Immunogenetics 30: 350-360, 1989 / / / 7 / / / / / / / 0 - geneO'cs

�9 Springer-Verlag 1989

Deletion, insertion, and restriction site polymorphism of the T-cell receptor gamma variable locus in French, Lebanese, Tunisian, a n d Black African populations

Nada Ghanem 1'*, Catherine Buresi 1'*, Jean-Paul Moisan z, Myi~ne Bensmana l, Paul Chuchana l, Sylvie HuckJ, G6rard Lefranc 1, and Marie-Paule Lefranc I

i Laboratoire d'Immunogrnrtique Molrculaire, UA CNRS 1191, Universit6 des Sciences et Techniques du Languedoc, Place E. Bataillon, F-34060 Montpellier Cedex 1, France

2 Laboratoire d'Immunologie Clinique, U 211 INSERM, Facult6 de Mrdecine, 1 rue G. Veil, F-44035 Nantes Cedex, France

Abstract. The human T-cell receptor gamma region spans 160 kb of genomic DNA and is densely populated by coding sequences. Restriction fragment length polymor- phisms have been previously documented for the constant region genes, the joining segments, and the variable genes belonging to subgroups I and IV. Here we further define the polymorphism of the V gamma I subgroup genes, based on complete mapping of the Eco RI and Taq I allelic restriction fragments. We describe seven haplotypes; five result from polymorphic restriction sites, the sixth cor- responds to a deletion of about 10 kb encompassing V4 and V5, and the seventh results from an insertion of an additional gene, V3P, between V3 and V4. As a conse- quence of the deletion or insertion polymorphism, the number of V gamma I subgroup genes vary from seven in haplotype TRGVI*3 to ten in haplotype TRGVI*4, whereas the most common haplotype, TRGVI*I, has nine V genes, five of them being functional. Frequencies of the different TRGVI haplotypes in French, Lebanese, Tunisian, and Black African populations are given.

Introduction

In human peripheral blood, about 3-5 % of the circulating T lymphocytes express the T-cell gamma/delta receptor. The human T-cell receptor gamma region has been characterized extensively (for review, see Lefranc 1988, Lefranc and Rabbits 1989). It comprises two constant region genes (TRGC) separated by a distance of 16 kilobases (Lefranc and Rabbitts 1985, Lefranc et al. 1986a, b) and at least 14 variable gamma genes (TRGV)

*Permanent address: Universit6 Libanaise, Facult6 des Sciences M&ticales, Beyrouth, Lebanon. Permanent address: Facult6 de M6decine de Tunis, Drpartement de G6nrtique, Tunis, Tunisia.

Address correspondence and offprint requests to: M.-P. Lefranc.

belonging to four subgroups and located upstream of the two C gamma genes (Lefranc et al. 1987; Fig. 1A). Nine Vgamma genes, five of them functional (V2, V3, V4, V5, and V8), and four pseudogenes (V1, V5P, V6, and V7) belong to subgroup I, whereas subgroups II, III, and IV each consist of a single gene, designated V9, VlO, and Vll , respectively (Lefranc et al. 1986c, Forster et al. 1987, Huck et al. 1988; Fig. 1A). Two pseudogenes, VA and VB, located upstream of V9 and Vll , respectively, belong to none of these subgroups (Forster et al. 1987, Huck et al. 1988). Five gamma joining segments (TRGJ) have been identified: J1 upstream of TRGC1; J2 upstream of TRGC2 (Lefranc et al. 1986a); JP in the region 5' of J1 (Lefranc et al. 1986c); JP1 and JP2 upstream of JP and J2, respectively (Huck and Lefranc 1987, Querter- mous et al. 1987, Tighe et al. 1987; Fig. 1A).

