Irnmunogenetics 22: 399-405, 1985

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geneucs

Springer-Verlag/985

Molecular Cloning and Partial Nucleotide Sequence of a 3.5 kb HLA-B27-Associated Fragment of Genomic D N A
Joseph A. Trapani, Claudia A. Mickelson, Nicholas J. Deacon, David J. Hooker, and Ian F.C. McKenzie Research Centre for Cancer and Transplantation, Department of Pathology, The University of Melbourne,Parkville,Victoria, 3052,Australia

Insight into the mechanism of involvement of HLA-B27 in ankylosing spondylitis (AS) (Brewerton et al. 1973, Schlosstein et al. 1973) has previously been hampered by the inability to directly examine the H L A - B 2 7 gene and/or contiguous genetic sequences. Accordingly, we investigated the HLA-B27 gene by generating restriction fragments of genomic DNA derived from healthy and diseased HLA-B27 subjects. Using an HLA class I-specific cDNA probe, we have previously reported the assignment of a restriction fragment length polymorphism (RFLP) which segregates strongly with the H L A - B 2 7 allele (Trapani et al. 1985). We now describe the molecular cloning and partial nucleotide sequence of this fragment of genomic DNA derived from a patient with AS. It is anticipated that further sequencing of this cloned fragment, which represents the first isolation of a segment of the HLA-B27 gene, may resolve the question of whether susceptibility to AS is related to a particular polymorphism of the B27 gene. High molecular weight genomic DNA derived from HLA-B27 + patients w i t h AS, healthy HLA-B27 relatives, and B27- control subjects was purified from peripheral blood leukocytes according to standard techniques (Wyman and White 1980), restricted with Taq I (New England Biolabs, Beverley, Massachusetts) according to the manufacturer's recommended assay conditions and electrophoresed in 0.8% agarose gels. Following transfer to nitrocellulose (Southern 1975), DNA sequences were probed with nick-translated pDP001 (Biro et al. i983), whose insert represents 85% of the mRNA sequence for HLA-B7. Examination of the resultant autoradiograph revealed multiple polymorphic bands (Fig. 1). The smaller of two bands which formed a doublet at approximately 3.6 and 3.5 kb was associated with the presence of the HLA-B27 allele as the 3.5 kb band segregated with the B27 status of nine of the ten individuals tested in this experiment. Of a total of 33 HLA=B27 + and B27- individuals examined thus far, all 17 HLA-B27 subjects have exhibited this band. Of the remaining 16 negative control subjects, 2 exhibited the 3.5 kb fragment (Z2 > 20; P < 0.0001). These studies have included two families, and in both, the polymorphic fragment was shown to segregate with the

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Fig. 1. Southern blot analysis of genomic DNA from ten individuals restricted with Taq I and probed with pDP001. An HLA-B27-associated band is indicated by the arrowhead at 3.5 kb. This band correlated with B27 status (shown as '+" or - " ) in nine of these ten subjects, the only exception being the individual second from the right, who is B27- but exhibits an apparently identical band. Molecular weight markers were derived from lambda restricted with Hind III.

B27-bearing h a p l o t y p e . Several i n d i v i d u a l s w h o b e a r serologically r e l a t e d specificities such as H L A - B 7 were e x a m i n e d a n d the 3.5 k b b a n d was absent ( T r a p a n i et al. 1985). It is thus clear t h a t this 3.5 k b f r a g m e n t consists, at least in part, of an H L A B27-associated sequence in g e n o m i c D N A , which is n o t characteristic of the genes e n c o d i n g serologically r e l a t e d class I molecules. T h e strategy e m p l o y e d for c l o n i n g this D N A f r a g m e n t was a n a l o g o u s to t h a t d e s c r i b e d p r e v i o u s l y ( S a k a n o et al. 1979, T a b a k a n d Flavell 1978), except that, because of the m u l t i p l i c i t y of D N A f r a g m e n t s g e n e r a t e d b y d i g e s t i o n with T a q I which were able to h y b r i d i z e with p D P 0 0 1 (even with very high stringency washing),

