Tissue Antigens ISSN 0001-2815

Novel alleles of the transforming growth factor 𝛃-1 regulatory region and exon 1 E. Arrieta-Bolaños1,2,3 , J. A. Madrigal1,2 & B. E. Shaw1,2 1 Anthony Nolan Research Institute, Royal Free Hospital, London, UK 2 Cancer Institute, University College London, Royal Free Campus, London, UK 3 Centro de Investigaciones en Hematología y Trastornos Afines (CIHATA), Universidad de Costa Rica, San José, Costa Rica

Key words +29T>C; −1347C>T; alleles; polymorphism; regulatory region; transforming growth factor β-1 Correspondence Esteban Arrieta-Bolaños Clinical Research Group Anthony Nolan Research Institute Royal Free & University College Medical School Pond Street Hampstead London NW3 2QG UK Tel: +44 20 7284 8327 Fax: +44 20 7284 8338 e-mail: [email protected]; [email protected] Received 27 October 2014; revised 30 January 2015; accepted 25 February 2015

Abstract Transforming growth factor β-1, encoded by the TGFB1 gene, is a cytokine that plays a central role in many physiologic and pathogenic processes with pleiotropic effects. Regulatory activity for this gene has been shown for 3.0 kb between positions −2665 and +423 from its translational start site. At least 17 TGFB1 regulatory region and exon 1 alleles have been defined on the basis of 18 polymorphic sites. Polymorphisms in TGFB1’s regulatory region have been associated with differential levels of expression of this cytokine and to genetic risk in cancer and transplantation. In this report, we present 19 novel TGFB1 regulatory region and exon 1 alleles: p018–p036. Amplification of TGFB1’s regulatory region was performed with an in-house protocol, and novel alleles were defined by either allele-specific amplification and/or molecular cloning of the amplicons, followed by sequencing in isolation. Three of these novel alleles (p018, p019, and p020) are shown to be formed by novel combinations of the aforementioned known polymorphic positions. Another 16 novel alleles are shown to carry additional known and unknown single-nucleotide polymorphisms. Polymorphism in TGFB1’s regulatory region could have an impact on important processes, including embryogenesis, hematopoiesis, carcinogenesis, angiogenesis, fibrosis, immune responses, and transplantation, making its characterization necessary.

doi: 10.1111/tan.12555

Introduction

Transforming growth factor β-1, encoded by the TGFB1 gene, is a cytokine that plays a central role in many physiologic and pathogenic processes, having pleiotropic effects on cell proliferation, differentiation, migration, and survival. The human TGFB1 gene is located in chromosome 19q13.1-q13.3 (1). TGFB1 has seven exons, and regulatory activity for this gene has been mapped to approximately 3.0 kb from positions −2665 to +423 (+1 being the translation start site). This region includes two promoter sites flanking the major transcription site, two negative regulatory elements, and two enhancers lying upstream of the first promoter in addition to its signal peptide and its exon 1. The TGFB1 promoters contain activator protein-1-binding sites, allowing for the binding of transcription factors such as c-jun and c-fos. In addition, stimulatory protein 1, early growth response gene 1, and upstream stimulating factor 1 are thought to also participate in TGFB1 gene regulation (1). 484

An important amount of polymorphism has been reported for this gene. According to GeneCards Human Gene Database (http://www.genecards.org/ v. 3.10.108, 24 October 2013) (2), 624 polymorphisms have been described in TGFB1 and its surrounding and regulatory regions, most of which (65%) are located in introns. At least 57 of these polymorphisms cause amino acid changes. Most of the polymorphisms are single-nucleotide polymorphisms (SNPs), and 18 of them that lie within its regulatory region and exon 1 have been shown to be arranged in at least 17 alleles (1, 3). Polymorphism in TGFB1 has been associated to differential levels of expression of this cytokine and to genetic risk in cancer and hematopoietic stem cell transplantation (HSCT) and organ transplantation. In this report, we extend this list by presenting novel alleles formed by new polymorphic positions and new combinations of the previously described polymorphisms. These novel alleles have been found during the typing of a cohort of unrelated HSCT patient–donor pairs, and of healthy volunteer donors. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2015, 85, 484–491

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Figure 1 TGFB1’s upstream regulatory region and exon 1 showing the subregions established for the allele-typing strategy. Each box represents one of the sub-regions and the known polymorphic positions enclosed (vertical bars) are their targets. E, enhancer; N, negative regulatory region; P, promoter; SP, signal peptide; UTR, untranslated region. Figure not to scale. Positions given according to the translation start site +1.

