Proc. Nati. Acad. Sci. USAVol. 82, pp. 3741-3745, June 1985Genetics

Deletion mapping of HLA and chromosome 6p genes(Southern blot analysis/restriction fragment length polymorphism/major histocompatibility complex)

FRED LEVINE*, HENRY ERLICHt, BERNARD MACH*, ROBIN LEACH§¶, RAY WHITE§, AND DONALD PIOUS**Departments of Genetics and Pediatrics, RD-20, University of Washington, Seattle, WA 98195; tCetus Corporation, 600 Bancroft Way, Berkeley, CA 94710;tDepartment of Microbiology, University of Geneva Medical School, 1205 Geneva, Switzerland; and §Howard Hughes Medical Institute, University of Utah,Salt Lake City, UT 84132

Communicated by A. H. Doerman, January 22, 1985

ABSTRACT We have analyzed a set of heterozygousmutants with deletions that encompass parts of HL4 andsurrounding regions of chromosome 6p. By a combination ofSouthern blotting, serologic, enzymatic, and cytogenetic analy-ses, we have ordered eight independent deletion break pointsinto a sequence that divides chromosome 6p into six regions.The deletion mutants have been used in conjunction with theSouthern blot technique to mapHLA and other 6p gene probesinto those regions. On the basis of these and other data wepropose a genetic and physical map of HLA and surroundingregions of chromosome 6p. We rind that for HL4 probes, mostofwhich hybridize with more than one gene, the multiple copiesrecognized by single probes map to single regions. Any chromo-some 6p gene can now be regionally mapped by using thesemutants.

The human major histocompatibility complex (HLA), locatedon the short arm of chromosome 6 (6p21), contains twoclasses of closely linked multigene families coding for highlypolymorphic, heterodimeric, cell surface proteins involved inimmune recognition and defense. It has been possible togenetically map the heavy chains ofHLA class I molecules byusing serologic methods and pedigree analysis because theclass I serologic polymorphisms reside solely in the heavychains. For class II antigens, the situation is more complex.There are currently three well-characterized class II cellsurface molecules, HLA-DR, HLA-DQ (formerly HLA-DCor -DS), and HLA-DP (formerly -SB). On the basis ofserologic analysis, cellular typing results, and pedigree stud-ies, the currently accepted order of the genes encoding thesemolecules is centromere, DP, DR/DQ, class I (summarizedin ref. 1). However, these approaches are limited because inmost cases it is not known which chain bears the determi-nants recognized by these methods. In addition, many of thegenes coding for these chains appear to be so close togetherthat pedigree analysis is unlikely to reveal any recombinationbetween them, Finally, Southern blot analysis and DNA andprotein sequencing indicate that there are two or more copiesof most class II genes (2, 3); the arrangements of the multiplecopies of the class II genes with respect to each other areunknown except in a few cases in which they have been foundon the same or overlapping genomic clones (4, 5).The use of deletion mutants in conjunction with the

Southern blot technique constitutes a powerful approach tothe problem of mapping human genes. In our first applicationof this approach, we used a single heterozygous deletionmutant of the human B-cell line T5-1 to map the HLA-DRagene to a small interval on the short arm ofchromosome 6 (6).In the present study we have generalized this approach byusing a set offour heterozygous deletion mutants with uniquebreakpoints as well as a number of gene probes believed to

map to the short arm of chromosome 6. The probes usedinclude cDNA clones for two HLA class I genes, six HLAclass II genes, one HLA class III gene, and several othergenes or genomic sequences known to map to chromosome6p. HLA class III genes code for components of the com-plement system, C2, C4, and factor B. Using serologicanalysis, cytogenetic analysis, and Southern blotting, wehave ordered eight independent deletion breakpoints into asequence that divides chromosome 6p into six regions intowhich the probes have been mapped. This approach permitsan evaluation of the complexity and relative locations of themultiple sequences recognized by most 6p gene probes.

