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* Immunology Program, Sloan-Kettering Institute for Cancer Research and
Allogeneic Bone Marrow Transplantation Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; and
Human Immunogenetics Program, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In humans, receptors that signal activation include the NK cytotoxicity receptors (4), whose ligands remain unclear, and NKG2D, which has been shown to recognize MHC class I chain-related proteins A and B and UL16-binding proteins (5). Inhibitory receptors include the heterodimer molecules CD94:NKG2A which recognize complexes of HLA-E and peptides encoded from the HLA-A, -B, -C, or -G leader sequences (6, 7). Fulfilling both inhibitory and activating roles are members of the CD158 gene family (8), commonly referred to as killer Ig-like receptors (KIR),3 which are found on all NK cells and on some CD8+ T cell subsets with activated or memory phenotype (9, 10, 11, 12, 13, 14, 15).
The strategy of regulating NK cells by pairs of activating and inhibitory KIRs is used not only in humans, but also in other primates such as apes and Old World monkeys in which orthologous KIR genes have been identified (16, 17, 18). In addition, KIR-like genes have also been found in nonprimate higher vertebrates, such as the cow (19). An intriguing finding has been that in rodents, a system of NK receptors has evolved in parallel with the human KIR, such that the C-type lectin Ly-49 receptor family in the mouse performs related functions to those of KIR in humans (20).
The Ly49 gene family is located within the NK complex on mouse chromosome 6 (21), which also contains other NK receptor genes such as CD94 and the NKG2 genes. The syntenic chromosomal region in humans is located on chromosome 12, but only a single, nonfunctional Ly49 gene has been identified (22, 23). The KIR gene family is located on human chromosome 19 within the leukocyte receptor complex in region 19q13.4 together with the genes encoding other Ig gene superfamily members such as the Ig-like transcripts (ILT) and leukocyte-associated Ig-like receptors (24). A similar complement of Ig superfamily members called novel immune-type receptor genes, have recently been described in cartilaginous and early jawed fish (25, 26, 27). These KIR/ILT-like gene clusters have been localized in the zebra fish to a syntenic region corresponding to mouse chromosome 7 and human 19q13. The novel immune-type receptor genes may thus represent early ancestors for the leukocyte receptor complex genes.
The ligand specificity for HLA-A, -B, -C, and -G has been demonstrated for certain KIR, while specificity for other KIR remains unknown (28). The KIR region exhibits an extensive degree of diversity, which it achieves through a combination of variable gene content and polymorphism (29, 30, 31, 32, 33, 34, 35, 36, 37, 38). Population studies have generally used KIR gene typing methods on genomic DNA to determine the presence or absence of each KIR gene and have demonstrated that between individuals, KIR gene content can vary widely. Recently, these studies were extended to include evaluation of allelic differences at polymorphic sites to document an additional dimension of KIR diversity achieved through polymorphism (36). Indeed, estimates of the extent of KIR genotype diversity within the population suggest that far <0.24% of unrelated individuals can expect to have identical genotypes (36).
However, underlying the diversity of the KIR genomic region
are patterns that appear conserved within the population. The
chromosomal arrangement of KIR genes, for instance,
maintains a certain regularity as exemplified by the regular spacing of
KIR genes 
2.4 kb from each other and the presence of the
framework genes 3DL3 and 3DL2 at either terminus
of the region and 2DL4 in the middle (24).
There is suggestion of conservation of haplotypes as well, with the
early description of two main haplotype groups A and
B found within the population (29, 30).
Estimated to be as frequent as 47–59% within the Caucasian
population, haplotype A comprises a common complement of
KIR genes 3DL3, -2DL3,
-2DL1, -2DL4, -3DL1, -2DS4,
and -3DL2. In contrast, haplotype B has been
defined as a more varied haplotype group that encompasses genotypes
containing more activating receptor genes, including
KIR2DS1, -2DS2, -2DS3, and
-2DS5. The definition of these haplotypes, particularly
haplotype B, has been limited due to an inability to
accurately resolve haplotypes, short of sequencing the genomic region,
which has only been achieved for three haplotypes (Refs.
24 and 39 and accession no.
AC011501.7). Family studies are a useful means by which to clarify
haplotypes, if the families are large and informative for haplotype
segregation.