Using a unique probe pH60, all the T-cell receptor gamma gene rearrangements in normal T cells, T-cell leukemias, and lymphomas can be related to known Vand J segments (Forster et al. 1987, Huck and Lefranc 1987, Lefranc 1988), indicating that most, if not all, gene segments of the human T-cell receptor gamma locus have been identified. Moreover, all the Vgamma genes are con- tamined in a unique 120 kb Xho I fragment detected by pulsed field gel electrophoresis (PFGE) which links the variable and constant regions (Lefranc et al. 1989; Fig. 1A). These variable and constant regions are remarkably close to each other since the distance between Vll, the most 3' variable gene, and JP1, the most 5' joining seg- ment, is only 16 kb (Lefranc et al. 1989). With its 14 V gamma genes, spanning 100 kb, the two C gamma geneS, and five joining segments covering less than 40 kb and only 16 kb separating the most 3' V gene from the most 5' J segment, the human T-cell receptor gamma locus spans 160 kb of genomic DNA and represents a particular- ly densely populated region when compared with the other rearranging loci (Lefranc et al. 1989).

N. Ghanem et al.: T-cell receptor gamma variable gene polymorphism 351

Restriction fragment length polymorphisms (RFLP) of the T-cell receptor gamma genes have previously been described using either the J1 probe pH60 or constant region probes (for review, see Buresi et al. 1989). It is noteworthy that the TRGC2 gene displays a polymorphism which results from the presence of either four or five distinct exons (Lefranc et al. 1986b, Littman et al. 1987, Buresi et al. 1989). RFLPs of the Vll gene (Ghanem et al. 1989a, b), as well as several polymorphisms of the V gamma I subgroup genes, including a deletion of the V4 and V5 genes (Font et al. 1988), have also been documented. In this paper, we further define the polymor- phism of the V gamma I subgroup genes, based on com- plete mapping of the Eco RI and Taq I allelic restriction fragments.

Materials and methods

Samples and DNA analysis. Blood samples were randomly collected from unrelated healthy French (35), Lebanese (40), Tunisian (37), and Black African (15) individuals. DNA extraction and analysis were per- formed as previously described (Ghanem et al. 1988a). Segregation of the different haplotypes was studied in informative families.

Probe. The V gamma I probe, clone pV3S (Lefranc et al. 1986c), is a 1.2 kb Sac I fragment containing TRGV3 from XSH4, cloned in pUC. That probe cross-hybridizes to the nine V gamma genes belonging to subgroup I (Forster et al. 1987; Fig. 1B).

Construction and screening of a cosmid library. A cosmid library was made from the DNA o1~ an alloreactive T-cell clone isolated from a re- jected kidney graft (Moisan et al. 1989). This T-cell clone has rearranged the gamma locus on both chromosomes (V2J2 and V8J2, respectively). This cosmid library made in vector Sp cos 2 (Heilig et al. 1987) was screened with the pV3S probe (Lefranc et al. 1986c). One clone, cosB6, was obtained which encompassed the region from V2 to V7 and cor- responds to the chromosome with a VRI2 rearrangement. The restriction map of the variable gamma I region has previously been published (Lefranc et al. 1986c) except for a gap between V4 and V5P. The cosmid clone, cosB6, allowed the linkage of the V4, VS, and V5P genes and mapping of that region. A similar linkage was obtained from phage clone analysis (Font et al. 1988). Note that the cosB6 clone has inserted about 10 kb of foreign DNA, probably during the steps of purification: this insertion was unambiguously demonstrated as not belonging to the T-cell receptor gamma locus. Except for that insertion, the map is in complete agreement with the previous results (Lefranc et al. 1986c) and the ex- pected map deduced from the germline and rearranged V4 and V5 fragments observed in Southern blot analysis (Forster et al. 1987). Nomenclature. "TRGVI" is used for T-cell receptor V gamma I subgroup genes. TRGVI*A and TRGVI*B corresponds to the Eco RI and Taq I restriction fragments, respectively. These fragments allowed us to define seven haplotypes designated TRGVI*I-TRGVI*7, as discussed in the text.

