alleles - Nature

Genes and Immunity (2003) 4, 547–558 & 2003 Nature Publishing Group All rights reserved 1466-4879/03 $25.00 www.nature.com/gene

Novel polymorphisms and the definition of promoter ‘alleles’ of the tumor necrosis factor and lymphotoxin a loci: inclusion in HLA haplotypes PE Posch1, I Cruz2, D Bradshaw2 and BA Medhekar2 Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC, USA; 2Department of Oncology, Georgetown University Medical Center, Washington, DC, USA 1

Tumor necrosis factor (TNF) and lymphotoxin alpha (LTA) influence a variety of cellular responses and play a complex role in the immune response. Several single nucleotide polymorphisms (SNPs) have been reported in these major histocompatibility complex (MHC)-linked loci; however, a comprehensive examination of polymorphisms in the promoter regions of TNF and LTA has not been carried out and was undertaken here. Seven novel SNPs in LTA were identified by sequence analysis of 69 samples. Eight novel TNF alleles and 16 novel LTA alleles were designated. The TNF alleles clustered into two closely related groups, while the LTA alleles clustered into three distinct groups using phylogenetic and percentage difference analyses. A total of 52 unique TNF-LTA-HLA haplotypes are reported. There appear to be some associations between TNF/LTA alleles and HLA haplotypes, but not with specific HLA alleles. The majority of the SNPs appear to be randomly associated within and between the two loci except for the LTA SNPs at 293, þ 81 and þ 369. These observations may provide an explanation for the oftentimes contradictory results of studies associating individual cytokine gene SNPs with expression level phenotypes, HLA and disease. Genes and Immunity (2003) 4, 547–558. doi:10.1038/sj.gene.6364023 Keywords: tumor necrosis factor; lymphotoxin alpha; single nucleotide polymorphism; promoter; allele; haplotype

Introduction Tumor necrosis factor (TNF[a]; cachectin) and lymphotoxin alpha (LTA; TNFb) are structurally homologous inflammatory cytokines produced primarily, but not exclusively, by macrophages and T cells, respectively.1,2 Initially identified for their cytotoxic and antitumor activity,3,4 it is now well documented that these cytokines play critical roles in a variety of immunologic processes including inflammation, secondary lymphoid organ development and the control of intracellular pathogens.1,5–7 TNF and LTA are expressed in various different forms2 and bind to multiple receptors to perform their functions.2,8,9 The TNF and LT loci are tandemly arranged in the class III region of the human major histocompatibility complex (MHC) (Figure 1a) approximately 220 kb centromeric to the HLA-B locus.10,11 These genes, each of which consists of four exons, are tightly linked. Only 1240 bases separate the polyadenylation site of LTA and the transcription start site of TNF. Like most cytokine genes, TNF and LTA gene expression is highly regulated.12–14

Correspondence: Dr PE Posch, Department of Microbiology and Immunology, Research Building E408, Georgetown University Medical Center, 3970 Reservoir Road, NW Washington, DC 20057, USA. E-mail: [email protected] Received 06 May 2003; revised 30 June 2003; accepted 05 July 2003

Several single nucleotide polymorphisms (SNPs) have been reported in these loci, although lack of a consistent numbering system has hampered comparison of studies. Multiple SNPs are found in the TNF locus15–19 (Figure 1b), including 10 located in the promoter upstream of the transcription start site at 1073 (C/T), 1031 (T/C), 863 (C/A), 857 (C/T), 575 (G/A), 376 (G/A), 308 (G/A), 244 (G/A), 238 (G/A) and 163 (G/A). Two SNPs are located in exon 1 within the 50 untranslated region (UTR) at þ 70 (C insertion)20 and þ 153 (C/T).21 Several SNPs also have been reported in the introns18 and a G/T dimorphism is present in the 30 UTR.10,22 Nine SNPs have been reported in the LTA locus23–28 (Figure 1c). Three are located in the promoter region upstream of the transcription start site at 626 (G/A), 294 (C/T) and 293 (G/A). In exon 1, which encodes the majority of the 5’UTR, there are two SNPs at þ 11 (G/A) and þ 81 (C/A). Two SNPs are found in the first intron, one at þ 253 (G/A) that is detectable by an NcoI restriction fragment length polymorphism (RFLP) and the other at þ 369 (G/C) that is detectable by an AspHI RFLP. There is an SNP located at þ 496 (T/C) in exon 2 that causes a cysteine to arginine change, respectively, at amino-acid position 21 in the signal sequence. Lastly, an SNP is located at þ 724 (C/A) in exon 3, which creates a threonine to asparagine change, respectively, at amino-acid position 26 of the mature protein. In addition, a (CT)n dinucleotide repeat (n¼9–10) is located in intron 1 and defines the TNFc microsatellite.29

TNF and LTA, novel SNPs, alleles and haplotypes PE Posch et al

548

Figure 1 The TNF and LT loci (a) encoded in the class III region of the human major histocompatibility complex on chromosome 6 in relation to the HLA-DR and HLA-B loci. Arrows indicate gene orientation and exons are numbered. Exon 1 (338 bp) of TNF encodes the 5’UTR and a majority of the signal sequence, exon 2 (46 bp) encodes the remainder of the signal sequence and the first amino acid of the mature protein, exon 3 (48 bp) encodes for part of the mature protein and exon 4 (1210 bp) encodes for the remainder of the mature protein and the 3’UTR. Exon 1 (163 bp) of LTA encodes the majority of the 5’UTR, exon 2 (99 bp) encodes the remaining nine bases of the 5’UTR and the majority of the signal sequence, exon 3 (106 bp) encodes the last codon of the signal sequence and part of the mature protein and exon 4 (1041 bp) encodes the remainder of the mature protein and the 3’UTR. (b) Enlargement of the TNF locus focusing on the fragment amplified (length indicated). Exons are boxed and numbered with shaded areas representing the 5’UTR. The positions of reported SNPs are shown. Arrows indicate the relative location of PCR primers. (c) Enlargement of the LTA locus focusing on the fragment amplified (length indicated). Exons are boxed and numbered with shaded areas representing the 5’UTR. The positions of SNPs, both previously reported and identified in this study, are shown. Arrows indicate the relative location of PCR primers. Figure is not drawn to scale.