MATERIALS AND METHODSSouthern Blot Analysis. This analysis was performed as

previously described (6) with minor modifications. DNAconcentrations were measured by a fluorometric assay (7).Each blot was repeated two to five times and the blots shownhere are representative. Densitometry was performed with anLKB Ultrascan densitometer. All band intensities reportedwere measured as the peak height normalized either to a bandon the same blot that was present only on the undeletedhaplotype and so was equally represented in both T5-1 andthe deletion mutant or to the same blot rehybridized with aprobe for invariant chain (not shown), which is geneticallyunlinked to HLA (8).

Cell Lines and Deletion Mutants. Deletion mutants werederived from the cell lines T5-1 and MIT-2, clonal derivativesof the B-lymphoid cell line PGLC33H, which has the HLAhaplotypes DQl, DR], B27, CWJ, andA2 and DQ2, DR3, B8,and Al (9). Deletion mutants have been diagnosed by HLAantigen typing, karyotyping, and glyoxalase I enzyme assay(9). Table 1 summarizes these data.

RESULTSRationale. The general strategy for the use of deletion

mutants in gene mapping depends on determining whether agene or DNA sequence is located within a deletion or isunaffected and therefore outside the deletion. This can bedone either by assaying for the presence or absence of thegene product or by Southern blotting on a set of overlappingdeletions. In using cloned gene probes, if the parental cellsare heterozygous for a restriction fragment length polymor-phism (RFLP), then the fragment in the Southern blot ofparental cells containing the relevant gene will be absent inthe blot from a heterozygous deletion mutant. On the otherhand, if the cell is not heterozygous for a RFLP, then the bandon the Southern blot from the deletion mutant will be reducedin intensity by approximately 50% (6).

Abbreviations: RFLP, restriction fragment length polymorphism;kb, kilobase(s).Present address: Norris Cancer Research Center, University ofSouthern California Medical Center, Los Angeles, CA 90033.

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Table 1. Gene marker and karyotypic data on deletion mutantsderived from T5-1

Deletion Deleted Visiblemutant haplotype Gene markers deletion

GLOJ DQJ DR] B27 A2

8.1.6 A + - - + + No

GLOI DQ2 DR3 B8 Al

9.28.6 B + ? - - - No6.3.6 B + - - - - 6p212-ter3.1.0 B - ND - - - 6p212-*terThe HLA haplotypes shown are those of T5-1 and were derived by

serologic analysis of the family of the donor of the cell line. Thedeletion mutants were isolated after mutagenesis with the followingagents: 8.1.6, ethyl methanesulfonate; 9.28.6, mitomycin C; 6.3.6,ICR-191; 3.1.0, y-irradiation. These data have been previouslydescribed (9), with the exception of the glyoxalase activity (encodedby GLOJ) of 8.1.6, which was measured as previously described (9)and had a value of 0.93 + 0.06 relative to T5-1. For each mutant, theundeleted haplotype is unaltered and expresses all of the markers. +,Present; -, absent; ND, not done.

Southern Blot Analysis. HLA-DRa. The DRa cDNA probe(10) hybridizes on Southern blots with a single gene that mapsto HLA (6) (Fig. 1A). T5-1 is heterozygous for a Bgl II RFLPin the DRa gene. The 3.8- and 0.4-kb DRa Bgl II fragmentsare missing in 8.1.6 and thus are in haplotype A, while the4.2-kb band is missing in 3.1.0, 6.3.6, and 9.28.6 and thus isin haplotype B. Therefore, the lesions in all four deletionvariants encompass the DRa gene.HLA-DRp8. Southern blots of T5-1 DNA digested with