Results and Discussion

Complete restriction mapping of the V gamma I subgroup. A series of overlapping phage clones spanning 130 kb of genomic DNA has previously been isolated in the human

A

Human TRG locus

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~ l I . L I t l I~r , , l i Ill I~ I I ~ l l f r , i ; , i ~,.~l , , , , , , . .,, V3 V5 V5P V6 V7 ',/8 VA

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ECO R I L ~ 58 I 18 I 7.0 I 2.4 T 31 t 3.7 - ~ I 9.3 I 3.6 I 3.8 I

T a q I ~ "~.0 i 6.0 i 42 i B.5 J L 3.7 _ _ J

Fig. 1. A Organization of the human T-cell receptor gamma region (Lefranc et al. 1989). B Restriction map of the TRGVI region. (Lefranc et al. 1986c and this report). Functional genes are shown as full rectangles and pseudogenes as open rectangles. Eco RI and Taq I restriction fragments hybridizing to the F j probe, pV3S (Lefranc et al. 1986c), are shown below the map. Asterisks indicate polymorphic sites discussed in the text. B, Barn HI; H, Hind III; K, Kpn I; S, Sac I; R, Eco RI; X, Xho I; T, Taq I.

352 N. Ghanem et al,: T-cell receptor gamma variable gene polymorphism

Fig. 2. A Southern filter hybridization of Taq I-digested T-ce!l clone DNAs with the pV3S probe. T-cell receptor 3' rear- rangements of these alloreactive T-cell clones have previously been characterized (Moisan et al. 1989). B Southern filter hybridization of Eco RI- (top panel) and Taq I- (lower panel) digested DNA from different individuals.

N. Ghanem et al.: T-cell receptor gamma variable gene polymorphism 353

T-cell receptor gamma locus. These clones encompass, on the one hand, the 14 known V gamma genes (Lefranc et al. 1986, Forster et al. 1987, Huck et al. 1988) (with two gaps, one between V4 and V5P and the other between V9 and VIO) and, on the other hand, all of the constant region genes and associated joining segments (Lefranc et al. 1986b, Huck and Lefranc 1987, Lefranc et al. 1989). In this report we completed the map of the V gamma I subgroup by linking the V4, V5, and V5P genes (Fig. 1B). Moreover , we localized the Taq I sites of the V gamma I region by fine restriction mapping of subclones derived from XSH5, XSH7 (Lefranc et al. 1986c), and cosB6. A Taq I site is located in the V8 gene, as shown by the nucleotide sequence (Lefranc et al. 1986c). The Taq I restriction mapping is in agreement with the gene deletion mapping obtained from a series of clones whose 3' rear- rangements are well characterized (Moisan et al. 1989; Fig. 2A). This analysis also pointed to a polymorphic Taq

I site located 1.4 kb downstream of J1. Assignment of the Taq I germline bands are shown in Figure 1B, com- pleting the Hind III, Eco RI (Lefranc et al. 1986c, Fors ter et al. 1987), and Kpn I (Moisan et al. 1989) data. Mapping of the Taq I (this report) and Eco RI sites (Forster et al. 1987) allowed us to correlate the Eco RI and Taq I restric- tion fragments detected by the V3`Iprobe pV3S (described in Materials and methods; Fig. 1B).

When DNAs from 35 French, 40 Lebanese, 37 Tuni- sians, and 15 Black Africans were digested with either Taq I or Eco RI and hybridized to the V3`I probe pV3S, no Eco RI and Taq I polymorphism was observed in the V5P-V8 region. In contrast, the V1-V5 region was characterized by a high degree of polymorphism, as revealed by both Eco RI and Taq I restriction fragments (Tables 1 and 2; Fig. 2B). Seven haplotypes were defined; five result from restriction site polymorphism, the sixth corresponds to a deletion of the V4 and V5 genes, and the

Table 1A. The Eco RI TRGVI restriction fragments (from Forster et al. 1987).