A limited number of studies have directly examined the effects of some of these polymorphisms on expression and transcription factor binding.27,28,30–35 On the other hand, there have been numerous studies, which have correlated specific SNPs to TNF and to LTA expression levels in groups of individuals and from cell lines, but with varying results.36–40 It is understood that the production level phenotype of an individual cytokine can have a major impact on immune responsiveness, and an abundance of studies have linked specific SNPs to disease.41,42 As examples, TNF and LTA expression levels and SNPs have been shown to correlate with an individual’s susceptibility to, severity of and/or prognosis of diseases such as HIV, multiple sclerosis and cerebral malaria43–46 and with prognosis or outcome of allograft transplantation and malignancies.7,47–49 Because of their linkage to HLA genes in the MHC and their biologic properties, it is speculated that TNF and/or LTA also may contribute to the etiology of MHC-associated autoimmune diseases such as systemic lupus erythematosus,50,51 insulin-dependent diabetes mellitus,52,53 myasthenia gravis54,55 and rheumatoid arthritis.56,57 Furthermore, TNF and LTA expression level phenotypes and individual SNP also have been associated with particular HLA types.58–61 Genes and Immunity

Although there are a plethora of studies suggesting an association between TNF and LTA SNPs, expression level phenotypes, HLA and disease, there are as many studies that are in conflict or that find no correlation.36,41,42,50,62,63 As these types of studies examine either individual SNP or a subset of the reported SNPs, the discrepent results are likely to be a result of the linked SNPs that were not examined. To our knowledge, no study to date has taken into account all of the SNPs at these loci when assessing the effects on expression or the linkage to disease and HLA. For these reasons, the extent of polymorphism in the TNF and LTA promoter regions was investigated. Promoter ‘alleles’ at each locus were determined and the relatedness of the alleles was examined by phylogenetic analysis. Finally, TNF and LTA alleles were included in HLA haplotypes and associations were examined.

Results Several novel SNPs in the LTA locus TNF and LTA were characterized by sequencing from 69 samples to determine the extent of polymorphism at these loci. The samples were chosen to include individuals from a variety of racial backgrounds and with a variety of HLA haplotypes. The TNF fragment (1236 bp) that was analyzed encompasses the region from þ 161 just upstream of the translation start site in exon 1 to 1075 in the promoter (Figure 1b). Primer sites were chosen for specificity, to include the largest promoter region fragment between the translation start site of the TNF gene and the 30 end of the LTA gene and to ensure coverage of all 12 of the previously reported promoter region SNPs. The transcription start site ( þ 1) of TNF has not been mapped and studies have been inconsistent as to its exact location. Therefore, we have positioned the transcription start site as to locate one of the most studied SNPs at 308. Base numbering and the position of the remaining SNPs coincide with our sequence data. No novel polymorphisms were identified in the TNF promoter region fragment from any of the samples that were examined; however, seven of the 12 previously reported TNF SNPs were present. The 3177 bp LTA fragment (2434 to þ 743) included all previously reported SNPs, the TNFc microsatellite and an extended promoter region (Figure 1c). The choice to examine an extended promoter region was based on a study of the interleukin-10 promoter, which showed SNPs associations with production level phenotypes in an additional 3.0 kb fragment upstream of the 1.0 kb proximal promoter region fragment that was originally analyzed.64 Designation of the transcription start site ( þ 1) was based on its mapping by S1 nuclease,22 although the authors noted that there may be some heterogeneity in its location. Base numbering and position of SNPs coincide with our sequence data. Seven novel or unreported SNPs were identified, as well as a 145 bp insertion. Five of these SNPs were found in the region upstream of the transcription start site: 2066 (C/T), 1816 (C/G), 1783 (G/A), 1563 (A/T) and 1051 (G/A). A novel SNP was identified at þ 633 (G/ C), which falls in the second intron. Finally, a novel SNP at position þ 697 (A/C) changes histidine to


proline, respectively, in the mature protein at amino acid 17. Two of the seven novel SNPs at positions 1816 and 1563 also are seen on alignment and comparison of all GenBank LTA sequences which overlap the fragment examined in this study (data not shown). Further, a 145 bp insertion was found in one sample (9075) at position 425. This insertion is actually a duplication of the promoter sequence between positions 570 and 426 and was present in one GenBank LTA sequence (accession #AF037255). Promoter ‘alleles’ of TNF and LTA Alleles of the TNF promoter and LTA promoter were determined by family segregation analysis and by sequencing of cloned DNA or MHC homozygous cell lines. Allele names were designated p* to denote that they were based on the sequence of the promoter region which was defined as the nucleotide positions upstream of the translation start site ( þ 181 for TNF and þ 460 for LTA). This is followed by a three-digit number to denote a unique allele. For example, TNFp*001 denotes promoter allele 1 of TNF. An additional two digits were added to allele names (TNFp*00101 and TNFp*00102, for example) to denote that the alleles have identical promoter regions, but differ downstream of the translation start site. GenBank sequences that fully overlapped the TNF and LTA fragments were included in the ‘allele’ assignment. Eight novel TNF alleles were present in the samples examined (Table 1). Allele TNFp*001 was designated as such because it was by far the most predominant (43 haplotypes) and was identical to three GenBank sequences (accession #AF129756, AL662801 and Y14768). The remaining alleles were named in the order that they were identified. Allele TNFp*002 (eight haplotypes)