Materials and methods Samples and subjects

Table 1 Primers used for the amplification of the four subregions of TGFB1’s upstream regulatory region and exon 1 Primer name

Sequence (5′ –3′ )a

RI-F RI-R RII-F RII-R RIII-F RIII-F-alt RIII-R RIV-F RIV-R RI-AGG-POS RI-AGG-NEG TGFB1 + 29T TGFB1 + 29C

M13F-GATGCTTCCAGATGCCAGGT M13R-TCGAAGTTGCGGAGCAGCAG M13F-CTGCTGCTCCGCAACTTCGA M13R-CACCGTCCTCATCTCGCGT M13F-ACGCGAGATGAGGACGGTG M13F-ATCTCCCTCCCACCTCCCT M13R-GTGTCTCAGTATCCCACGGA M13F-TCCGTGGGATACTGAGACAC M13R-CCAGTTTCTTCTGCCAGTCA M13F-AGGGCAGGGACATGAGGAGG M13F-AGGGCAGGGACATGAGGGAA AGCAGCGGTAGCAGCAGCA GCAGCGGTAGCAGCAGCG

Volunteer healthy donors

Volunteer healthy donors were used to obtain blood cells and nucleic acids for experiments. These were recruited among laboratory staff and gave informed written consent. Blood, saliva, or buccal swabs were obtained, and samples were given a unique identifier. Patients and stem cell donors

HSCT patient and donor samples are part of Anthony Nolan Research Institute’s sample biobank. This bank receives preconditioning blood samples from all recipients of an HSCT performed at a transplant center in the UK who received a stem cell graft from an unrelated donor registered in Anthony Nolan’s Stem Cell Donor Registry. Similarly, a sample from the respective donor is also obtained.

TGFB1, transforming growth factor β-1. a M13, M13 sequence tag; forward (F), 5′ -GTAAAACGACGGCCAGT and reverse (R) 5′ -CAGGAAACAGCTATGAC. Bold and underlined letters show polymorphic positions.

Sample collection and DNA extraction

DNA was extracted from peripheral blood from healthy donors and HSCT patients by an in-house salting out protocol based on Miller’s method (4). In some cases, saliva samples from healthy volunteer donors were also collected for DNA extraction. For this purpose, the Oragene OG-500 sample collection kits (DNA Genotek Inc., Ontario, Canada) were used for the collection of 2 ml of saliva as indicated by the manufacturer. DNA extraction from epithelial cells contained in human saliva was carried out as indicated by the manufacturer. For some subjects, a buccal swab was collected using a nylon bristle cytology brush. DNA was extracted from epithelial buccal cells collected with a brush using the Gentra Purgene Buccal Cell Kit (Qiagen, Manchester, UK), following the manufacturer’s instructions. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2015, 85, 484–491

Sequencing of the regulatory region of TGFB1

The ∼3.0 kb region of the genome known to exert regulatory functions over TGFB1 expression was analyzed for polymorphism by sequencing. Based on the studies by Shah et al. (1, 3), the region extending from −2664 to +423 according to this gene’s translation start site was divided into four subregions (called RI, RII, RIII, and RIV) that were amplified separately by polymerase chain reaction (PCR) (Figure 1). The primers used for each region are shown in Table 1. The basic PCR mix included 2.5 μl of 10× NH4 buffer, 0.4 μl of 50 mM dNTPs, 0.5 μl of 50 mM MgCl2 , 0.5 μl of each relevant primer at 25 μM, 0.2 μl of Taq polymerase, 19.4 μl of distilled H2 O, and 1.0 μl of the target DNA (50–100 ng). 485