In this study, we report the gene content evaluation of 12 families, 6 of which are multigenerational with large sibships, and the elucidation of specific haplotypes within these families. In addition, the investigation presented in this study includes typing for the two major subtypes of a pseudogene, for the expressed and nonexpressed forms of KIR2DL5, and for a new KIR gene that previously was included in the typing for KIR2DS4. Typing for these loci provides additional insight into distinct haplotypic gene combinations based on gene content. These findings were then extended to a set of 49 sibling pairs and a panel of unrelated donors. The studies indicate the existence of a limited number of gene combinations centromeric to KIR2DL4 and greater variability telomeric to this anchor gene. We present a model for the genomic organization of the human KIR region where the gene content can be defined by six major haplotypic gene combinations, each with multiple permutations.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The normal Caucasian population consisted of 85 randomly selected unrelated individuals. Sixty-one of these individuals were relatives of patients from the New York tristate area referred to Memorial Sloan-Kettering Cancer Center (New York, NY) for allogeneic hemopoietic stem cell transplantation, and the remaining 24 individuals were parents from the Center d’Etude du Polymorphisme Humain (CEPH) cell bank. An additional 49 unrelated Caucasian individuals, each being a sibling of one of the panel donors from the New York tristate area, were included for comparison of KIR genotypes between siblings. These 49 individuals were not included in the analysis of the unrelated panel. Family studies were performed on B-lymphoblastoid cell lines (BLCL) derived from the family members of 12 CEPH families (consisting of at least two parents and two children). Six CEPH families, consisting of multiple generations and sibships containing at least eight children, were selected for extended typing studies for a total of 98 individuals. Seventeen BLCLs derived from offspring of consanguineous parents and obtained from the International Histocompatibility Workshop Repositories were included as a potential source of KIR homozygous cells (40). BLCL were maintained in RPMI 1640 supplemented with 10% FBS at 5% CO2, 37°C.
Sample preparation
Genomic DNA was extracted from 5 x 106 PBMCs, bone marrow mononuclear cells, or BLCL tissue culture cells using the Puregene DNA Isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturer’s instructions.
Polymerase chain reaction
PCR-sequence-specific primers (SSP) for the detection of
KIR gene loci in genomic DNA are detailed in Table I
. To detect the gene encoding all known
alleles at a given locus and to achieve consistent results, alternative
primer sets to those previously published (29, 32) were
designed for the detection of KIR2DS1, 2DS5, 2DL1, 2DL4, 2DL5,
3DS1, 3DL1, 3DL2, 3DL3, and pseudogenes 3DP1 and
2DP1. Pseudogene KIR3DP1 has previously been
designated KIRX (24), KIR2DS6
(39), KIR48 (41), and
CD158c (8). Pseudogene KIR2DP1 has
previously been designated KIRZ (24),
KIR15 (41), and KIRY
(20). KIR genes are reported in this study
according to the guidelines by the Human Gene Nomenclature Committee
(HGNC) (42). Results obtained with these primer sets were
compared with results using primers designed by other groups (29, 32). Primers were also designed in the 5'-untranslated
region (UTR) and 3'-UTR as forward or reverse primers,
respectively, for use with a KIR gene-specific primer
partner (Table I). For many KIR genes, two different primer
sets were used to establish the presence or absence of the gene. The
primer pair for pseudogene 3DP1 amplifies two
different-sized amplicons (344 and 1817 bp) corresponding to
KIR3DP1 and KIR3DP1v. In addition, primer sets
were designed to accurately identify 2DS4 and distinguish it
from a novel gene (designated KIR1D), which contains a 22-bp
deletion in D2, leading to a frame shift and premature stop codon, with
a predicted protein product truncated just within the transmembrane
domain. Primers were also designed for KIR2DL5 subtyping to
distinguish between expressed and nonexpressed variants of
2DL5 (44). Amplifications using primers labeled
2DL5.1 identify the expressed variants 2DL5.1 and
2DL5.3, and amplifications using primers labeled 2DL5.2
identify the nonexpressed variants 2DL5.2 and
2DL5.4 (nomenclature 2DL5.1-2DL5.4 as previously described;
Ref. 44). All primers were confirmed to be KIR
gene-specific by basic local alignment sequence tool searches
and verification with the National Center for Biotechnology
Information KIR database
(http:// www.ncbi.nlm.nih.gov/IEB/Research/GVWG/MHC/MHC.cgi)
(3) and were subsequently validated by amplicon
sequencing. Nucleotide sequencing of amplicons was performed using dye
terminators and automated sequencing (ABI 377 instrument; PE Applied
Biosystems, Foster City, CA) in the Sloan-Kettering Institute
Sequencing Core Facility (New York, NY).