Cloning an HLA-B27-Associated Genomic DNA Fragment

401

Fig. 2. Southern blot analysis of fractions of genomic DNA purified from an HLA-B27+ individual with AS (lanes A, B, and C) and a B27- control subject (lanes D, E, and F). The fraction of B27 DNA of approximately 3.2-3.6 kb (lane B) clearly exhibits a band at 3.5 kb which hybridizes with pDP001 and is absent from the corresponding negative control DNA (lane E). The upper band of the 3.5 and 3.6 kb doublet (see Fig. 1) is seen faintly in lane A and is largely absent from lane B. Fig. 3. Southern blot analysis of plasmid DNA derived from four negative clones (lanes 1-4) and four isolates of S14.t3 (lanes 5-8). Approximately 1 gg of plasmid DNA was electrophoresed in a 0.8~oagarose gel, following digestion to completion with Taq I. A specific band at 3.5 kb appears in tracks representing the positive clones following probing with pDP001. Molecular weight markers at left are derived from Eco RI digests of iambda DNA.

we decided to size-restrict the D N A fragments used to construct the library. D N A from an H L A - B 2 7 spondylitic patient was restricted with T a q I, and fragments in the approximate size ranges of 2.7 to 3.2 kb, 3.2 to 3.6 kb, and 3.6 to 4.2 kb were purified by elution from an agarose gel. It was shown that the pDP001 insert detected a b a n d in the 3.2 -3.6 kb fraction of B27 D N A which was abSent from the corresponding fraction of B 2 7 - D N A , which had been similarly purified. Fortuitously, the upper b a n d of the 3.5-3.6 kb doublet was found principally in the higher molecular weight fraction (Fig. 2). Thus it was possible to enrich B27 D N A for the relevant B27-associated 3.5 kb species approximately 20-fold while markedly reducing the presence of non-B27-associated D N A species which were able to bind to pDP001. After construction of a library in the plasmid vector pBR322 using the 3.2-3.6 kb fraction, a unique clone, designated S14.13, was selected which hybridized strongly with the pDP001 insert c D N A . Binding of the pDP001 insert was specific for a 3.5 kb insert derived from four separate isolates of the positive clone, while no such binding was apparent with inserts from the negative clones (Fig.

3).
To ensure that the S14.13 insert contained sequences which are specific for HLA-B27, we used the nick-translated insert to probe a panel of T a q I-digested B 2 7 - and B 2 7 - genomic DNAs. A b a n d at approximately 3.5 kb appeared in each of the lanes containing B27 D N A but was absent from the remaining lanes (Fig. 4). Interestingly, it was also noted that another p o l y m o r p h i c b a n d of approximately 4.3 kb segregated with the Bw44 allele in nine of the ten members of this test population. T w o other principal bands were noted, one of approximately 1.5 kb which was

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Fig. 4. Southern blot analysis of genomic DNA from ten individuals restricted with Taq I and probed with the 3.5 kb insert from S14.13 plasmid DNA. A 3.5 kb polymorphic band indicated by the closed arrowhead appears which segregates with B27 status as shown at the foot of the figure. Another polymorphic band at approximately 4.3 kb (indicated by " < ' ) segregates with HLA-Bw44 in nine of these teri individuals, the only exception being the individual at the extreme right side of the autoradiograph. Molecular weight markers at left are derived from Eco RI digests of lambda DNA. present in each D N A sample a n d a n o t h e r of 7.4 k b which was polymorphic. Neither, however, could be assigned to a specific H L A - A or -B allele. R e c o m b i n a n t S14.13 clones in M 1 3 m p l 8 were sequenced according to the Sanger dideoxy m e t h o d (Sanger et al. 1977). A c o m p a r i s o n of the nucleotide sequence of the 1.1 k b Sac I fragment of the S14.13 insert (which included part of the

Cloning an HLA-B27-AssociatedGenomic DNA Fragment Table 1. Sequence comparisons of selected HLA genest

403

HLA-B27
A. Noncoding regions HLA-B27 pHLA-12.4 HLA-A2 HLA-A3 HLA-Cw3 HLA-B27 pHLA-12.4 HLA-A2 HLA-A3 HLA-Cw3 *

pHLA-12.4 HLA-A2 HLA-A3 HLA-Cw3

46 *

38 75 *

36 73 93
*

52 55 60 62 91 83 87 85

Intron 7 (56 bases)

86 *

86 92 *

84 94 96 *

Intron 6 (78 bases)