Table 1

differs from TNFp*001 at 308 (A vs G, respectively) and was a match (100%) to one GenBank sequence (accession #AL662847). TNFp*002 is the only allele to encode 308A, which has been associated with a high TNF expression level phenotype. All GenBank sequences that fully overlapped the TNF fragment matched alleles reported in this study. TNFp*003 (five haplotypes), TNFp*004 (two haplotypes) and TNFp*007 (four haplotypes) each also differ by only one SNP from TNFp*001, a cytosine to thymine change at 857 (TNFp*003), a thymine to cytosine change at 1031 (TNFp*004) and a guanine to adenine change at 244 (TNFp*007). TNFp*006 (9 haplotypes) and TNFp*008 (1 haplotype) each differ by two SNPs from TNFp*001. TNFp*006 encodes adenine instead of cytosine (TNFp*001) at 863 and TNFp*008 encodes adenine instead of guanine (TNFp*001) at 238, while both encode cytosine at position 1031 (TNFp*001 encodes thymine). The most distinct allele is TNFp*005 (three haplotypes), which differs from TNFp*001 by three SNPs: 1031 (T-C), 376 (G-A) and 238 (G-A) (TNFp*001-TNFp*005). A total of 16 novel LTA alleles were identified (Table 2) with 15 of these encoding unique promoter regions. Allele LTAp*001 was designated as such because it matched (100%) two GenBank sequences (accession #AP000505 and Y14768), one of which was the first LTA sequence reported. Allele LTAp*00201 was named as such because it also was identical (100% match) to one GenBank sequence (accession #AF129756). All GenBank sequences that fully overlapped the LTA fragment match alleles reported in this study. The remaining alleles were named in the order that they were identified. When compared to allele LTAp*001, the remaining 15 alleles differed by as few as a single SNP (LTAp*009 and LTAp*010) to as many as nine SNPs (LTAp*008). Three alleles predominated in the samples examined,

549

TNF promoter alleles Positiona

Promoter allele b,c p*001d p*002e p*003 p*004 p*005 p*006 p*007 p*008

1073

1031

863

857

575

376

308

244

238

163

+70

+153

C C C C C C C C

T T T C C C T C

C C C C C A C C

C C T C C C C C

G G G G G G G G

G G G G A G G G

G A G G G G G G

G G G G G G A G

G G G G A G G A

G G G G G G G G

C C C C C C C C

C C C C C C C C

a Position relative to transcription start site (+1). All positions reported to be polymorphic are included, although the alternate nucleotide may not have been present in the samples examined. b Promoter defined as nucleotide positions upstream of the translation start site (+181, ATG codon) which includes the 5’ untranslated region (UTR) encoded in exon 1. TNF promoter allele sequences can be accessed from GenBank: TNFp*001 (accession #AY274889 and AY274896), TNFp*002 (accession #AY274893 and AY274901), TNFp*003 (accession #AY274890 and AY274895), TNFp*004 (accession #AY274898), TNFp*005 (accession #AY274891 and AY274900), TNFp*006 (accession #AY274894 and AY274899), TNFp*007 (accession #AY274892 and AY274902) and TNFp*008 (accession #AY274897). c GenBank sequence accession #Z15026, which encompassed both TNF and LTA, was excluded. This sequence was derived from IHWS reference cell 9031 (BOLETH), which was examined in this study. The Z15026 sequence contained numerous nucleotide differences that were not seen in the TNF and LTA sequences obtained in this study from 9031 or any other sample, as well as in any of the other GenBank sequences. d This allele was a match (100%) to three GenBank sequences (accession #AF129756, AL662801 and Y14768) over the region examined. e This allele was a match (100%) to one GenBank sequence (accession #AL662847) over the region examined.

Genes and Immunity


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Table 2 LTA promoter alleles Positionb Promoter allele a 2066 1816 1783 1563 1051 626 425c 294 293 +11 +81 +253 +369 +496d +633e +697f +724f TNFcg p*001h p*00201i,j p*00202j p*003 p*004 p*005 p*006 p*007 p*008 p*009 p*010 p*011 p*012 p*013 p*014 p*015

C C C C C C C C T C C C C C C C

C C C C G C C C C C C C C C C G

G G G G G G A G G G G G G G G G

T A A T A A T T A T A T A A T A

G G G G G A G G G G G A G G A G

A A A A A A A A A G A A A A A A

+

C C C C T C C C C C C C T C C C

A G G G G G G G G A A G G G G G

G A A G G G A A A G G G G A G A

A C C C C C C C C A A C C C C C

A G G A A A G G G A A A A G A G

C G G G G G G G G C C G G G G G

T T C C C C T T C T T C C T C T

G G G G G G G G G G G C G G G G

A A A A A A A A A A A A C A A A

C A A C C C A A A C C C C C C A

9 9 9 10 10 10 9 9 9 9 9 10 10 9 10 9

a

Position relative to transcription start site (+1). Promoter defined as nucleotide positions upstream of the translation start site (+460). This includes exon 1, intron 1 and the first nine bases of exon 2. LTA promoter allele sequences can be accessed from GenBank: LTAp*001 (accession #AY274908 and AY274915), LTAp*00201 (accession #AY274910 and AY274918), LTAp*00202 (accession #AY274903 and AY274925), LTAp*003 (accession #AY274909 and AY274916), LTAp*004 (accession #AY274913 and AY274920), LTAp*005 (accession #AY274905 and AY274914), LTAp*006 (accession #AY274904), LTAp*007 (accession #AY274911 and AY274912), LTAp*008 (accession #AY274926), LTAp*009 (accession #AY274906 and AY274923), LTAp*010 (accession #AY274922), LTAp*011 (accession #AY274907), LTAp*012 (accession #AY274917), LTAp*013 (accession #AY274919), LTAp*014 (accession #AY274921) and LTAp*015 (accession #AY274924). c Plus (+) indicates the presence of a 145 bp insertion that consists of a duplication of the promoter region from 570 to 426. d Located in exon 2. The T to C change at +496 causes a cysteine to arginine change at amino-acid position 21 in the signal sequence. e Located in intron 2. f Located in exon 3. The A to C change at +697 causes a histidine to proline change at amino acid 17 in the mature protein. The C to A change at +724 causes a threonine to asparagine change at amino acid 26 in the mature protein. g Microsatellite consisting of a (CT)n repeat (n¼9–10). h This allele was a match (100%) to two GenBank sequences (accession #AP000505 and Y14768) over the region examined. i This allele was a match (100%) to one GenBank sequence (accession #AF129756) over the region examined. j These alleles have identical promoter regions. The difference between them occurs after the translation start site and is located in intron 2. b

