ala/9Ala31 33 F − ⁎ + − − + − 9Ala/6Ala32 40 M − ⁎ + − − + − 9Ala/9Ala33 45 M − ⁎ + − − + − 9Ala/9Ala34 39 F − ⁎ + − − + − 9Ala/6Ala35 20 F − + + − + − 9Ala/9Ala36 15 M − ⁎ + − − + − 9Ala/6Ala37 40 M − ⁎ + − − + − 9Ala/9Ala38 9 M − + + − − − 9Ala/9Ala39 28 M − ⁎ + − − + − 9Ala/9Ala40 25 M + + − − + − 9Ala/9Ala41 7 M − § + − + − − 9Ala/9Ala42 32 M + + − − + − 9Ala/9Ala43 54 F − § + − + + − 9Ala/9Ala44 44 M + + + − + − 9Ala/9Ala45 22 M + + − − + − 6Ala/6Ala46 43 M + + + + + − 9Ala/6AlaWt = wild type; yrs = years; M = male; F = female; CNS = central nervous system; CV = cardiovascular system; skel = skeletal system; Pneum = pneumothorax;⁎ = presence of dural ectasia, required for diagnosis of Marfan; § = diagnosis of MFS2. Data in bold indicates patients with the polymorphism.nor translational regulatory sequences; therefore, this maybe considered a “silent” and private rare polymorphism.The frequencies of three known polymorphisms appearedto be higher in patients than in controls (Table 4).In vitro studies on TGFβ type I receptor showed that themolecular differences between 6Ala and 9Ala are due toaltered 6Ala signal sequence; this mechanism may resulteither from direct transcriptional inactivation or fromalterations of TGFβ signalling pathways [14]. The 6Alasignal sequence could directly interact with regulatoryelements of the signalling pathway genes [14].A highly significant association between the 6Ala alleleof TGFBR1 gene and patients with Marfan syndrome is heredemonstrated for the first time; moreover, homozygosity for283Letters to the EditorTable 3aModifier mutations in TGFBR1 gene in Marfan patients and healthy controlsEX/IVS Position (nt) Status Patients (n=160) Genotype distribution Controls (n=237) Genotype distributionEx1-5′UTR ins 20nts (22/23) Novel Hetero 1 ins=0.006 0 ins=0Ex1 del 9nts (146-154) Known a Homo 4 6Ala/6Ala=0.025 0 9Ala/6Ala=0.16 bHetero 35 9Ala/6A=0.219 38a [13].b Frequency from literature (9Ala/6A=0.14).Table 3bTGFBR1 6Ala genotype distribution and allele frequency in MFS patients versus controlsGenotype distribution MFS patients (n=160) Controls (n=237) pTGFBR1 6Ala polymorphism9Ala/9Ala 121 (75.6%) 199 (84.0%)9Ala/6Ala 35 (21.9%) 38 (16.0%)6Ala/6Ala 4 (2.5%) 0 0.014 aTGFBR1 6Ala allele frequency 0.13 0.08 0.013 ba Genotype distribution.b Allele frequency.Table 4Polymorphisms in TGFBR1 gene in Marfan patients and healthy controlsEX/IVS Position (nt) Status Patients (n=46) Genotype distribution Controls (n=92) Genotype distributionIVS7 +24 GNA Novel Hetero 16 A=0.35 26 A=0.28IVS8 del 5nts+(90–94) Novel Hetero 16 del=0.35 25 del=0.27Ex9-3′UTR +69 ANG Known a Hetero 13 G=0.28 25 G=0.27nt = nucleotide; ins = insertion; del = deletion.a [5].the 6Ala allele was found only in patients. For this reason, atthe moment it may be defined as a low penetrance allele. Alarger number of patients is required to investigate thepossibility that 6Ala allele may act as modifier mutation.References[1] Judge DP, Dietz HC. Marfan's syndrome. Lancet 2005;366:1965–76.[2] Collod-Beroud G, Le Bourdelles S, Ades L, et al. Update of the UMD-FBN1 mutation database and creation of an FBN1 polymorphismdatabase. Hum Mutat 2003;22:199–208.[3] Mizuguchi T, Collod-Beroud G, Akiyama T, et al. HeterozygousTGFBR2 mutations in Marfan syndrome. Nat Genet 2004;36:855–60.[4] Matyas G, Arnold E, Carrel T, et al. Identification and in silico analysesof novel TGFBR1 and TGFBR2 mutations in Marfan syndrome-related disorders. Hum Mutat 2006;27:760–9.[5] Singh KK, Rommel K, Mishra A, et al. TGFBR1 and TGFBR2mutations in patients with features of Marfan syndrome and Loeys–Dietz syndrome. Hum Mutat 2006;27:770–7.[6] Lu SL, Kawabata M, Imamura T, Miyazono K, Yuasa Y. Two divergentsignaling pathways for TGF-beta separated by a mutation of its type IIreceptor gene. Biochem Biophys Res Commun 1999;259:385–90.[7] Pannu H, Fadulu VT, Chang J, et al. Mutations in transforming growthfactor-beta receptor type II cause familial thoracic aortic aneurysmsand dissections. Circulation 2005;112:513–20.[8] LoeysBL,Chen J,NeptuneER, et al.A syndrome of altered cardiovascular,craniofacial, neurocognitive and skeletal development caused by mutationsin TGFBR1 or TGFBR2. Nat Genet 2005;37:275–81.[9] Loeys BL, Schwarze U, Holm T, et al. Aneurysm syndromes causedby mutations in the TGF-beta receptor. N Engl J Med 2006;355:788–98.[10] Habashi JP, Judge DP, Holm TM, et al. Losartan, an AT1 antagonist,prevents aortic aneurysm in a mouse model of Marfan syndrome.Science 2006;312:117–21.[11] Pepe G, Giusti B, Evangelisti L, et al. Fibrillin-1 (FBN1) geneframeshift mutations in Marfan patients: genotype–phenotype corre-lation. Clin Genet 2001;59:444–50.[12] Giusti B, Lucarini L, Pietroni V, et al. Dominant and recessiveCOL6A1 mutations in Ullrich scleroatonic muscular dystrophy. AnnNeurol 2005;58:400–10.[13] Pasche B, Luo Y, Rao PH, et al. Type I transforming growth factor betareceptor maps to 9q22 and exhibits a polymorphism and a rare variantwithin a polyalanine tract. Cancer Res 1998;58:2727–32.[14] Pasche B, Knobloch TJ, Bian Y, et al. Somatic acquisition andsignaling of TGFBR1⁎6A in cancer. JAMA 2005;294:1634–46.0167-5273/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.ijcard.2007.07.048284 Letters to the Editor

May TGFBR1 act also as low penetrance ellele in marfan syndrome?

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May TGFBR1 act also as low penetrance ellele in marfan syndrome?

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