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2DS4 variant identification and cloning
Amplification of KIR2DS4 using previously published
primer pairs (29) was performed on genomic DNA samples.
PCR products were gel-purified using the GFX PCR DNA and Gel Band
Purification kit (Amersham Pharmacia Biotech, Piscataway, NJ) and were
subsequently cloned into pCR2.1-TOPO using the TOPO-TA Cloning kit
(Invitrogen, Carlsbad, CA). Plasmids were isolated from individual
bacterial clones using the QiAmp Spin Miniprep kit (Qiagen, Chatsworth,
CA), and vector-specific primers were used for sequence analysis of
plasmid inserts using dye terminators and automated sequencing (ABI 377
instrument; PE Applied Biosystems) for identification of a 22-bp
deletion. Primer pairs were then designed to specifically identify the
intact KIR2DS4 gene and KIR1D from genomic DNA
samples (Table I
). NK cells isolated from individuals homozygous for
KIR1D and individuals heterozygous for 2DS4 and
KIR1D were used for mRNA isolation (MicroFastTrack 2.0 Kit;
Invitrogen) and cDNA isolation (cDNA Cycle kit; Invitrogen).
KIR2DS4-specific primers (forward
5'-CCATGTCGCTCATGGTCATCAT-3', reverse 5'-ACATGTTCTGATTGGGACC-3')
were then used to generate a 730-bp 2DS4 amplicon spanning
from the ATG start codon to the premature TGA stop codon. PCR products
were gel-purified using the GFX PCR DNA and Gel Band Purification kit
and cloned into pCR2.1-TOPO. Plasmids were isolated from individual
bacterial clones using the QiAmp Spin Miniprep kit, and KIR1D cDNA
clones were distinguished from 2DS4 clones by sequencing and by PCR
analysis.
Haplotype analysis
KIR haplotypes were determined by segregation patterns in families. In assigning genes to specific haplotypes, the following assumptions were made: 1) all haplotypes contained KIR3DL3, 2DL4, and 3DL2; 2) haplotypes contained either 2DL2 or 2DL3, but not both; and 3) haplotypes contained either pseudogene 3DP1 or 3DP1v, but not both. All assumptions were supported by previous analyses of linkage disequilibrium (LD) (29, 31, 32, 34, 35) and by sequenced KIR haplotypes (Refs. 24 and 39 and accession no. AC011501.7).
Haplotype deduction for sibling pairs not included in the family studies was performed by first applying the haplotypes defined in the family studies and only defining "new" haplotypes when this approximation was not possible. The haplotypes in the second sibling were then deduced using the assumption that 75% of siblings will share one KIR haplotype and 25% of siblings will share two haplotypes. Haplotype deductions in the unrelated Caucasian panel and in the panel of consanguineous DNA samples was performed using first the KIR haplotypes defined in the family studies, then adding "new" haplotypes derived from the sibling pair analysis and finally adding extra haplotypes if needed.
Statistical analysis
The observed KIR Ag frequency was determined by the ratio of
gene presence within the population to the total population number.
Gene frequency was calculated by the formula: gene
frequency = 1-2
(1-
), where is
the observed Ag frequency in the population. Two-locus LD parameters
(
) were calculated according to Mattiuz et al.
(45).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A PCR-SSP typing method was devised for the identification
of 16 known KIR genes and pseudogenes, KIR2DS1-5,
2DL1-5, 3DS1, 3DL1-3, and pseudogenes
3DP1 and 2DP1 (Table I). The method includes
alternative primer sets designed for inclusion of all known alleles for
KIR2DS1, 2DS3, 2DS5, 2DL4,
2DL4, 2DL5, 3DS1, 3DL1, and
3DL2. Confirmation of new primer set specificity was
achieved in several ways: positive amplification reactions were
compared with results obtained using previously published primer sets
(29, 32); amplification products were sequenced for
specificity; and finally, typing of 85 unrelated Caucasian individuals
yielded comparable estimated gene frequencies to those in previously
published findings (29, 32, 34, 35). In this cohort, 36
different genotypes were identified, 23 of which were unique, with the
remaining genotypes each observed from 2 to 14 times (Fig. 1). The most common genotype (observed 14
times) was KIR3DL3-2DL3-2DP1-2DL1-3DP1-2DL4-3DL1-1D-3DL2,
corresponding to homozygosity for the major subtype of the previously
reported A haplotype (see below). Observed nine times was
the genotype
KIR3DL3-2DL3-2DP1-2DL1-3DP1-2DL4-3DL1-2DS4-1D-3DL2,
corresponding to heterozygosity for the two major subtypes of haplotype
A. Genotypes represented more than once accounted for 73%
of all genotyped samples. Included in this typing is the detection of a
new gene KIR1D and typing for the two different variants of
pseudogene 3DP1.