LTAp*001 (16 haplotypes) LTAp*00201 (19 haplotypes) and LTAp*004 (13 haplotypes). Seven of the alleles were found in only one haplotype (LTAp*006, LTAp*008, LTAp*010, LTAp*011, LTAp*012, LTAp*014 and LTAp*015). The remainder of the alleles were present in low to moderate numbers: LTAp*00202 (two haplotypes), LTAp*003 (four haplotypes), LTAp*005 (six haplotypes), LTAp*007 (eight haplotypes), LTAp*009 (two haplotypes) and LTAp*013 (two haplotypes). The length of the TNFc microsatellite did not factor into the allele designations, as each was unique with regard to SNP content. Relatedness of TNF alleles and of LTA alleles The relatedness of the TNF alleles and of the LTA alleles was determined by the construction of phylogenetic trees using the neighbor-joining method with Kimura twoparameter correction. All of the TNF allelic sequences form a relatively tight cluster (Figure 2). This was expected due to the high degree of homology (X98%; Table 3) among the TNF promoter sequences. However, two groups of TNF sequences can be identified that appear to be based on the SNP at position 1031. TNF group 1031C includes TNFp*004, TNFp*005, TNFp*006 and TNFp*008. TNF group 1031T includes the remaining alleles. Genes and Immunity

The LTA allelic sequences clustered into three groups in the evolutionary tree based on the TNFc microsatellite and shared blocks of SNPs (Figure 3). The LTA sequences with the TNFc (n¼9) microsatellite clustered into two distinct groups. TNFc (n¼9) group 1 consisted of alleles LTAp*001, LTAp*009 and LTAp*010. TNFc (n¼9) group 2 included the remaining TNFc (n¼9) sequences such as alleles LTAp*00201, LTAp*007 and LTAp*013. The split appears to be based on a shared block of SNPs from 293 to þ 369 (see Table 2). Group 1 members encode the block A-G-A-A-C, while group 2 sequences encode the block G-A-C-G-G. Members of group 1 also share an upstream block of SNPs from þ 496 to þ 724 (T-G-A-C), which is variable in group 2. The third group of LTA sequences was composed of all alleles with the TNFc (n¼10) microsatellite. These alleles share a block of SNPs from 293 to þ 496 (G-G-C-A-GC). This block of SNPs is equidistant from the same block of SNPs in each group of TNFc (n¼9) sequences (see Table 2); TNFc (n¼9) group 1 shares þ 11G, þ 253A and þ 496C, while TNFc (n¼9) group 2 shares 293G, þ 81C and þ 369G. Calculation of percentage difference using an allele from each group of LTA alleles as a reference sequence (Table 4) showed that each cluster of LTA alleles is equidistant from the others. Members of each


LTA sequence group were 490% homologous (o0.10% different) to each other when compared to the reference sequence from that group. When the designated LTA reference sequence from each group was compared to each of the LTA sequences in the other two groups, the

homology ranged from B72 to 88% (0.12–0.28% different) for each group of LTA alleles. There also appears to be two distinct subgroups of TNFc (n¼10) LTA sequences based solely on the SNP at 294. TNFc (n¼10) group 1 (LTAp*003, LTAp*005, LTAp*011 and LTAp*014) encodes 294C, while TNFc (n¼10) group 2 (LTAp*004 and LTAp*012) encodes 294T.

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Inclusion of TNF and LTA alleles in HLA haplotyes Haplotypes including TNF, LTA and HLA-A, -C, -B and -DRB1 were evaluated because the TNF and LT loci are located in the MHC, have been associated via SNPs to particular HLA alleles and have been implicated in Table 3 Percentage difference among TNF promoter alleles TNF promoter allele

Figure 2 Relatedness of the TNF promoter alleles. The tree was constructed using the neighbor-joining method with Kimura twoparameter correction as described in Materials and methods.

Designated reference sequence p*001

1031T group p*001 p*002 p*003 p*007

0.00 0.01 0.01 0.01

1031C group p*004 p*005 p*006 p*008

0.01 0.02 0.02 0.02

Figure 3 Relatedness of the LTA promoter alleles. The tree was constructed using the neighbor-joining method with Kimura two-parameter correction as described in Materials and methods. Genes and Immunity


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Table 4 Percentage difference among LTA promoter alleles Designated reference sequence LTA promoter allele