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The PCR amplification products of the regions of interest were purified with either the column-based Illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK) or the enzyme-based illustra ExoStar PCR and Sequence Reaction Clean-Up Kit (GE Healthcare). The purified products were then sequenced using M13 tail-targeted primers in two directions and Sanger sequencing chemistry. The sequencing reactions were prepared with BigDye Terminator (BDT) sequencing technology v.3.1 (BDT, PE Applied Biosystems, Foster City, CA). The reaction mix included 5.5 μl of distilled H2 O, 1 μl of 5× BDT buffer, 1 μl of BDT Ready Reaction mix, 1 μl of either M13 forward or reverse primers at 1.6 μM, and 1.5 μl of the purified template. The general cycle sequencing protocol was carried out on either a Research PTC-200 thermal cycler (Alpha Laboratories Ltd., Hampshire, UK), an AB GeneAmp® PCR System 2700 thermalcycler (PE Applied Biosystems) or an Eppendorf Mastercycler gradient thermalcycler (Eppendorf, Stevenage, UK) and consisted on an initial denaturation at 96∘ C for 1 min, followed by 25 cycles of 96∘ C for 10 s, 50∘ C for 5 s and 60∘ C for 2 min, followed by a final cooling at 4∘ C. After the template samples had been subjected to cycle sequencing, the products were purified by alcohol precipitation. All sequencing data were generated by capillary electrophoresis on an ABI GA-3730xl genetic analyzer (Applied Biosystems). The sequencing data were analyzed with either Sequencing Analysis 5.2 or with the help of a purpose-built TGFB1 polymorphism library in SEQSCAPE 2.5.0 software (PE Applied Biosystems). The sequenced fragments were then aligned onto a template sequence of the complete genomic region set up on SEQSCAPE software, and the polymorphic positions, both known and unknown, were recorded. Based on the genotypes for 18 known polymorphic positions and on previous work by Shah et al. (1, 3), in which the authors defined the phase of these polymorphisms into 17 haplotypes, the sequencing data were used to assign a TGFB1 regulatory region and exon 1 allelic genotype. The sequencing assay was validated by typing of six International Histocompatibility Workshop (IHW) cell lines (5) previously typed for TGFB1 regulatory region and exon 1 by Shah et al.: IHW9065 (p003/p006), IHW9077 (p001/p008), IHW9089 (p002/p003), IHW9050 (p012/p013), IHW9058 (p003/p005), and IHW9016 (p001/p007) (1). In addition, 10% of the samples from the HSCT patient–donor cohort were randomly selected for retyping. All typings agreed with previous results. Samples found to have previously unreported polymorphisms or unresolvable genotypes that did not match any of the previously reported alleles were confirmed by either (1) whole-region amplification (using primers RIF and RIVR) followed by molecular cloning (Zero Blunt TOPO PCR Cloning Kit, Invitrogen, Life Technologies, Paisley, UK, see below) and/or (2) phase-specific whole-region amplification using primers specific for the −2389AGG duplication 486

(RI-AGG-POS or RI-AGG-NEG with RIVR) or the +29T>C (RIF with TGFB1 + 29T or TGFB1 + 29C), both followed by sequencing in isolation using versions of the primers in Table 1 without M13 tails.