TRGVI genes Restriction fragments (in kb)

V1 5.8 V2 1.8 V2 + V3 7.0* V4 2.4* V4 3.1 V5 3.7 V5P-V6 9.3 V7 3.6 I/8 3.8

* Polymorphic bands discussed in the text and Table lB.

Table 1 B. The Eco RI polymorphic (TRGVI*A) restriction fragments and their frequencies in French (F), Lebanese (L), Tunisian (T), and Black African 03) populations.

TRGVI genes Restriction fragments (in kb) Polyrnorphism Frequencies

V2-V5 V2-V3 5"V4 V4 V5 TRGVI*A1 (7.0+ 2.4+ 3.1 +3.7) RS 0.693 (F)

TRGVI*A2

TRGVI*A3

0.609 (L) 0.608 (T) 0.500 03)

( 9.4 + 3.1+3.7) RS 0.096 (F) 0.187 (L) 0.190 (T) 0.234 (B)

(7.0 d d d ) D 0.211 (F) 0.203 (L) 0.162 (T) 0.133 03)

I 0.040 (T) 0.133 03)

TRGVI*A4 (7.0+6.4 + 3.1+3.7)

d, deleted. RS, polymorphic restriction sites; D, deletion; I, insertion. The polymorphic TRGVI*A (Eco RI) restriction fragments are underlined. For a complete definition of the haplotypes, the 3.1 kb V4 and 3.7 kb V5 fragments have to be considered since these fragments are absent in the haplotype characterized by the deletion of the V4 and V5 genes.

354 N. Ghanem et al.: T-cell receptor gamma variable gene polymorphis m

seventh corresponds to an insertion of 6 kb with an addi- tional Vgene between V3 and V4. "Deletion" and "inser- tion" are indicated by reference to the most frequent haplotype which includes nine Vgamma I subgroup genes (Forster et al. 1987), but are not meant to convey informa- tion on the chronological order of the events during evolu- tion of that region.

Haplotypes TRGVI*I and *2 defined by polymorphic Eco RI and Taq I sites. As previously described, the V2 and V3 genes are usually located on a 7 kb Eco RI fragment, whereas the 5' V4 region, downstream of V3, corresponds to a 2.4 kb Eco RI fragment (Table 1A; Lefranc et al. 1986c, Forster et al. 1987), these two fragments being defined as TRGVI*A1 (Figs. 3 and 4; Table 1B). The absence of the polymorphic Eco RI site at 2.4 kb in 5'

of V4 results in a 9.4 kb fragment (TRGVI*A2; Figs. 3 and 4, Table 1B) which can be distinguished from the 9.3 kb germline V5P-V6 band on long run digests (Fig. 3). In all samples we studied, there was a correlation bet- ween the presence of this Eco RI site and the presence of a Taq I site located between V3 and V4 (Figs. 1B and 4). Consequently, the adjacent 7 .0+2.4 kb Eco RI fragments (TRGVI*A1; Table 1B) are correlated with the contiguous 16.0+6.0 kb Taq I fragments (TRGVI*B1 Figs. 3 and 4, Table 2); conversely, the only 9.4 kb Ec0 RI fragment (TRGVI*A2) is associated with the 22.0 kb Taq I fragment (TRGVI*B2), due to the simultaneoug absence of the polymorphic Eco RI and Taq I sites (Figs i 3 and 4). These results allow us to define two haplotypes i TRGVI*I and TRGVI*2, present in the four populationS studied (Table 3, Fig. 3).

Table 2 A. The Taq I TRGVI restriction fragments.

TRGV1 genes Restriction fragments (in kb)

V1-V3 16" V2 6* I/5 4.2 V5P-V6 8.5 V7 3,7 V8 0.8+1.0

* Polymorphic bands discussed in the text and Table 2B.

Table 2B. The Taq I TRGVI polymorphic (TRGVI*B) restriction fragments and their frequencies in French (F), Lebanese (L), Tunisian (T), and Black African (B) populations.