p*001

p*013

p*003

TNFc (n¼9) group 1 p*001 p*009 p*010

0.00 0.03 0.03

0.18 0.21 0.15

0.12 0.15 0.15

TNFc (n¼9) group 2 p*013 p*00201 p*00202 p*006 p*007 p*008 p*015

0.18 0.21 0.24 0.21 0.18 0.27 0.24

0.00 0.03 0.06 0.09 0.06 0.09 0.06

0.12 0.15 0.12 0.15 0.12 0.15 0.18

TNFc (n¼10) cluster p*003 p*004 p*005 p*011 p*012 p*014

0.18 0.27 0.24 0.24 0.28 0.21

0.18 0.21 0.18 0.24 0.22 0.21

0.00 0.09 0.06 0.06 0.09 0.03

many MHC-related autoimmune diseases. A total of 57 haplotypes were determined from MHC homozygous cell lines or from family segregation (Table 5), with 52 of these being unique. No association was noted between TNF or LTA alleles and specific HLA-B or HLA-DRB1 alleles. For example, HLA-B*1801 was associated with haplotypes carrying TNFp*001, TNFp*003 and TNFp*005, and HLA-B*5301 was associated with LTAp*00201, LTAp*00202 and LTAp*008. HLA-DRB1*0301 was found in haplotypes carrying TNFp*001, TNFp*002 and TNFp*005 and in haplotypes carrying LTAp*00201, LTAp*003 and LTAp*007. Some associations between HLA and TNF and LTA alleles were noted with HLA haplotypes. For example, the 8.1 ancestral haplotype (A*0101, C*0701, B*0801 and DRB1*0301) present in three International Histocompatibility Workshop (IHWS) reference cell lines (9022, 9023 and 9086) was only associated with the TNFp*002 and LTAp*007 alleles, whereas, the individual HLA alleles of this haplotype were found associated with multiple TNF and LTA promoter alleles in other haplotypes. However, the association of specific HLA haplotypes with specific TNF and LTA alleles is not fixed. For instance, the haplotype A*0201, B*0501, C*4402 and DRB1*0401 present in cell lines 1060 and 9090 were associated with TNFp*001/LTAp*00201 and with TNFp*001/LTAp*013, respectively. Although the sample size was small, there does appear to be a relatively tight association between specific TNF and LTA alleles. For example, TNFp*002 was always found associated with LTAp*007 (four haplotypes) and LTAp*00201 was always found associated with either TNFp*001 or TNFp*007 (15 haplotypes). In fact, each LTA allele was strongly associated with either one or two TNF alleles in all of the haplotypes identified. This would be expected because of the very short distance (1240 bp) between the TNF and LTA genes. There was only one exception noted to the association between TNF and LTA Genes and Immunity

alleles. From the perspective of the TNF locus, TNFp*001 was found associated with at least 10 of the 16 LTA alleles. This observation is not surprising since TNFp*001 was by far the most predominant TNF allele and was present in over 50% of the haplotypes that were examined.

Discussion The goals of this project were to explore the extent of polymorphism in the promoter regions of the TNF and LTA genes, to designate ‘alleles’ at each locus and to examine associations among SNPs, alleles and HLA. The TNF and LTA loci were characterized in 69 samples from various racial backgrounds with a variety of HLA haplotypes. Polymorphism in the TNF promoter was limited within the sample group. Of the 12 previously reported SNPs, only seven were present and no novel SNPs were identified. However, eight highly related novel TNF promoter alleles were designated. LTA proved to be highly polymorphic. Seven novel SNPs and an insertion were identified in addition to the nine SNPs that were previously reported. The abundance of SNPs led to the designation of 16 novel LTA alleles which clustered into three distinct phylogenetic groups. Finally, this sample group yielded 52 unique TNF-LTA-HLA haplotypes. Multiple alleles of TNF and LTA were reported here. Others also have predicted ‘alleles’ or ‘haplotypes’ of TNF based on associations of a subset of SNPs.34,59,62,65 In each case, some of the reported ‘allelic’ SNP associations were contained in multiple TNF alleles identified here (Table 6). For instance, ‘allele’ TNFA-U01 identified by Matsushita et al59 is included in TNFp*001, TNFp*002 and TNFp*007, while ‘allele’ TNFA-U04 identified by Tsuchiya et al65 is included in TNFp*005 and TNFp*008. There were three alleles in one report that were not seen in this study. This was not surprising as it is expected that additional alleles will be identified. These observations support the importance of comprehensive nucleotide sequence characterization at the TNF and LTA loci. There have been numerous reports of associations among individual SNPs within and between the TNF and LTA loci and their relationship to expression levels, HLA and disease.18,23,40,58,63,66 When associations of individual SNPs among the TNF and LTA alleles were examined in this study, only a single association was observed. In each of the alleles described, the LTA SNP 293A was always found in association with LTA þ 81A and LTA þ 369C, while the LTA SNP 293G was always found associated with LTA þ 81C and LTA þ 369G. Another tight, but not absolute, LTA SNP association (15 of the 16 alleles) was noted between the SNP at þ 253 (NcoI RFLP) and þ 724 ( þ 253A with þ 724C and þ 253G with þ 724A). The remainder of the SNPs within and between the TNF and LTA loci appear to be randomly associated. Other associations could be noted, but are dependent on the context. For example, an association could be assigned between the SNPs TNF 308A, LTA þ 253G (NcoI RFLP; TNFB*1) and LTA þ 724A, which has been observed by others.23,25,27,28,40 Interestingly, each of these SNPs generally have been associated with higher production level phenotypes of both TNF and LTA, with the 8.1 ancestral HLA haplotype


Table 5

553

TNF-LTA-HLA haplotypes HLAa

Cell line

Raceb

A*

Cw*

B*

DRB1*

TNFp*

LTAp*

9001 9047 9050 9051 9038 9065 DS396 DS396 1261 9008 9082 1061 9016

API C C C C C Cam Cam AA C C LI AI

2402 0301 2902 2902 0201 0301 0202 0301 7401 2501 0301 110101 0204

0702 0602 1601 1601 0701 0702 1403 1403 0701 1203 0702 0501 1502

0702 4701 440301 440301 1801 0702 440301 440301 070201 1801 0702 440201 5101

0101 0701 0701 0701 1201 1301 130101 130101 130201 1501 1501 150101 1602

001 003 001 001 003 001 001 001 001 003 001 001 003

001 001 001 001 001 001 001 001 001 001 001 001 001

2090 1061 1081 9021 9021 1060 9101 9071c 9077 1183 9077 2011 2011 9053 2006

AA LI LI AA AA LI AI AI API AA API AA AA API AA

6802 020101 020101 3001 6802 020101 3101 3101 0207 3002 0207 1101 020101 3303 2301