Molecular cloning

In some experiments, allele separation in order to define polymorphism phase was performed by molecular cloning and sequencing in isolation of the region of interest. This was achieved by following an in-house Anthony Nolan Research Institute protocol normally used for cloning of human leukocyte antigen (HLA) class-I PCR products. For this, an amplification of the region of interest was carried out with the relevant primers and using Finnzyme Phusion polymerase (Thermo Scientific, ABgene House, Surrey, UK), generating blunt-end PCR fragments. All amplification reagents were obtained from Thermo Scientific, ABgene House, unless stated otherwise. The amplification reaction was carried out in duplicate for each sample of interest and consisted of 4.0 μl of 5× Buffer GC, 0.4 μl of each relevant primer at 25 μM, 0.6 μl of 100% dimethyl sulfoxide, 0.4 μl of dNTP, 0.2 μl of the Phusion polymerase, and 50 ng of target DNA with the necessary amount of sterile distilled H2 O (usually 13 μl) to complete a 20 μl reaction. This PCR mix was subjected to cycling on a Research PTC-200 thermal cycler (Alpha Laboratories Ltd.). The cycling program consisted of an initial activation at 98∘ C for 30 s, followed by 10 cycles of 98∘ C for 15 s, 70∘ C for 30 s, and 72∘ C for 3 min, these in turn followed by 20 cycles of 98∘ C for 15 s, 68∘ C for 30 s, and 72∘ C for 3 min, and a final extension at 72∘ C for 30 min. The PCR products were then checked for purity, yield, and specificity by agarose gel electrophoresis and were then purified by GFX merging of the twin amplifications in 30 μl of sterile H2 O. The PCR products were cloned into a Blunt TOPO PCR vector (Zero Blunt TOPO PCR Cloning Kit, Invitrogen). The selection for these vectors is based on kanamycin resistance conferred by the plasmid. The cloning reaction was performed as instructed by the manufacturer. The plasmid with insert was then transfected to the competent Escherichia coli (supplied with the kit) using heat shock. The bacteria were then enriched in a short-term culture in S.O.C. medium, followed by plating of this medium on Luria-Bertani agar with kanamycin (Sigma-Aldrich Ltd., Gillingham, Dorset, UK) and overnight incubation at 37∘ C. The transformed, kanamycin-resistant E. coli that grew were then picked (typically 24 per sample) and cultured in 800 μl of a liquid culture in Luria-Bertani broth culture media (Sigma-Aldrich Ltd.) with kanamycin overnight at 37∘ C. After this second culture, 200 μl of the broth were used to create a glycerol-preserved stock stored at −20∘ C, whereas the remaining 600 μl was used for the plasmid DNA extraction. The extraction of plasmid DNA from each individual clone culture was done with the PureYield™ Plasmid Miniprep © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2015, 85, 484–491

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System (Promega, Madison, WI), following the manufacturer’s instructions. After the plasmid DNA was extracted for all clones, restriction enzyme digests for each were set up with EcoRI in order to identify fragment incorporation. The digested plasmids were run on an agarose gel and their digestion pattern recorded so that clones that had incorporated the fragment of interest would be identified. Once the right clones had been identified, these were subjected to sequencing as described before but using versions of the primers in Table 1 without M13 tails. The sequencing reaction was prepared with 2.5 μl of sterile H2 O, 1.0 μl of Ready Reaction mix, 0.5 μl of 5× buffer, 1.0 μl of the relevant primer at 1.6 μM, and 5.0 μl of plasmid DNA. Ethical approval

The Patient–Donor project has been granted ethical approval by the United Kingdom’s National Research Ethics Service (www.myresearchproject.org.uk, application number MREC 01/8/31).

Results A novel TGFB1 regulatory region and exon 1 allele found in a healthy volunteer donor

A healthy volunteer sample, HVD28, showed genotypes for the 18 basic polymorphic positions that were not congruent with any of the possible allelic combinations based on the work by Shah et al. (1, 3). After repeating the sequencing and confirming the results, the sample was deemed to carry a novel allele and selected for further studies. In order to confirm this, we used two strategies to identify this potentially novel allele. First, saliva samples from relatives from HVD28 were sought. These samples were typed for TGFB1’s upstream regulatory region and exon 1 alleles with the four-subregion protocol. The typing results for these experiments are presented in Table 2. The family study of the unsolvable genotype revealed that the novel allele was carried by the subject’s father, whereas the mother was homozygous for allele p001. The subject and the sibling had the same genotype, further confirming the suspicion of the presence of this novel allele. As is evident from the results, HVD28’s father bears what appears to be the novel allele and a p003 allele. Secondly, the samples from HVD28 and their father were subjected to molecular cloning in order to isolate the novel allele and to be able to generate a sequence from a single fragment of this genomic region. The DNA samples were amplified using primers RI-F and RIV-R, thus spanning all the subregions. Sequencing of the clones was performed with a different set of primers, which were versions of the primers included in Table 1 that lacked M13 tags. The analysis of the sequencing data allowed the confirmation of the presence of the new allele in a number of clones. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2015, 85, 484–491

Novel alleles in TGFB1

The allele was then shown to be similar to the p001, but had the −2389 AGG duplication and also a C at position −1347, therefore differing from all other previously reported alleles. As shown in Table 2 and confirmed by sequencing in isolation, the new allele is formed by a combination of variants at positions −2410(A), −2391(AGG+), −1347(C), and +29(C), without polymorphism at positions −768(−) and +74(G). Novel alleles within TGFB1’s regulatory region and exon 1 in a cohort of stem cell transplant patients and donors