TRGVI genes Restriction fragments (in kb) Polymorphism Frequencies

V1-V3 V4 V5 V1-V5 TRGVI*B1 (16 + 6 + 4.2) RS

TRGVI*B2 ( 22 + 4.2) RS

TRGVt*BS (17 d d ) D

TRGVI*B4 (17 +11 + 4.2) I

TRGVI*B5 (15 + _7 + 4.2) RS

TRGVI*B6 (12.8+4.2+ 4.2) RS

0.693 (F) 0.594 (L) 0.594 (T) 0.500 (B)

0.096 (F) 0.187 (L) 0.162 (T) 0.167 (B)

0.211 (F) 0.203 (L) 0.162 (T) 0.133 (B)

0.040 (T) 0.133 (B)

0.014 (T) 0.067 (B)

0.015 (L) 0.028 (T)

d, deleted. RS, polymorphic restriction sites; D, deletion; I, insertion. The polymorphic TRGVI*B (Taq I) restriction fragments are underlined. For a complete definition of the haplotypes, the 4.2 kb 115 fragment has to be considered since this fragment is absent in the haplotype characterized by the deletion of the V4 and V5 genes.

N. Ghanem et al.: T-cell receptor gamma variable gene polymorphism 355

Fig. 3. Southern DNA analysis from individuals homozygous or heterozygous for the haplotypes TRGVI* to *4. The Eco RI and Taq I fragments were hybridized to the Vr/probe, pV3S (Lefranc et al. 1986c). Assignment of the Eco RI restriction fragments to given V is according to Forster and co-workers 1987. Assignment of the Taq I fragments is from this report (See also Tables 1 and 2). The haplotypes are defined by the correlation of the polymorphic Eco RI (TRGVI*A) (top) and Taq I (TRGVI*B) (bottom) fragments. Between brackets are the 114 and 1,15 fragments necessary for a complete definition of the haplotypes (discussed in legend of Tables 1B and 2B). The 5' V2 and 5' 114 Eco RI fragments are due to the existence of an Eco RI site in the leader intron of these genes (Lefranc et al. 1986c). The two V8 Taq I fragments result from the presence of a Taq I site in the II8 gene sequence (Lefranc et al. 1986c). These 0.8 and 1 kb V8 fragments are not visible on long run digests (*B2/B2, *B3/B3, and *B1/B4 lanes).

The Mende l i an inher i tance of the Eco RI and Taq I restr ict ion fragments has been demonstra ted in infor- mat ive families (Figs. 5 and 6). As seen in F igure 5, fami- ly W is characterized by the TRGVI*A1 and *A2 restric- t ion fragments. The father and two chi ldren are homozygous for *A2, whereas the mother and a daughter are heterozygous *All*A2, as shown by the reduced inten- sity of the 2.4 kb band and the presence of the two allelic 9 .4 and 7 .0 kb Eco RI fragments. In family S (Fig. 6), both parents are heterozygous *B1/*B2, whereas two

chi ldren are homozygous *B1/*B1 and two others are homozygous *B2/*B2.

Haplotype TRGVI*3: deletion o f 114 and 115. The 5" 114(2.4 kb), 3 ' II4(3.1 kb), and 115(3.7 kb) Eco RI fragments , on the one hand, and the 1/"4(6.0 kb) and 115 (4.2 kb) Taq I bands , on the other hand, were absent in the pV3S hybr id iza t ion pattern obtained in five in- dividuals (Figs. 3 and 4; Fig. 5, father in family A). These results suggest a delet ion of the II4 and I15 genes on both