HindIll and probed with a DR/3 cDNA (3) have 12 bands (Fig.1B). The 10.2-, 6.6-, 6.0-, 3.0-, 2.6-, 2.5-, 1.6-, and 1.2-kbbands are absent from 3.1.0, 6.3.6, and 9.28.6 and cantherefore be assigned to haplotype B. The 6.9-, 3.6-, and3.2-kb bands are absent from 8.1.6 and can therefore beassigned to haplotype A. The 8.0-kb band is present in all ofthe mutants but is too faint to permit a determination ofdosage. Three features from these results are noteworthy.One is the large number ofDRB-like sequences that hybridizewith the DRP probe. Another is the high degree of restrictionenzyme polymorphism in and around the DRJ3 sequences inT5-1. The third is the disparity in the number of bands fromthe two haplotypes. Haplotype B has 8 haplotype-specificHindIII restriction fragments, while haplotype A has only 3.A similar disparity is evident when Msp I is used in place ofHindIII, with 9 Msp I fragments from haplotype B and 5 fromhaplotypeA (Fig. 1C). Either there are large differences in thenumbers of HindIII and Msp I sites between the twohaplotypes or there must be a larger number of DRP se-quences in haplotype B than in haplotype A.p25#4. p25#4 is a genomic 6.2-kb BamHI fragment that

contains the leader sequence of one of the DR,8 genes plusapproximately 4 kb of 5' flanking sequence and 2 kb of theintron between the leader sequence and the exon coding forfirst structural domain (unpublished results). As expectedfrom the results with the DRJ3 cDNA clone, most sequenceshybridizing with this probe are deleted in all of the mutants(Fig. 1D). However, the 4.6-kb band, which is reduced inintensity in 3.1.0, 6.3.6, and 9.28.6, is not detectably reducedin intensity in 8.1.6. There are several possible explanationsfor this result. As with the DR/3 genes, the number of p25#4sequences may differ in the two haplotypes, with the 4.6-kbfragment overrepresented in haplotype B. This might makethe reduction in band intensity in 8.1.6 undetectable. Alter-natively, the 4.6-kb fragment could be deleted in 3.1.0, 6.3.6,and 9.28.6, but not in 8.1.6. Thus, 8.1.6, although it appearsto be deleted for all of the DRJ3 sequences in haplotype A

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FIG. 1. Southern blot analysis of deletion mutants with HLADRa, DRJ3, and p25#4 gene probes. To eliminate hybridization witha repetitive sequence, hybridizations with the p25#4 probe containedhuman placental DNA at 250 Ag/ml. The normalized intensity of the4.6-kilobase (kb) band in D relative to T5-1 is 0.49 for 3.1.0, 0.39 for6.3.6, 0.94 for 8.1.6, and 0.41 for 9.28.6. For 3.1.0, 6.3.6, and 9.28.6,normalization was to the 5.4-kb band; for 8.1.6, to the 5.0-kb band.

when probed with a DR/ cDNA clone, may not be deletedfor all of the sequences hybridizing with p25#4.HLA-DQa. The DQa cDNA clone hybridizes with two

DQa-like genes (11). In HindIII digests of T5-1 DNA (Fig.2A), these are represented by 7.9-, 6.0-, and 2.7-kb bands.The 7.9- and 6.0-kb bands each represent one of the twoalleles of the DQa gene, while the 2.7-kb band representsanother DQa-like sequence designated DXa (11). Either the7.9- or the 6.0-kb band is absent in each of the mutants (Fig.2A), and thus DQa is deleted in all of them. The 2.7-kb DXaband has a decrease in intensity in all of the mutants relativeto T5-1, thus they all have heterozygous deletions of thissequence.HLA-DQ/3. Southern blots of T5-1 DNA digested with

HindIII and probed with a DQB cDNA clone (3) have eightmajor bands (Fig. 2B). The 12.9-kb band is absent in 3.1.0,6.3.6, and 9.28.6 and is therefore from haplotype B, while the6.9-kb band is absent in 8.1.6 and thus from haplotype A. The9.4-, 7.6-, 5.7-, 2.7-, and 1.1-kb HindIII bands are present inall of the mutants but are too weak to allow gene dosageestimates. The intensity ofthe 3.2-kb band is reduced in 8.1.6to approximately 20% of the intensity in T5-1 as determinedby densitometry. This reduction is considerably greater thanthe expected 50% reduction. On the other hand, the intensityof this band in 3.1.0, 6.3.6, and 9.28.6 is about 80% that ofT5-1. An Msp I digest reveals the same disparity, with a3.3-kb band being severely reduced in intensity in 8.1.6 (Fig.2C). One possible explanation for this is that the number ofDQl3 sequences may be equal in the two haplotypes but that