During the typing of 1024 samples from the Anthony Nolan Research Institute’s patient–donor cohort, a total of 16 new TGFB1 regulatory region and exon 1 alleles formed by additional polymorphic positions were identified (Figure 2). In addition, three novel TGFB1 regulatory region and exon 1 alleles formed by new combinations of the 18 basic polymorphic positions were discovered. The additional polymorphic positions were caused by SNPs in all cases. These novel SNPs were discovered when the sequences for regions previously considered to be invariant were scanned during sample analysis. All potential additional SNPs were confirmed by reamplifying and sequencing the relevant subregion. If all other positions in the sample were homozygous (i.e. TGFB1 regulatory region and exon 1 allele homozygosity), then the SNP was deemed to be carried by this previously known allele. If the sample had a heterozygous allelic genotype (e.g. p001/p003), then further studies were carried out to define the phase of this novel polymorphism. For this purpose, the samples were amplified with allele-specific primer combinations using the −2389dupAGG- and the +29T>C-specific primers. Most of the allelic phases could be defined using this strategy. However, in a small number of cases full (RI-RIV) amplification and subsequent cloning of the alleles was required to identify which one bore the novel SNP. Overall, 16 additional SNPs were identified in 37 samples. Consequently, 3.6% of the samples in the cohort carried SNPs other than the basic 18 polymorphisms. Six of these SNPs had been reported in the dbSNP database, whereas the remaining 10 SNPs are novel. Six of these additional SNPs were seen in more than one sample, and always in the context of the same allele, thus confirming their presence in the population, albeit at low frequencies. Of note, one of these additional SNPs, −1569C>T, had a frequency of 1.36% among the 1024 samples. The 16 additional SNPs are presented in Table 3. Their location in the context of TGFB1’s regulatory region and exon 1 is shown in Figure 2. Six additional SNPs were found in a p001-like allele, whereas seven were in the context of a p003-like allele, and two were carried by a p014-like allele. Of note, one of the novel SNPs, −1189C>T, was seen twice in the context of a previously unknown combination of TGFB1 487

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Table 2 Results for the typing of TGFB1 regulatory region and exon 1 polymorphisms for HVD28 and their relatives Sample

−2410A>G

−2389dupAGG

−1347C>T

−768insC

+29T>C

+74G>C

Alleles

HVD28 Father Mother Sibling

AA GA AA AA

AGG/— AGG/AGG −/− AGG/—

C/T CC TT C/T

−/− −/− −/− −/−

CC T/C CC CC

GG GG GG GG

p001/N p003/N p001/p001 p001/N

dup, duplication; HDV, healthy volunteer donor; ins, insertion; N, novel allele. All other positions were not polymorphic. –2001C>G –535C>T

–2316C>A

–265C>G

–73G>A

–1922C>T –498C>G –1569C>T

–2069G>A

–1189C>T –2219G>C

–2389_–2391dupAGG

–1826C>A -1985C>G

–768–769insC

–1740C>T

–1347C>T

– 778G>A –1287G>A

+149G>C

–144G>A –328C>T

–469C>A

+74G>C +122T>G

–1169_–1170delTT

+348C>T

+29T>C

–387C>T –229C>G

–14G>A

+91T>G

–827G>C –2410A>G –1154C>T

–1638G>A

SP –2665

+423

–2204 –2201

E2

–1570

–1971

N2

E1

–1292

N1

–1161

P1

–839

–568

+1

+354

P2

Exon 1

Figure 2 Additional polymorphisms in TGFB1’s regulatory region and exon 1. The gene’s regulatory region and exon 1 analyzed in this study is shown schematically and not to scale. The translation start site is indicated as +1. Previously known variants that define known alleles are shown in ( ), whereas additional variant positions grouped in novel alleles discovered in this study are shown in ( ). The locations of enhancers (E), negative regulatory regions (N), and promoters (P) are shown. 5′ UTR, 5′ untranslated region; SP, signal peptide.