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PCR-SSP typing for gene 2DS4 using a
previously published primer set (29) revealed a nearly
ubiquitous gene frequency within the population. However, sequencing of
several genomic DNA amplicons using this primer set revealed the
presence of a 2DS4 gene variant, characterized by a 22-bp
deletion in the second extracellular Ig-like domain (D2). Although the
remainder of the nucleotide sequence is identical to
KIR2DS4, the amino acid sequence resulting from
the deletion-generated frame shift is comprised of a novel
stretch of 88 aa, loss of the D2 Ig domain, and termination at a final
length of 239 aa, one amino acid into the putative transmembrane
domain, with no discernible cytoplasmic domain (Fig. 2). Search of GenBank nucleotide
sequences revealed the presence of this gene in the genomic DNA
sequence of a human chromosome 19 haplotype (accession no. AC011501.7),
placing this novel KIR gene in the KIR gene
cluster on human chromosome 19q13.4. A GenBank amino acid search
identified significant amino acid homology (72%) to a variant of the
Mm-KIR1D receptor found in rhesus monkeys (accession no.
AF334633) which has nucleotide homology to Mm-KIR2DL4, but
contains only one complete Ig domain and no cytoplasmic tail as a
consequence of a frame shift (17). Because of the amino
acid homology between this novel human gene and Mm-KIR1D, we
propose designation of the human gene as KIR1D.
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). From a separate individual, a
KIR1D variant was identified; while exhibiting the same
22-bp deletion, this variant lacked the stem region (corresponding to
exon 5), indicating its likely identity as a splice variant (accession
no. AY102633). A GenBank search also revealed a recently cloned
KIR gene that is identical to human KIR1D except
for a single nucleotide substitution at position 156 giving rise to a
codon change from arginine to lysine in the exon 4-encoded gene product
(accession no. AF417554).
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The percentage of Caucasian individuals exhibiting
KIR1D is high (78.8%) with an estimated gene frequency of
0.54. Accurately revised typing for KIR2DS4 revealed a lower
Ag frequency (35.3%) than previously determined with an estimated gene
frequency of 0.20. LD analysis identified strong negativity between the
two genes, supporting their possible identities as alleles. Strong
positive LD with KIR1D was noted with KIR3DL1 and
2DL3, whereas there was only a very weak LD between
KIR2DS4 and these genes, indicating that the previously
noted positive LD between KIR2DS4 and KIR3DL1 and
KIR2DS4 and 2DL3 was likely due to lack of
distinction between KIR1D and 2DS4. Strong
negative LD between KIR1D and 2DS1 is also seen.
Other LD relationships are indicated in Table II.
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KIR typing for pseudogene 3DP1 revealed the
presence of two predominant subtypes. These can be identified as two
different amplicons, whose 1.5-kb size difference is due to the
presence or absence of exon 2 and its flanking intron sequences.
KIR3DP1, characterized by the absence of exon 2, was found
in the genomic DNA sequence of a fully sequenced human chromosome 19
KIR haplotype (accession no. AC011501.7);
KIR3DP1v, characterized by the presence of exon 2, was found
in a separate chromosome 19 KIR haplotype genomic sequence
(accession no. AL133414). LD for KIR3DP1 and
KIR3DP1v reveal the two exhibiting strong negative LD,
supporting a possible allelic relationship. Whereas KIR3DP1
has a high gene frequency within the population (0.72),
KIR3DP1v exhibits a significantly lower gene frequency
(0.17). KIR3DP1 displays a strong positive LD with
pseudogene KIR2DP1 and with 2DL1, supporting
their inclusion in a partial haplotype
KIR2DP1-KIR2DL1-KIR3DP1. In contrast, KIR3DP1v
displays an equally strong negative LD with KIR2DP1 and
2DL1; moreover, the identical frequency of these gene pairs
in relation to each other further supports their definition of a
KIR3DP1v partial haplotype distinguished by the absence of
KIR2DP1 and 2DL1. In other words, when
KIR3DP1v is present, KIR2DP1 and 2DL1
are absent. This partial haplotype lacking KIR2DP1-2DL1 en
bloc can be observed in PCR-based genotyping only when the
individual is homozygous for KIR3DP1v, a relatively rare
combination noted in five of the genotypes from the Caucasoid panel
studied (see Fig. 1, genotypes Z, AA, AB, AC, and AD) and in extended
family study haplotype elucidation. In addition, KIR3DP1v
was found to be in strongly positive LD with KIR2DL2 and
KIR2DS2 extending the partial haplotype to resemble
KIR2DL2-(absent 2DP1-absent 2DL1)-3DP1v.