3742 Genetics: Levine et al.

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A OQDa B DQf3n iD i

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FIG. 2. Southern blot analysis of deletion mutants with HLADQa, DQ,8, DPa, and DP,8 gene probes. (A) The normalizedintensity ofthe 2.7-kbDXa band relative to T5-1 is 0.55 for 3.1.0, 0.42for 6.3.6, 0.31 for 8.1.6, and 0.48 for 9.28.6. Normalization is to the6.0- or 7.9-kb DPa bands. (B and C) The densitometric analysis of the3.2-kb HindIII and 3.3-kb Msp I bands is presented in the text. (D)The normalized intensities of the 4.2- and 3.0-kb DPa bands relativeto T5-1 are, respectively, 0.49 and 0.56 for 3.1.0, 0.47 and 0.56 for6.3.6, 2.48 and 1.18 for 8.1.6, and 0.52 and 0.50 for 9.28.6. Hybridiza-tion with the invariant chain probe was used to normalize thesevalues. The increased intensity of the 4.2-kb DPa band in 8.1.6 overT5-1 was not seen in other blots. (E) The normalized intensities ofthe5.6- and 8.3-kb bands relative to T5-1 are, respectively, 0.45 and 0.33for 3.1.0, 0.44 and 0.29 for 6.3.6, 0.90 and 0.81 for 8.1.6, and 0.43 and0.39 for 9.28.6 with normalization to invariant chain.

the 3.2-kb HindIII fragment is present in more copies inhaplotype A than B because of a greater conservation ofrestriction sites in haplotype A. This would predict an excessof restriction fragments from haplotype B, which is not thecase. Therefore, it seems more likely that the numbers ofDQ/3 sequences in the two haplotypes differ, with more DQ/3genes in haplotype A than B.HLA-DPJ3. Cell surface expression of DP and its poly-

morphisms has been mapped between DR and GLOJ (12,13).Human class II 0-chain cDNAs corresponding to DP havealso been mapped to this region (14, 15). Southern blots ofT5-1 DNA cut with six restriction enzymes have not revealedany DP/3 RFLPs that are heterozygous in T5-1. Band intensi-ties were therefore used to determine whether the genesrecognized by this probe are affected in the deletion mutants.The 8.3- and 5.6-kb bands in 8.1.6 are of equal intensity tothose in T5-1, whereas those bands in 3.1.0, 6.3.6, and 9.28.6are reduced in intensity (Fig. 2E), indicating that the DPBsequences are deleted in these mutants. This places thebreakpoints in 6.3.6 and 9.28.6, in which GLOI has not beendeleted, between DPB and GLOJ. The 14.5-kb band is toolight to permit conclusions about gene dosage.HLA-DPa. This cDNA probe hybridizes to three HindIII

fragments, all of which are reduced in intensity and thereforedeleted in 3.1.0, 6.3.6, and 9.28.6 but are of normal intensityand so retained in 8.1.6 (Fig. 2D). Therefore, DPa maps to thesame interval as DPB, between DR/DQ and GLOJ. Ourfinding that DPa and -,B sequences map to the same intervalis consistent with the findings ofDPa and -,B sequences on thesame genomic clone (5).

C4. C4, the fourth component of the human complementsystem, has been mapped by family studies to the regionbetween HLA-DR and the class I loci (16). Recently, genomicclones for all four class III major histocompatibility complexgenes, C4A, C4B, C2, and FB (the gene encoding factor B),have been found to be located within 30 kb of one another(17). Neither of the two bands detected by the C4 cDNAprobe (18) (Fig. 3D) is reduced in intensity in 8.1.6, but theyare reduced and therefore deleted in 3.1.0, 6.3.6, and 9.28.6.Thus, 8.1.6, which retains all of the class I markers but haslost virtually allDR andDQa and -f3 genes, must have a distalbreakpoint between the class II and class III gene families.HLA-1.3. HLA-1.3 is an HLA-B7 class I gene clone (19).