regulatory region and exon 1 basic positions (i.e. allele p020, see below), confirming the existence of both. In addition, three novel combinations of the basic 18 polymorphic positions were identified in this cohort. These samples showed genotypes that were incompatible with any of the possible combinations between known TGFB1 regulatory region and exon 1 alleles. As it was mentioned before, this was the case for nine of the 1024 samples typed in this study. These samples were then subjected to allele-specific amplifications as explained above. Among these nine samples (five donors and four patients) two of the three novel combinations were seen in more than one sample. Novel allele p018 was observed in six samples, whereas alleles p019 and p020 were seen in one and two samples, respectively. Their novel combinations are shown in Table 4. Of note, allele p020 is equivalent to that discovered in the family study explained above, thus confirming the previous results in a completely unrelated sample. As shown in Table 4, allele p018 is most similar to allele p001, except for its +29T. The p019 allele is also similar to p001 but shows a −2410G in addition to a +29T. The p020 allele seems 488

to have an upstream half similar to p014, but its proximal half lacks the −768insC and the +74C variant. All the novel TGFB1 regulatory region and exon 1 alleles (p018–p036) have been deposited in the European Nucleotide Archive at the European Molecular Biology Laboratory and given the consecutive accession numbers LM651041–LM651059.

Discussion

The identification of a genotype in one healthy volunteer donor that could not be explained by any combination of the previously described alleles prompted the study of this sample. A typing error was excluded by repetition of the assay and analysis of samples of the relatives of this donor added evidence to the predicted sequence of this novel allele. Final proof came from sequencing in isolation of the allele following molecular cloning of an amplicon spanning all of TGFB1’s regulatory region and exon 1. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2015, 85, 484–491

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Table 3 Novel alleles formed by additional SNPs within TGFB1’s regulatory region and exon 1 discovered during the typing of 1024 patient and donor samples in the unrelated donor-HSCT cohort Novel TGFB1 allele p021 p022 p023 p024 p025 p026 p027 p028 p029 p030 p031 p032 p033 p034 p035 p036

Closest known allele

Additional variant

Variant ID

Times observed

Region

Coding effect

Position in chromosome 19a

p001 p003 p001 p003 p001 p003 p020 p003 p003 p003 p001 p001 p014 p003 p014 p001

−2316C>A −2219G>C −2069G>A −2001C>G −1922C>T −1569C>T −1189C>T −535C>T −498C>G −328C>T −265C>G −144G>A −73G>A +122T>G +149G>C +348C>T

NEW NEW rs41445948 NEW NEW rs4987025 rs186958149 NEW NEW rs45457101 NEW NEW rs56155294 NEW NEW rs56281462

1 1 4 1 1 13 2 1 1 3 1 1 3 1 1 3

Enhancer 2 Enhancer 2 Negative regulatory region 2 Negative regulatory region 2 Enhancer 1 Negative regulatory region 1 Promoter 1 5′ UTR 5′ UTR 5′ UTR 5′ UTR 5′ UTR 5′ UTR Exon 1 Exon 1 Exon 1

N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Codon 41, Val->Gly Codon 150, Arg->Pro Codon 116, silent

41355360 41355263 41355113 41355045 41354966 41354613 41354233 41353579 41353542 41353372 41353309 41353188 41353117 41352923 41352896 41352697

HSCT, hematopoietic stem cell transplantation; SNPs, single-nucleotide polymorphisms; transforming growth factor β-1. a Based on assembly GRCh38, build 106. Table 4 Novel TGFB1 regulatory region and exon 1 alleles formed by new combinations of previously known variants Positionb Allelea −2410 −2389 dupAGG −1985 −1638 −1347 −1287 −1169c −1154 −827 −778 −768c ins C −469 −387 −229 −14 +29 +74 +91 p001 p018 p019 p020

A — G —

— — — AGG

C — — —

G — — —

T — — C

G — — —

TT — — —

C — — —

G — — —

G — — —

— — — —

C — — —

C — — —

C — — —

G — — —

C T T —

G — — —

T — — —

a Promoter allele defined by the sequence of TGFB1 regulatory region and exon 1. Sequences are compared with allele p001 (GenBank accession no. AY871232). b Nucleotide position relative to the major translation start site (+1) as described in Shah et al. (1, 3). Only known polymorphic positions are included. c Indicates deletion (del) or insertion (ins) of nucleotide(s).