Family segregation studies define haplotypes
Family studies performed to date have not included typing for
KIR1D or for the pseudogenes 3DP1,
3DP1v, and 2DP1, whose typing significantly
helped to define distinct haplotypes. Twelve families consisting of
both parents and two children were analyzed for gene content; of these,
six families were selected for extended family analysis based on
heterozygosity at the KIR3DL1-3DS1 locus and the likelihood
of haplotype resolution. Through typing of multiple generations and
sibships numbering no fewer than eight and in several cases more than
10, KIR haplotypes could be reliably resolved with nearly no
ambiguities. Examples of genotype analysis for individuals from two
families and the resolved parental and grandparental haplotypes are
shown in Fig. 4. From 12 families, 11
different haplotypes were resolved by gene content (Fig. 5A), with 2 representing
haplotype A (haplotypes 1 and 2), as previously defined by
Shilling et al. (36) as the presence of
KIR2DL3 and the lack of all activating genes with the
exception of 2DS4, presented in this study as comprising
both 2DS4 and KIR1D.
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Segregation analysis of haplotypes supported the following
findings: as predicted by their negative LD relationship,
KIR2DL2 and 2DL3 segregated exclusively onto
separate chromosomes. In none of the families could both KIR
genes be assigned to the same haplotype, supporting their relationship
as possible alleles. Pseudogene 2DP1, KIR2DL1,
and pseudogene 3DP1 segregated en bloc and could be
associated with either KIR2DL3 or 2DL2, although
more often with the former (Fig. 5A, haplotypes 1, 2, 5, 6,
7 vs haplotypes 8 and 9). In contrast, the presence of the pseudogene
variant 3DP1v was associated with the absence of pseudogene
2DP1 and KIR2DL1 and was observed exclusively
with KIR2DL2 in the family studies. This "deletion"
partial haplotype was observed in three of the resolved haplotypes and
occurred in 9 of the 24 parents (37.5%) (Fig. 5A,
haplotypes 3, 4, and 10). Haplotype 11 is an unusual
KIR haplotype, seen only in one family.
Haplotype analysis-telomeric half-KIR1D redefines haplotype A
Telomeric to KIR2DL4, the following findings were
observed: KIR1D and 2DS4 segregated exclusively
onto separate chromosomes (e.g. Fig. 4, C and D).
Typing for KIR1D revealed that what was previously defined
as haplotype A can be divided into two groups, one
containing KIR1D, designated as haplotype A-1D
(Fig. 5A, haplotype 1), and the other containing
KIR2DS4, designated as haplotype A-2DS4 (Fig. 5A, haplotype 2). In the family analyses, haplotype
A-1D was the more frequently observed, representing 20 of a
total of 48 parental haplotypes (41.7% gene frequency). In contrast,
haplotype A-2DS4 was present in only 6 of a total of 48
haplotypes (12.5%). These frequencies are comparable to the
frequencies in the panel of 85 unrelated Caucasians (38.8% and 11.8%,
respectively). Homozygosity for haplotype A-1D was seen in
14 of 85 unrelated Caucasian individuals (16.67%), whereas
homozygosity for haplotype A-2DS4 was seen in only 1 of 85
unrelated individuals (1.1%). From the same panel, haplotype
A-1D had the highest haplotype frequency of 38.8%, while
haplotype A-2DS4 had a haplotype frequency of 11.8%.
In one extended family, the exclusive segregation of KIR2DS1
from haplotypes containing KIR1D or 2DS4 was
unequivocally observed (Fig. 4, C and D). This
mutual exclusion is supported by the highly significant negative LD
between the genes, suggesting their possible relationship as alleles.