356

A Eco R I

*tM I "131 I

Taq I

*A2 I * B 2 I

*A3 I "133 I

*A4 I

B

TRGVI*I *2

TRGVI*3

TRGVI*4

VI

5.8 R

5.8 N

5.8 FI

5.8 13

Vl V2

m

Vl V 2

FI m

V2 V3 V4 V5

t8 7.0 2.4 3.1 3.7 k �9 ) km I �9

~ o i 6 . 0 I 4.2

N. Ghanem et al.: T-cell receptor gamma variable gene polymorphism

V5P V6

n fq

1.e 9 .4 3.1 3.7 [ I= �9 k �9 I n n

22 .0 I 4.2

ts 7.0 V3 V5P 9.3 V6 I k �9 I I n n

17.0 ~ 1 8.5 I

t8 7 .0 V 3 6.4 V3P V4 3.1 V53.7 I w �9 I I w , . i i

17.0 e 11.0 t I

V3 V4 V5 V5P V6P V7 V8

I I I ~ ~ I I

V1 V 2 V 3 V5P V6P V7 V 8

I I ~ ~ I I

V 3 V3P V4 V5 V5P

I ~ I I

V6P V7

I

Fig. 4A. Schematic representation Of N u m b e r the Eco RI (TRGVI*A) and Taq ! of V ~ (TRGVI*B) restriction fragments cor- g o n o s responding to the haplotypes TRGVI*I-

TRGVI*4. The first haplotypes res/lt from the presence (TRGVI*I) or the

O absence (TRGVI*2) of the Eco RI and Taq I polymorphic sites in 3' of I,'4. The TRGVI*3 haplotype corresponds to ;:a deletion of the I/4 and I/5 genes and the TRGVI*4 haplotype to an insertion 9f

7 �9 i 6 kb between 113 and I/4, corresponding to the additional V3P gene. B Schematic representation of the haplotyp~s

V8 TRGVI*I-TRGVI*4. Functional genes are shown as black boxes and

m 10 pseudogenes as white boxes�9

chromosomes, as previously postulated (Font et al. 1988). The deletion of the I14 and II5 genes encompasses about 10 kb, as shown by the shortened size (31 kb) of the Bam HI fragment hybridizing to the pV3S probe instead of the usual 41 kb fragment which contains the nine V gamma I subgroup genes (Forster et al. 1987); the two Bam HI sites which limit that fragment have been located 3 kb upstream of V1 and 3 kb downstream of I18, respectively (Lefranc et al. 1986c and unpublished data). It should be noted that the Taq I fragment containing the I,'1, II2, and V3 genes in this seven-gene haplotype designated as TRGVI*3 is 17 kb long; this is 1 kb longer than the usual 16 kb (TRGVI*B1) Taq I fragment (Fig. 3). Indeed, the 3' site of that 17 kb (TRGVI*B3) fragment corresponds to the Taq I site upstream of V5P as a consequence of the deletion (Fig. 4A). Therefore, in Taq I long run gels, the TRGVI*3 haplotype can be readily distinguished in heterozygous individuals (Fig. 6, family CHA), where decreased intensity of the bands may sometimes be dif- ficult to evaluate.

Haplotype TRGVI*4: insertion of 6 kb containing an addi- tional V gene, V3P. In four Black African and three unrelated Tunisian individuals, among them the individual LAT (Lefranc et al. 1979, van Loghem et al. 1982), a new 6.4 kb Eco RI (TRGVI*A4) band and 11 kb Taq I

(TRGVI*B4) band were observed (Fig. 3), Interestingly, the presence of the 6.4 kb Eco RI band is associated with the absence of the 5' 1,14 2.4 kb Eco RI fragment. Indeed, in family K (Fig. 5), the 2.4 kb band is absent in the father who is heterozygous *A3/*A4 and is present as a single copy in the mother and daughter with the genotype *A1/*A4. Since the 7.0 kb I12-II3 as well as the 3.1 kb 3' I/4 Eco RI fragments are present, this suggests that an insertion occurred between 1,I3 and I,'4. Judging from the restriction map, the size of that insertion must be 6 kb (Fig. 4A). Hybridization of double digests Kpn I-Eco RI and Kpn I-Taq I to the pV3S probe enables the localization o fa Kpn I site 6.7 kb downstream of I/3 and 4 kb upstream of I/4. This site is internal to an additional V gamma ger~e belonging to subgroup I and designated as V3P in Figure 4 (N. Ghanem and M.-P. Lefranc, unpublished data). The presence of an additional Vgamma gene in the TRGVlocus is not surprising since our data on the organization of the variable gamma I subgroup locus shows that the evolution of that locus proceeded by a series of duplications Or amplifications of a = 5 kb unit (Lefranc et al. 1986c),.