The number of new TGFB1 regulatory region and exon 1 alleles was further enlarged by the identification of additional SNPs and novel combinations of the basic 18 polymorphic positions during the typing of a cohort of unrelated stem cell transplant patients and donors. The fact that 3.6% of the typed samples in this cohort bore additional SNPs suggests the presence of a considerable degree of polymorphism only recognized when large groups of samples are studied. This becomes more relevant considering that most of these samples are likely to belong to a Northwestern-European population, and the increased diversity among other human populations such as Sub-Saharan Africans might hide several other novel polymorphisms. However, it must be stressed that both in Shah et al.’s publications and in our own experience there is a predominance of certain alleles. Specifically, three alleles dominated this healthy volunteer donor group: p003 (60%), p001 (30%), and p014 (10%). These alleles were also dominant in Shah et al.’s reports. This could mean that, despite a significant amount of polymorphism yet to be discovered, it is likely that for this gene the vast © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2015, 85, 484–491

majority of the individuals bear common alleles, in a situation that could resemble, albeit in a reduced way, the situation found for the HLA system (6). Regarding the consequences of these additional SNPs, eight of 16 lie within a known regulatory region within TGFB1 upstream region and could thus have a potential impact on gene expression. Two of the remaining SNPs are predicted to cause amino acid changes in the mature protein (Table 3). Altogether, these polymorphisms could have functional consequences for those people carrying them. Many of the polymorphisms found within TGFB1’s regulatory region appear to influence the expression of TGF-β1 and to explain inter-individual differences in the levels of this cytokine. The −1347C>T (also known as −509C>T if the transcriptional start site is considered as +1; rs1800469) located in the proximal negative regulatory region, has been shown to mediate differences in TGF-β1 plasma and transcription levels (7, 8). In addition, this polymorphism, as well as −2726G>A and −2389_−2391dupAGG, is predicted to alter DNA-protein complex formation in electrophoretic mobility shift assays (9). 489

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Another two SNPs in the regulatory region of TGFB1, −1287G>A, and −387C>T, which are common in African Americans, have been shown to modify the binding affinity of transcription factor complexes and, consequently, the expression levels of this gene in reporter vector experiments (1). Two polymorphisms that lie within TGFB1’s signal peptide region, +29T>C (rs1982073) and +74G>C (rs1800471), cause changes to the amino acid sequence of the pre-pro-TGF-β1 and have also been associated with differential production of this cytokine (10–13). The functional effect of other polymorphisms has not been sufficiently addressed yet. Polymorphisms in TGFB1’s regulatory region and exon 1 have been associated with the development or modification of several diseases, most probably because of the diverse involvement of TGF-β1 in physiological processes. These mostly relate to fibrotic (14–16), immune (8, 17–20), endocrine (21, 22), neurologic (23), vascular (11, 24–26), or neoplastic processes (27–29). TGFB1 polymorphism has also been associated with pregnancy loss (30). In conclusion, our results show a considerable degree of unreported variation within TGFB1’s regulatory and exon 1 regions. Most of the new SNPs found lie in either known regulatory regions such as enhancers, promoters, or negative regulatory elements or in the coding translated region of exon 1 causing amino acid changes in two cases. All these variations could potentially have an effect in the regulation of this important gene. Knowledge of the extent and consequences of this variation are fundamental for understanding the immunogenetics of TGF-β1 and its possible applications in medicine. We are currently continuing our analysis of the effect of polymorphisms within the regulatory region of TGFB1 on the outcome of unrelated donor HSCT and function of regulatory T cells. These data will hopefully help to identify the risk factors associated with TGFB1 expression in patients and donors and generate clinically useful algorithms of genetic risk assessment in HSCT.

Acknowledgments

This work was supported by grants from the University of Costa Rica and the Costa Rican National Council for Scientific and Technologic Research (CONICIT).

Conflict of interest

The authors have declared no conflicting interests. References 1. Shah R et al. Allelic diversity in the TGFB1 regulatory region: characterization of novel functional single nucleotide polymorphisms. Hum Genet 2006: 119: 61–74. 2. Safran M et al. GeneCards version 3: the human gene integrator. Database 2010: 2010: baq020.

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Novel alleles of the transforming growth factor β-1 regulatory region and exon 1.

Transforming growth factor β-1, encoded by the TGFB1 gene, is a cytokine that plays a central role in many physiologic and pathogenic processes with p...
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