0701 0102 0501 1701 1701 0501 0102 0102 0304 0102 0804 0102 0704 040101 1403 040101

5801 270502 440201 4201 4201 440201 1501 1501 1520 4601 8101 4601 1801 3502 440301 5301

010201 030101 030101 0302 0302 040101 080201 080202 090102 1201 120201 1301 1302 1302 1401

001 001 001 007 007 001 001 001 001 001 001 001 001 001 001

00201 00201 00201 00201 00201 00201 00201 00201 00201 00201 00201 00201 00201 00201 00201

1261 S21d

AA IC

030101 ND

020204 ND

3901 5301

1001 ND

001 ND

00202 00202

9020 9085 9052

C C C

2601 3002 0201

0501 0501 0602

1801 1801 5701

0301 030101 0701

005 005 008

003 003 003

9031 9032 2015 1179 1193 9058 9097 9097 9064 DT23c

C C AA AA AA AA C C AI Cam

0201 0201 020101 020101 3303 0201 0201 0301 0217 2301 3004

0304 0304 1601 1601 1601 1601 0304 0304 0303 040101 0802

1501 1501 4501 4501 4501 4501 4001 4001 1501 1403 3501

0401 0401 110102 1102 110102 1301 1302 1302 1402 1114 1322

006 006 006 006 006 004 006 006 006 001

004 004 004 004 004 004 004 004 004 00201 004

1083 2015 9042 1100 1183 1100

AA AA C AA AA AA

3001 3303 2402 2301 3201 2902

0701 030402 0401 0701 0802 020204

5801 2703 3508 1503 1405 5801

090102 1102 1103 130201 130301 1503

001 001 001 001 001 001

005 005 005 005 005 005

2006

AA

680101

0602

5802

1103

001

006

9022 9023 9086 1060 1083 DT29

C C C LI AA Cam

0101 0101 0101 2402 0102 7401

0701 0701 0701 0701 0701 020204

0801 0801 0801 0801 4901 1503

0301 0301 0301 030101 130201 1503

002 002 002 002 002 002

007 007 007 007 007 007

S21d

IC

ND

ND

5301

ND

ND

008

Cam AA

2301 030101

ND 0804

1403 5801 1510

030201 010201 110402

001 007 001

00201 009 009

DT3c,d 2090

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Table 5 (continued) HLAa Cell line

Raceb

A*

Cw*

B*

DRB1*

TNFp*

LTAp*

DT24c

Cam

0205

040101 0706

5301 440301

1503 130201

001

004 010

2093

AA

6802

0701

1510

130201

001

011

9075

C

2402

0304

4001

090102

006

012

9090

C

0201

0501

4402

0401

001

013

DT16c

Cam

0201 2301

0802 1601

1403 4501

030201 1503

001 007

00201 014

DT32c

Cam

0201 0202

040101 0706

4703 440301

1503 080401

ND

004 015

a

Allele level HLA typings for IHWS reference cell lines can be found at the website http://www.ashi-hla.org/storefiles/repository/ ASHIAllelesREV.html. Allele level HLA typing on the remaining cell lines was performed in house as described in Materials and methods. Some alleles cannot be distinguished over the commonly sequenced exons; possible alternative allelic designations can be found at the website http://www.ebi.ac.uk/imgt/hla/ambig.html. b Race/ethnicity: C¼Caucasian, AA¼African American, API¼Asian/Pacific Islander, AI¼American Indian, LI¼Lumbee Indian, Cam¼Cameroon and IC¼Ivory Coast. c Haplotypes could not be resolved due to heterozygosity at two or more loci. These cell lines were included because they carried a less frequent or unique TNF or LTA allele or because they carried a unique combination of TNF and LTA alleles. These cell lines were not included in the count of unique haplotypes, but were included in the count of haplotypes carrying specific TNF and LTA alleles. d In most cases, there was insufficient sample to complete typings (ND¼not determined). These cell lines were included because they carried a less frequent or unique TNF or LTA allele. These cell lines were not included in the count of unique haplotypes, but were included in the count of haplotypes carrying specific TNF and LTA alleles.

Table 6 Comparison of predicted TNF promoter ‘alleles’ TNF SNP position Allele (haplotype)

1031

863

857

308

238

TNF promoter allelea

Matsushita et al59 TNFA-U01 TNFA-U02 TNFA-U03 TNFA-U04

T T C C

C C A C

C T C C

NDb ND ND ND

ND ND ND ND

p*001, p*002, p*007 p*003 p*006 p*004, p*005, p*008

van Heel et al34 TCCG TCCA TCTG CACG CCCG

T T T C C

C C C A C

C C T C C

G A G G G

ND ND ND ND ND

p*001, p*007 p*002 p*003 p*006 p*004, p*005, p*008

Tsuchiya et al65 TNFA-U01.1 TNFA-U01.2 TNFA-U02 TNFA-U03 TNFA-U04

T T T C C

C C C A C

C C T C C

G A G G G

G G G G A

p*001, p*007 p*002 p*003 p*006 p*005, p*008

Ubalee et al62 TNFP-A TNFP-B TNFP-C TNFP-D TNFP-M1c TNFP-M4c TNFP-M7c

T C C T T C T

C A C C C C A

C C C T C T T

G G G G G A A

G G A G A G G

p*001, p*007 p*006 p*005, p*008 p*003 Not found Not found Not found

a

TNF promoter ‘alleles’ identified in this study that contain the ‘allelic’ sequence previously reported. ND¼not determined. c These SNP combinations were not seen in the alleles identified in this study. b