B. Coding regions (exon 7)

HLA-B7
HLA-B27 pHLA-12.4 HLA-A2 HLA-A3 HLA-Cw3 * 95 * 98 93 * 95 90 98 * 90 88 88 86 * 98 98 95 93 90

t All figures are expressed as percentage homology. intron preceding exon 7, the whole of this exon, and approximately 1.0 kb of downstream sequences) with the corresponding portions of other cloned H L A genes demonstrated a high degree of homology in the sequences for exon 7, which encodes the carboxy terminal end of the B27 polypeptide (Table 1 and Fig. 5). There was only one nucleotide difference compared with the sequences ofHLA-B7 (Biro et al. 1983) and HLA-A2 (Koller and Orr 1985), and two differences from that of HLA-A3. The 14 amino acid residues encoded by this exon are predicted to be identical for all of these alleles; however, as did HLA-B7, the HLA-B27 polypeptide terminated 3 amino acids short of the heavy chains of HLA-A2, -A3, and -Cw3, as each of the latter three genes possesses two more amino acids encoded by exon 7 and a single amino acid from exon 8. The degree of homology among these genes in the intron preceding exon 7 was highest between allelic genes (HLA-A2 and -A3); however, a comparison of the B27 sequence in this region with other genes revealed closer homology with I-ILA-Cw3 than with the I t LA - A alleles (91~ versus 86~o and 84~, respectively). When the sequences downstream from exon 7 were compared, the HLA-B27 gene was found to have a donor splicing site at a position equivalent to that of I-ILA-A2, -A3, and -Cw3, despite the fact that the latter three genes encode two additional amino acids in exon 7. The sequence for pHLA-12.4 lacked this consensus sequence (Fig. 5). A comparison of the first 56 bases of intron 7 revealed that the degree of similarity between HLA-B27 and the other alleles was markedly diminished in this region, the degree of homology being most preserved at the 5' end. For example, the initial 18 bases of HLA-B27 and HLA-Cw3 were highly homologous (15 identical bases) in this intron; however, beyond this point the concordance was far lower. The same

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A

J.A. Trapani et al.

EXON 7

Sac I
HLA-B27 prILA-12.4 HLA-A2 ULA-A3 HLA-Cw3

30 60 ,I, 90 GAGCTCACCCACCCC CTAATTCCT CCTGTC*CCACGTCT CCTGC GGGCTCTGAC CAGGTCCTGTTTTTGTTCTACTCCAC-GCAGCGACAGTGCC CAG AC C C A*G A A T A A A AC C AG A T T A T C T C T AC C AG A T T A T T A T T * A TT T C A HLA-B7[

120 HLA-B27 pHLA-12.4 HLA-A2 HLA-A3 HLA-Cw3 HLA-B7

"~INTRON 7

150

180

GGCTCT GTGTCTCCACAGCTTGAAAAGGTGAGATCTGGGGTcTAGAGTGGGTGGGTGGCAGGTCTGGGGGTGGGTGGGGCA GAT G C~" CCT G G CT TGT TG GATGI-[ GGG AACATG ACA T GC G A G CT TGT TGTT G TGTT GGC A,~CATGACACAG C T G T G A A C A T T TGTT G TGTT G C AACA T G C C G C T G G AAA TC T G GG GA C TC G TGT* GGCA A'CA AAAA GC

a~

B
HLA-B27 HLA-B7 HLA-A2 HLA-A3 HLA-Cw3 pHLA-12.4 SSDSAQGSDVSLRA-C K V C K V C K A

N N

I T

Fig. 5A and B. Nucleotide sequence of HLA class I genes (A), consisting of the 78 bases at the 3' end of intron 6, the whole of exon 7, and a portion of intron 7. Only exon informationis availablefor HLA-B7. The predicted amino acid sequences arising from exon 7 (B) show close similarities for these genes. Deletions in the sequences are indicated by *