Likewise, KIR2DS3 and 2DS5 were never identified
on the same haplotype in our studies. Accompanying KIR2DS3
and 2DS5 is 2DL5, which was not found to
segregate separately from either of the two activating receptor genes,
except in one haplotype (Fig. 5B, haplotype 12), which was
later found in a sibling pair that displayed full KIR
identity. Subtyping for the expressed (labeled 2DL5.1) and nonexpressed
forms of KIR2DL5 (labeled 2DL5.2) identified a haplotype
containing both forms (Fig. 4, A and B, and Fig. 5A, haplotype 9). Other haplotypes contained either the
nonexpressed or the expressed KIR2DL5 subtype, paired with
either 2DS3 or 2DS5. Finally, KIR2DL2
and 2DS2 were found to segregate together for all resolved
haplotypes. To address the issue of nonspecific amplification or
coamplification, >20 amplicons for both KIR2DL2 and
KIR2DS2 were sequenced, repeatedly confirming gene
specificity of the primers.
Sibling studies
KIR genotyping of 49 sibling pairs allowed for the informed
deduction of 10 additional haplotypes (Fig. 5B). Of the 49
pairs, 17 pairs (34.7%) yielded identical genotypes, which could imply
identical KIR haplotype pair combinations. Although this
ratio was higher than the 25% expected from normal Mendelian
segregation, it could be explained by high frequency of haplotype
A-1D in these sibling pairs (12 of 17). Given the high
frequency of this haplotype in the population, it might be expected
that in some cases where both siblings exhibit this haplotype, they may
not be identical haplotypes by descent. Eleven pairs (22.5%) exhibited
no shared haplotypes, while 21 pairs (42.9%) exhibited one shared
haplotype. Of the 10 new KIR haplotypes deduced among the
sibling pairs, one haplotype contained both expressed and nonexpressed
variants of KIR2DL5 (Fig. 5B, haplotype 22).
Combinations from the deduced sibling haplotypes in addition to the 11
identified through family studies could account for all 36 genotypes
seen in the unrelated Caucasian panel (Fig. 1). Fifteen unrelated
individuals among the sibling group were found to be homozygous for 1
of the 24 identified or deduced haplotypes. This included eight
instances of haplotype 1, two of haplotype 5, and one each of
haplotypes 2, 4, 8, 14, and 17.
Haplotype frequency in the Caucasian panel
The unrelated Caucasian panel described in Fig. 1 was analyzed for
deduced KIR haplotypes based on the haplotypes characterized from
family studies and sibling pair analysis (Fig. 5, A and
B). All 36 genotypes could be resolved into corresponding
pairs of haplotypes as shown in Fig. 1. Of the 170 haplotypes exhibited
in 85 individuals, the most commonly observed haplotype was haplotype
1, occurring 66 times (38.8%). In contrast, haplotype 2 was found 20
times (11.8%). Comprising the "classical haplotype A"
frequency, the sum of frequencies for haplotype 1 (haplotype
A-1D) and haplotype 2 (haplotype A-2DS4) yielded
a total haplotype A frequency of 50.6%, consistent with
previous estimates (29, 36). Less frequent, but very
common, were the haplotypes characterized by the deletion partial
haplotype (KIR2DS2-2DL2-3DP1v): haplotype 3 had a frequency
of 7.7% while haplotype 4 had a frequency of 6.5%. In total,
"short" haplotypes with 3DL1-2DS4 or 3DL1-1D
accounted for 64.7% of all KIR haplotypes in this Caucasian
population.
Consanguineous individuals
Consanguineous cell lines have proved useful for HLA
allele identification and HLA haplotype analysis. A panel of
17 cell lines derived from individuals from consanguineous families was
analyzed for KIR gene content (Fig. 6). Among these individuals, two more
unique genotypes were found, yielding two additional unique haplotypes
(Fig. 5B, haplotypes 18 and 23). Based on the conservative
estimates for the haplotypes described in this study, five of the
consanguineous individuals appear to be homozygous for haplotype
A-1D, while one is homozygous for haplotype
A-2DS4. These cell lines could very well be KIR
homozygous by descent. For cell lines with larger gene content, it was
not possible through this analysis to document homozygosity.
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Haplotype B has been defined as containing more
variable KIR gene combinations and characterized by
KIR2DS genes other than KIR2DS4. According to
this definition, 21 different B haplotypes were resolved.