Other haplotypes defined by polymorphic Taq I sites. Three other haplotypes corresponding to polymorphic Taq I sites have also been observed in the Tunisian popula-

i

N. Ghanem et al.: T-cell receptor gamma variable gene polymorphism

Table 3. The TRGVI haplotypes as defined by the Eco RI (TRGVI*A) and Taq I (TRGVI*B) polymorphic restriction fragments.

357

TRGV1 haplotypes *A-*B combination Polymorphism Frequencies

TRGVI*I TRGVI*AI*B1 RS 0.693 (F) 0.594 (L) 0.594 (T) 0.500 (B)

TRGVI*2 TRGVI*A2*B2 RS 0.096 (F) 0.187 (L) O. 162 (T) 0.167 (B)

TRGVI*3 TRGVI*A3*B3 D 0.211 (F) 0.203 (L) 0.162 (T) 0.133 (B)

TRGVI*4 TRGVI*A4*B4 I 0.040 (T) 0.133 (B)

TRGVI*5 TRGVI*A2*B5 RS 0.014 (T) 0.067 (B)

TRGFI*6 TRGVI*AI*B6 RS 0.015 (L) 0.014 (T)

TRGFI*7 TRGVI*A2*B6 RS 0.014 (T)

RS, polymorphic restriction sites; D, deletion; I, insertion.

Fig. 5. Mendelian inheritance of the Eco RI (TRGVI*A) restriction fragments. Tunisian family W: TRGVI*A1 and *A2; Tunisian family A: TRGVI*A1 and *A3; Tunisian family CHA: TRGVI*A1, *A2, and *A3; Tunisian family K: TRGVI*A1, *A3, and *,44.

358 N. Ghanem et a l . : T-cell receptor gamma variable gene polymorphism i

S [] t

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m - 8.s =,~- ,.s .,, ~

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Fig. 6. Mendelian inheritance of the Taq I (TRGVI*B) restriction fragments. French family S: TRGVI*B1 and *B2; Tunisian family CHA: TRGVI*B1, *B2, *B3, a n d *B5; Tunisian family AJ: TRGVI*B1, *B2, and *B6; Tunisian family WO: TRGVI*B2, *B3, a n d B6.

tion. The first one results from the presence of a Taq I site in the 22 kb fragment yielding two fragments of 15 and 7 kb (TRGVI*B5; Table 2B). The Mendelian in- heritance of these fragments is shown in family CHA (Fig. 6), where one child has inherited the 15 +7 kb Taq I fragments from his mother, heterozygous *B1/*B5. The TRGVI*B5 fragments and the associated Eco RI TRGVI*A2 (9.4 kb) fragment define the TRGVI*5 haplotype (Table 3) found in the heterozygous state in this Tunisian family.

The other two haplotypes are both characterized by a 12.8 kb Taq I fragment (TRGVI*B6) due to two new Taq I polymorphic sites. This polymorphism also results in a new 4.2 kb fragment which comigrates with the V5 Taq I band, as shown by the increased intensity of the 4.2 kb band on Southern blots. These fragments were found in one Lebanese family (not shown) and two related Tuni- sian families (AJ and WO; Fig. 6). The 12.8 kb Taq I fragment is associated with either the TRGVI*A1 (7.0 + 2.4 kb) or the TRGVI*A2 (9.4 kb) Eco RI restriction fragments defining the TRGVI*6 and the TRGVI*7 haplotypes, respectively (Table 3). The TRGVI*6 haplotype has been observed in one Tunisian and one Lebanese family, the TRGVI*7 in one Tunisian and one Black African individual.