finding was evident When HLA-B27 was compared with the equivalent regions of the two H L A - A alleles (Table 1). Overall, in the comparison of the noncoding nucleotide sequences of these four genes and that of pHLA-12.4 which encodes a gene, the mapping of which is unclear (Malissen et al. 1982), HLA-B27 was the most dissimilar; it should be noted however, that the noncoding sequence of no other HLA-B region allele was available for comparison. S 14.13 represents the first isolation of a portion of HLA-B27 (or a closely related gene) from an individual with AS. The technique described, when applied in the same way to the Taq I-digested D N A from a healthy subject, should provide a means of direct comparison of the HLA-B27 gene in health and disease. Initial nucleotide sequence analysis has indicated the isolation of a portion of an H L A gene, but the size of this cloned insert and the presence of a considerable amount of 3' untranslated sequence preclude the possibility of having isolated the entire HLAB27 gene. Limited restriction mapping and comparison to the restriction map of pHLA-12.4 suggest that the 5' end of S14.13 coincides with the Taq I site either at position 539 or at position 771 (Malissen et al. 1982). The 5' end of the insert would thus map either to e x o n 2 or to the following intron, while the 3' end has been shown to be approximately 1.0 kb downstream from the stop c o d o n (not shown). However, the precise position of the 5' end of the molecule remains to be confirmed by further nucleotide sequencing. Similarly, the possibility that S14.13 encodes a pseudogene in close linkage disequilibrium with HLA-B27 cannot be excluded by the data; though such a class I pseudogene has not been previously described in association with HLA-B27, such genes are k n o w n to occur in the mouse system (Steinmetz et al. 1981). Final identification of this clone as HLA-B27 will depend on comparison of

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405

the predicted amino acid sequence with that published for HLA-B27 (Ezquerra et al.

1985).
In summary, this paper describes the first isolation of a portion of the H L A - B 2 7 gene from a patient with AS. It is anticipated that the techniques described herein may eventually allow a direct comparison of this gene in health and disease, thus addressing some of the possible mechanisms which link class I H L A alleles with disease susceptibility.

References
Biro, P., Pan, J., Sood, A. K., Kole, R., Reddy, B., and Weissman, S. M.: The major histocompatibility complex. Cold Spring Harbor Symp. Quant. Biol. 47: 1082, 1983 Brewer ton, D. A., Hart, F. D., Nicholls, A., Caffrey, M., James, D. C. O., and Sturrock, R. D.: Ankylosing spondylitis and HL-A27. Lancet 1 : 904.907, 1973 Ezquerra, A., Bragado, R., Vega, M. A., Strominger, J. L., Woody, J., and Lopez de Castro, J. A.: Primary structure of papain-solubilised human histocompatibility antigen HLA-B27. Biochemistry 24: 1733-1741, 1985 Koller, B. H. and Orr, H. T.: Cloning and complete sequence of an HLA-A2 gene: Analysis of two HLAA alleles at the nucleotide level. J. Immunol. 134: 2727-2733, 1985 Malissen, M., Malissen, B., and Jordan, B.R.: Exon/intron organisation and complete nucleotide sequence of an HLA gene. Proc. Natl. Acad. Sci. U.S.A. 79: 893-897, 1982 Sakano, H., Rogers, J. H., Huppi, K., Brack, C., Traunecker, A., Maki, R., Wall, R., and Tonogawa, S.: Domains and the hinge region of an immunoglobulin heavy chain are encoded in separate DNA segments. Nature 277: 62%633, 1979 Sanger, F., Nicklen, S., and Coulson, A. R.: DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467, 1977 Schlosstein, L., Terasaki, P. I., Bluestone, K., and Pearson, C. M.: High association of an HLA antigen, W27 with ankylosing spondylitis. N. Engl. J. Med. 288: 704.706, 1973 Southern, E.M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517, 1975 Steinmetz, M., Moore, K.W., Frelinger, J. G., Sher, B.T., Shen, F.W., Boyse, E.A., and Hood, L.: A pseudogene homologous to mouse transplantation antigens: Transplantation antigens are encoded by eight exons that correlate with protein domains, Cell 25: 683-692, 1981 Tabak, H. F. and Flavell, R. A.: A method for the recovery of DNA from agarose gels. Nucleic Acids Res. 5: 2321, 1978 Trapani, J.A., Mickelson, C.A., and McKenzie, I. F. C. : A 3.5 kilobase Taq I restriction fragment of genomic DNA segregates with HLA-B27. Immunogenetics 21 : 189-192, 1985 Wyman, A. R. and White, R.: A highly polymorphic locus in human DNA. Proc. Natl. Acad. ScL U.S.A. 77: 6754-6758, 1980

Received June 7, 1985; revised version received July 16, 1985