Genes and Immunity


and with MHC-linked autoimmune diseases.24,37,67 This apparent association of SNPs results from the linkage of the TNFp*002 (308A) allele with the LTAp*007 ( þ 253G, þ 724A) allele in several haplotypes, including the 8.1 ancestral haplotype (A1, B8, DR3). However, this association only holds from the perspective of the TNF 308A SNP, as it is not possible to type for either of the LTA SNPs and assume linkage. Both LTA þ 253G and LTA þ 724A were found associated more frequently with TNF 308G (found in TNFp*001 and TNFp*007). The LTA þ 253G SNP also can be associated with LTA þ 724C (found in LTAp*013). One study also placed LTA þ 11A (also found in LTAp*007) within the TNF 308A, LTA þ 253G and LTA þ 724A SNP association,27 but this SNP also can be associated with TNF 308G and LTA þ 724C. Therefore, the context in which SNP associations are reported is of consequence. It is well documented that differences exist in cytokine expression levels between individuals and that these differences are due to some of the SNPs carried by the individual at that locus. Consequently, a multitude of studies have linked cytokine expression level phenotypes with either individual SNPs or combinations of SNPs, oftentimes with the reporting of contradictory results.41,42 The LTA SNP at þ 253 (NcoI RFLP; TNFB) is a classic example of these types of studies as this SNP has been associated with both TNF and LTA expression levels. In the case of TNF, there have been several conflicting studies correlating TNF expression levels with the LTA NcoI RFLP.23,37,38,60,68 Some light may be shed by using examples from a study by Abraham et al,37 who measured TNF levels from cell lines (also examined in this study) carrying various ancestral haplotypes and typed for the LTA NcoI RFLP. Although a range of TNF levels were noted, higher mean TNF levels were reported with LTA þ 253G (NcoI RFLP; TNFB*1) particularly in the 8.1 ancestral haplotype, but there were intermediate and low TNF producing cell lines that also possessed the þ 253G SNP. In our study, LTA þ 253G (TNFB*1) was contained in seven distinct alleles which were linked to three distinct TNF alleles and LTA þ 253A (TNFB*2) was contained in nine unique alleles which were linked to five different TNF alleles. The TNF expression level results obtained by Abraham et al37 are more readily explained by the TNF allele each cell line carried, instead of the LTA SNP at þ 253. The high TNF producing cell lines 9022 and 9023 each carry TNFp*002 and encode LTA þ 253G. Cell lines 9082 and 9090 were intermediate TNF producers. Both carry the TNFp*001 allele, but one encodes LTA þ 253A (9082) and the other encodes LTA þ 253G (9090). The remaining cell lines were all low producers, carried TNF alleles other than TNFp*001 and TNFp*002 and were variable with respect to the LTA þ 253 SNP. These include 9008 (TNFp*003; LTA þ 253A), 9085 (TNFp*005; LTA þ 253A), 9031 (TNFp*006; LTA þ 253A), 9021 (TNFp*007; LTA þ 253G) and 9052 (TNFp*008; LTA þ 253A). Studies correlating LTA expression levels with the LTA NcoI RFLP have produced similarly contradictory results.41,42 For example, the TNFB1 allele (LTA þ 253G, NcoI RFLP) has been correlated with both increased23 and decreased39 LTA expression levels, while a third study found no correlation between this SNP and LTA expression levels.60 Each of these studies were very similar in design with the exception of the HLA types of

the individuals examined. The variety of HLA types of the subjects in the first two studies was limited and also differed. It is possible that the subjects in these two studies carried different subsets of LTA alleles and that, by chance, the LTA alleles, which were represented, skewed the expression level results. However, the latter study, which found no correlation between the LTA SNP at þ 253 and LTA expression levels, measured LTA levels from subjects with a wider variety of HLA types likely representing a broader set of LTA alleles. Collectively, these observations again suggest that allelic, not SNP, typing at the TNF and LTA loci would be more informative for these types of studies. Interestingly, when the LTA þ 253 SNP was examined with more direct methods for its effect on LTA expression,27,28 conflicting results also were obtained. Multiple studies have associated specific SNPs in either the TNF or LTA locus with HLA-B and/or HLADR as these loci flank the MHC class III region which contains the TNF and LTA genes.58,66,68,69 Although a definitive answer to linkage will likely require more comprehensive population studies, this study found no association of individual HLA-B or HLA-DRB1 alleles with specific SNPs in either TNF or LTA. For example, both DR2 (DRB1*1501) and DR3 (DRB1*0301) were linked with various TNF promoter alleles that encode 308G and that encode 308A.60,68 On the other hand, there were three instances in which different cell lines that carried the same HLA haplotypes also carried the same TNF and LTA alleles. These results lend support to previous reports of associations between HLA haplotypes and specific TNF and LTA SNPs.24,25,67,70 However, there was an instance in which cell lines carrying identical HLA haplotypes were found associated with different combinations of TNF and LTA alleles. The difference between these cell lines was in the LTA allele that was present in the haplotype, LTAp*00201 (1060) and LTAp*013 (9090). These LTA alleles differ only at the commonly studied SNP þ 724 C/A (Thr26Asn). These observations are quite significant when one considers how often this SNP has been linked to other SNPs in both LTA and TNF, to HLA and to expression levels. Thus, linkage should not automatically be assumed. This study was undertaken to begin to explore the extent of polymorphism in TNF and LTA promoters. It was shown that polymorphism in the promoter regions of TNF and LTA is quite complex. Multiple TNF and LTA promoter alleles were identified and associations were assessed. The data reported here hopefully may provide a resolution to the often ambiguous and contradictory results of studies investigating the associations among SNPs, expression levels, HLA and disease. Studies are currently underway to examine the expression level phenotype of each of the TNF and LTA alleles.