As is typical of class I probes, it cross-hybridizes with a largenumber of class I sequences. Southern blots of a HindIIIdigest of T5-1 hybridized with this probe (Fig. 3A) show aband at 1.4 kb, a cluster of bands at about 5.8 kb, andprominent bands at 20.2 and 25.1 kb. The 5.8-kb band ispresent in T5-1 and 8.1.6 but not in 3.1.0, 9.28.6, or 6.3.6,which has previously been shown to be deleted for all classI sequences in haplotype B (6). Since the 3.1.0 and 9.28.6lanes appear to be the same as that of 6.3.6, they must alsobe deleted for all class I sequences detected by the class Iprobe. Mutant 8.1.6, on the other hand, has no detectabledeletion of class I sequences.pCH6. pCH6 (20) is a genomic clone containing a class I

sequence that is 0.7 + 0.7 map unit distal to HLA-DQa (21).T5-1 is heterozygous for a Taq I RFLP in the pCH6 gene. Fig.3B shows that 8.1.6, with a band pattern identical to T5-1, isnot deleted for pCH6; 3.1.0, 6.3.6, and 9.28.6, which aremissing the 5.5-kb band, are deleted for this gene. Fig. 3C

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FIG. 3. Southern blot analysis of deletion mutants with HLAclass I and class III gene probes. The intensities of the 3.1- and 2.1-kbC4 bands inD relative to T5-1 are respectively 0.38 and 0.50 for 3.1.0,0.45 and 0.37 for 6.3.6, 0.78 and 0.89 for 8.1.6, and 0.58 and 0.32 for9.28.6, with normalization to invariant chain.

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A HPGK B Insulin-like0P D toa

ur _. rn -- cU _; N0a)0 d0a

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FIG. 4. Southern blot analysis of deletion mutants with non-HLA6p geneprobes. The intensities of the 2.6-, 4.8-, 5.5-, 7.9-, and 8.2-kbHPGK bands in A relative to T5-1 are respectively 1.04, 1.11, 0.91,1.31, and 1.07 for 3.1.0 with normalization to invariant chain.

shows the same result with a restriction enzyme that does notrecognize an RFLP that is heterozygous in T5-1.HPGK. HPGK is a cDNA clone derived from the autoso-

mal phosphoglycerate kinase (PGK) gene that is expressedonly in testis (22). The HPGK probe hybridizes to sevenbands on a Southern blot ofHindIII-digested T5-1 DNA (Fig.4A), which is expected because PGK cDNA clones cross-hybridize with both the X-linked and the autosomal PGKgenes (23). The autosomal gene has been mapped by usingsomatic cell hybrids (23) and in situ hybridization (24) to6ql2-6p23 and 6pl26p21, respectively. However, 3.1.0, themutant with the most proximally extending deletion, is notreduced in intensity for any of the fragments hybridizing tothis probe. The fact that the autosomal PGK gene is notdeleted in 3.1.0 maps it proximal to GLOI and pAGB6 (seebelow), both of which are deleted in 3.1.0, and places itbetween 6p12 and 6p211.pAGB6 (insulin-like). pAGB6 is a cDNA clone derived

from an insulin gene-like gene (25) that has been mapped byfamily studies 8.0 ± 5.6 map units proximal to GLOJ (22).T5-1 is heterozygous for an Msp I RFLP, with the two allelesbeing represented by 3.3- and 2.8-kb bands (Fig. 4B). The3.3-kb band is absent in 3.1.0; the other mutants retain bothbands. Therefore the lesion in 3.1.0, which is the only mutantdeleted for GLO), extends through pAGB6 but does notinclude HPGK or p7H4, which thus must be more proximal.