Frequencies of the different TRGVI haplotypes in French, Lebanese, Tunisian, and Black African populations. Three haplotypes (TRGVI*I, *2, and *3) have been fouiad in the French population at the following frequencies: 0.69 (TRGVI*I) and 0.09 (TRGVI*2) for the two haplotypes defined by the presence and the absence of the Eco RI and Taq I polymorphic sites, respectively, and 0.21 for the third haplotype (TRGVI*3) corresponding to the deletion of V4 and V5 (Table 3). These three haplotypes are observed in the Lebanese, Tunisian, and Black African populations with a decreased frequency for the TRGVI*I haplotype (0.59 in Lebanese and Tunisiah, 0.50 in Black African populations) and, reciprocally, an increased frequency for the TRGVI*2 haplotype (0.18 for the Lebanese and 0.16 for the Tunisian and Black African populations). The Tunisian population is the most polymorphic with the presence of the seven haplotyp~s; the haplotypes TRGVI*5, TRGVI*6, and TRGVI*7 hage been observed, each in one heterozygous individual. Family members have subsequently been studied to ascer- tain the Mendelian inheritance of these haplotypes (Figs. 5 and 6). The haplotype TRGVI*4 (insertion of an addi- tional V gene, V3P, between V3 and V4 genes) is present at a frequency of 0.04 in the Tunisian population and 0.13 in our sample of Black Africans. A Negroid admixture

N. Ghanem et al.: T-cell receptor gamma variable gene polymorphism 359

might account for the presence of this expanded ten-gene haplotype and for the frequency observed in Tunisia, as previously demonstrated for numerous other genetic markers (Lefranc et al. 1979, Ghanem et al. 1988b, Buresi et al. 1989).

Our results show that the number of V gamma I subgroup genes may vary from seven to ten. The most frequent haplotype in the four populations studied, TRGVI*I, contains nine genes, of which five are func- tional (Lefranc et al. 1986c, Forster et al. 1987). The dele- tion of F4 and V5, which results in a seven-gene haplotype (TRGVI*3; 21% of the haplotypes in the French popula- tion), does not seem to have a deleterious effect since it has been observed in the homozygous state in healthy in- dividuals. It is likely that the loss of the F4 and 115 genes is compensated by V2 and F3 due to the high degree of homology existing between V2 and V4, on the one hand (95 % at the nucleotide level and 93 % at the protein level; Lefranc et al. 1986c, Huck et al. 1988), and between F3 and F5, on the other hand (95 % at the nucleotide level and 92% at the protein level; Lefranc et al. 1986c, Font et al. 1988); this, moreover, suggests a recent duplication of these genes. Evolution of the Vgamma I subgroup gene region by contraction/expansion is further supported by the insertion of an additional V gene, F3P, between F3 and V4, thus determining the ten-gene haplotype TRGVI*4 observed with the highest frequency in Black African populations (13 % of the haplotypes) or in populations with Negroid admixture. Similar duplication/deletion events have occurred also in the constant region and in associated joining segments (Lefranc et al. 1986b, Huck and Lefranc 1987): the TRG1 region is characterized by the insertion of an additional J segment, JP (although it cannot be ex- cluded that an equivalent segment was lost by the TRG2 region after the duplication of the constant region); duplication and triplication of exon 2 of the TRGC2 gene has been well documented and shown to correspond to allelic polymorphism (for review, see Buresi et al. 1989).

Acknowledgments. We wish to thank J. Ghanem and G. Merhege for collecting the blood samples under very hard conditions in Lebanon, and the family members for their cooperation. We are grateful to all the blood donors of UA CNRS 1191 and to the Lebanese and Black African students of Montpellier for their contribution. This work was supported by the Fondation pour la Recherche Mtdicale, the Association pour la Recherche sur le Cancer, the Centre National de la Recherche Scientifique, the Ligue Nationale Franqaise contre le Cancer, and the Minist~re de la Recherche et de l'Enseignement Suptrieur.

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Received June 16, 1989.