555

Materials and methods Cells and sequence-based typing Cells were chosen from IHWS reference cell lines, families and unrelated individuals to include various HLA types and races/ethnicities. In total, 69 samples were characterized for TNF and LTA. ‘Alleles’ of the TNF promoter and LTA promoter were determined by family segregation analysis and by sequencing (see below) of Genes and Immunity


556

cloned DNA or homozygous cell lines. Allele level HLA sequence-based typing was performed on the family samples and the unrelated individuals as follows: (1) HLA-DRB1 typing using the HLA-DRB BigDye Terminator Sequence-Based Typing Kit (PE Applied Biosystems, Foster City, CA, USA) as per the manufacturer’s protocol, (2) HLA-A and HLA-B typing was performed as previously described at http://www.ihwg.org/components/sbtover.htm and (3) HLA-C genomic DNA amplification was performed as previously described71 and sequencing was performed as described below. Haplotypes were deduced from MHC homozygous cell lines and from family segregation analysis. The total number of unique TNF/LTA-HLA haplotypes was determined from the samples for which complete haplotypes could be resolved. The number of haplotypes that carry each allele of TNF and of LTA was determined from all samples. Homozygous cell lines were counted as two haplotypes with the exception of consanguineous cell lines72 whose haplotypes were considered identical by descent and counted only once. Genomic DNA extraction Genomic DNA was extracted with the DNA Isolation Kit (Pel-Freez; Brown Deer, WI, USA) as per the manufacturer’s protocol. Genomic DNA was resuspended in H2O (200–500 ml) and stored at 201C. DNA concentration was determined by absorbance at 260 nm. Amplification of TNF and LTA promoter regions TNF and LTA were amplified from genomic DNA (500 ng) by polymerase chain reaction (PCR) with the primers (20 pm each) TNF5-1 (gatggactcaccaggtgag) and TNF3-1 (ggtgtcctttccaggggag) and with the primers (20 pm each) LTA5-4.2. (ctgacctatctcatctgatagtagg) and LTA3-2 (aggtggatgtttaccaatgag), respectively. Reactions (100 ml) were performed in a Gene Amp 9600 (PerkinElmer, Norwalk, CT, USA) and included Platinum TaqHi Fi (2.5 U) (Invitrogen, Carlsbad, CA, USA), MgSO4 (2 mM), dNTP mixture (0.2 mM each) and 10 buffer (1 ). For amplification of the B1.2 kb TNF fragment, the reaction was denatured (10 min, 951C) for one cycle and amplified (30 s, 951C; 30 s, 601C; 1.5 min, 681C) for 35 cycles which was followed by a final extension step (5 min, 681C) for one cycle. Amplification of the B3.2 kb LTA fragment was performed under the following conditions: denaturation (10 min, 951C) for one cycle, amplification (45 s, 951C; 45 s, 581C; 3.5 min, 681C) for 35 cycles and final extension (5 min, 681C) for one cycle. Amplification products were confirmed by agarose gel electrophoresis with DNA size standards. Amplified DNA was purified using the Wizard PCR Preps DNA Purification System (Promega, Madison, WI, USA) as per the manufacturer’s protocol with the exception that the DNA was eluted from the column with 70 ml H2O. Sequencing Purified PCR products (400 ng) were sequenced with the Big Dye Terminator sequencing kit (PE Applied Biosystems, Foster City, CA, USA) in a series of reactions (10 ml) containing primer (5 pm) and Ready Reaction Mix (4 ml). Reactions were purified by ethanol precipitation. Briefly, each reaction was combined with a mixture (25 ml) of ethanol (70%) and sodium acetate pH 4.6 (1 M) and centrifuged (3000 r.p.m., 30 min). Pellets were washed

Genes and Immunity

with 70% ethanol (50 ml) and centrifuged (3000 r.p.m., 5 min). Dry pellets were stored at 201C. Pellets were resuspended in 2 ml of a mixture of deionized formamide (100 ml) plus gel loading dye (20 ml), heated (951C, 2 min) and placed directly on ice prior to loading (0.5–1.0 ml). The remaining sample was stored at 201C. Reactions were run on an ABI377 automated DNA sequencer (PE Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. Data were analyzed with Sequencher 4.1 software (Gene Codes Corporation, Ann Arbor, MI, USA). Sequences were reported only when confirmed from data obtained on amplified DNA from at least two separate PCR amplifications.

Cloning TNF and LTA ‘alleles’ were confirmed by cloning and sequencing of multiple clones. Amplified products were cloned into the TA cloning vector (Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s protocol. Individual clones were harvested in 50 ml of water and lysed (951C, 5 min). Lysed clones were centrifuged and lysates were stored at 41C. Lysate (10 ml) was amplified by PCR with corresponding primer sets (as described above). Amplified products were sequenced as described above.

Phylogenetic analysis and percentage difference Phylogenetic trees were constructed with PAUP software (version 4.0.0b10; Sinauer Associates, http://www.sinauer.com) using the neighbor-joining method with a Kimura two-parameter correction. The branch length of the unrooted phylograms shows relative genetic distance between alleles. Percentage difference analysis was performed using PercentDif, a program developed inhouse using Perl, version 5.6.1. This program was based on an analysis by Rajalingam et al.73 Sequences were aligned in Sequencher 4.1 software (as above) and imported into the PercentDif program. The percentage difference is calculated between each member of the alignment as compared to a designated reference sequence. The program calculates percentage difference between two sequences by comparing the number of gaps and mismatches over the length that the two sequences overlap. Sequences LTAp*012 and AF037255 have a 145 bp insertion that was not taken into account in either analysis so as to better ascertain its relatedness to the remaining ‘alleles’.

Acknowledgements The authors would like to thank Carolyn Katovich Hurley, PhD for all of her support in this research project and William Klitz, PhD at UC Berkeley for his thorough analysis of our data. This study was supported by funding from the Office of Naval Research N00014-001-0898 (CK Hurley) to the CW Bill Young Marrow Donor Recruitment and Research Program. The views expressed in this article are those of the authors and do not reflect the official policy of the Department of the Navy, the Department of Defense or the US government.


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