DISCUSSIONWe have used the Southern blotting technique to analyze aset of somatic cell mutants with deletions on the short arm ofchromosome 6. The mutants possess deletions on only onehomologue and consequently retain an intact chromosome 6in addition to any sequences remaining on the affectedchromosome. Fig. 5 summarizes the derived map locations ofall of the probes used in this study relative to the deletionbreakpoints of the various mutants. The four deletion mu-

tants described here have breakpoints that divide the shortarm of chromosome 6 into six regions. We have mappedgenes or DNA sequences into five of those regions. Region1, the most proximal to the centromere, contains HPGK.Regioti 2 contains an insulin gene-like gene and GLO).Region 3, which extends into HLA, contains the DPa andDP.8 genes. Region 4 contains all of the a and genes for DRand DQ; one of the breakpoints in region 4 may lie within one

HPGK Insulin OPC EDjJlike GLO DR- E FB C2 B C A pCH6

CIAr-2 3 t_8 4 58.1.6

16.3.63.1.0

FIG. 5. Genetic map of HLA and chromosome 6p based ondeletion mutants. This map is based primarily on data reported in thispaper but also incorporates results from other studies as cited in thetext. The boxes denote probes that recognize multiple genes. I, II,and III refer to the three classes of genes located within HLA. Thearabic numbers between the deletion breakpoints denote the sixregions described in the text. The proximal breakpoints in 9.28.6 and6.3.6 are shown as different because the deletions occurred inde-pendently of each other, but they have not as yet been distinguishedby markers or gene probes. The location of pCH6 has not beendetermined relative to HLA-A, -B, or -C. The orientation of the classIII genes with respect to the centromere is not known. The uppergenetic map is drawn to scale; the lower (deletion) map indicatesorder but not scale. -o-, centromere.

of the DR/3 genes. We do not yet know the orientation ofregion 4 with respect to the centromere. Region 5 contains allof the HLA class I and class III genes. Region 6 contains thepart of6p distal to HLA; none of the probes tested map to thisregion.We, like other investigators, find that the complexity of the

class II genes, in particular the ,( genes, is greater than wouldbe suspected from serological studies, which to date havedefined only three distinct cell surface class II antigens (1).The HLA-DRf3, -DQf3, -DPa, and -DPf cDNA probes allhybridize to a larger number of bands on Southern blots thanwould be expected for single genes. Seven distinct P-chainprotein sequences have been reported in a single possiblyhomozygous cell line (26); results from cDNA cloning alsosupport the idea that more genes are expressed than thus faraccounted for by serological studies (27). Overall, the humanclass II region probably possesses at least seven P genes andsix a genes. It is interesting that for all cases described here,including DR/3, DQa, DQP, DPa, DPA, and class I genes, allmembers of a single multigene family are found in a singleregion. Our results also raise the possibility that there may bedifferent numbers of DRP and DQf3 sequences on the twoHLA haplotypes. None of the major bands hybridizing withthe HLA class I, II, or III probes lies outside of the region on6p encompassed by the deletion in 9.28.6, which has a short,karyotypically undetectable deletion. This places an upperlimit on the size of HLA of about 10% of 6p. However, wecannot exclude the possibility that some of the weak, seem-ingly nonpolymorphic, bands seen with some of the probesmap outside of the deletions. The question of which of themultiple ,-chain sequences in T5-1 are expressed is animportant one. Cloning of class II genes from wild-type andsingle gene mutants will permit correlations between alteredanti-class II antibody binding and altered class II genesequences. In this way, it should be possible to betterunderstand the organization and expression of the class IIgene family.By using the mutants described here, any 6p probe can be

mapped into one of the six regions of6p defined ih this study.This approach can provide both a genetic location relative toother genes or probes as well as a physical location relativeto cytogenetically visible deletions. This provides an alterna-tive to in situ hybridization, which provides only a physicallocation, or "chromosome walking," which provides a ge-

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netic map but only over relatively short distances. As such,the deletion mutants described here provide a powerful toolfor gene mapping.

We thank Drs. Jack Stominger, Sherman Weissman, AllanNickelson, Stuart Orkin, Derek Woods, Harvey Colten, and LydiaVilla-Komaroff for providing gene clones. This work was supportedby National Institutes of Health Grants A17705, A116689, andGM07266.

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