These haplotypes included most of the partial haplotypes described by
other groups (31, 36); however, in contrast to one
previous report (31), there was no representation of the
2DS3-2DS5-2DS1 complement in our studies. Evaluation of the
haplotypes revealed specific patterns that are supported by
KIR locus LD analysis. Although KIR3DS1 is most
commonly associated with the presence of multiple 2DS genes,
KIR3DL1 could occasionally, but not as commonly, also be
found to be associated with these genes. KIR2DS3 and
2DS5 were each almost always paired with 2DL5,
whose subtyping revealed the nonexpressed 2DL5.2 variant
exhibiting a stronger positive LD with 2DS3
(p << 0.0005) in comparison to 2DS5
(p < 0.05). In addition, the nonexpressed
2DL5.2 variant exhibited a strong positive LD
(p << 0.0005) with 2DL2. The expressed
2DL5.1 variant showed strong positive LD with
3DS1 (p << 0.0005), 2DS3
(p < 0.0005), and 2DS5
(p << 0.0005) and no significant LD with
2DL2. These analyses support earlier reports of the
placement of KIR2DL5.2 adjacent to 2DL2 on the
chromosome and 2DL5.1 adjacent to 3DS1
(44). Although pair KIR2DL5-2DS3 could be seen
with KIR1D, 2DS4, or 2DS1, pair
KIR2DL5-2DS5 was seen only with 2DS1 in this
analysis, consistent with the strongly positive LD between
2DS5 and 2DS1. KIR2DS2 displayed a
strong positive LD with 2DS3, but its main association
appeared to be with 2DL2. Although not exclusive, these
patterns were supported by the overwhelming majority of identified and
deduced haplotypes. Only rarely would haplotypes be identified that did
not adhere to these patterns (see Fig. 5, haplotypes 11 and 12).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Haplotype A has been defined as containing KIR3DL3, -2DL2, -2DL1, -2DL4, -3DL1, -2DS4, and -3DL2, and haplotype B has been defined as having more KIR genes than the A haplotype; generally, these additional genes are the activating KIR genes characterized by short cytoplasmic tails. The ability to unambiguously define haplotypes has previously been approached by family studies; however, these studies have typically included fairly small family cohorts, consisting of three to six members. In this study, we report the elucidation of haplotypes through gene content analysis of extended family pedigrees whose large sibships and multiple generations could reliably identify specific haplotypes. Eleven different haplotypes were resolved through these studies, many of which have not previously been described. Contributing significantly to the resolution of these haplotypes was the typing for two KIR loci in particular, the 2DS4/KIR1D locus and the pseudogene 3DP1 locus. In this study, we report for the first time the cDNA isolation of human KIR1D, whose typing has not previously been distilled from that of 2DS4 and which appears to be the more prevalent of the two putative alleles of the 2DS4 locus. In addition, typing for the two known variants of pseudogene 3DP1 has revealed the presence of a "deletion" partial haplotype that is distinguished by the presence of the pseudogene 3DP1v with KIR2DS2 and 2DL2, and the absence of 2DL1 and pseudogene 2DP1.
KIR1D has not before been identified in humans and was recently identified in the rhesus monkey as Mm-KIR1D (17). Although the nucleotide sequences do not show significant homology, the amino acid sequences between the two gene products display a high degree of homology, suggesting they may be functional orthologs. In addition, while the Mm-KIR1D gene appears to be a deletion variant of Mm-KIR3DL7, the human KIR1D is likely a deletion variant of 2DS4. Interestingly, the respective deletions in the rhesus and human genes result in frame shifts that yield homologous predicted protein products. As in the rhesus monkey, it also appears that several putative splice variants of human KIR1D are present, with at least one isolated clone having a deletion of the exon encoding the stem region. KIR1D encodes a predicted protein product that has a complete D1 Ig domain and a portion of the D2 domain, before the frame shift deletion abrogates any transmembrane or cytoplasmic region. Splice variants of other KIR/ILT genes have been identified (46, 47), some of which are lacking the sequences encoding the transmembrane and cytoplasmic regions, leading one to speculate about the possibility of a secreted truncated protein product. However, the presence of an encoded secreted protein such as KIR1D has not heretofore been described. The ligands for the KIR1D proteins, both in the human and the rhesus monkey, remain unknown; however, the likelihood that that it is an MHC-like molecule is remote. Of interest is the difficulties in isolating KIR1D relative to 2DS4 transcripts in a heterozygous individual, despite the high prevalence of the gene within the population. The significance of this apparent transcriptional discord remains unclear. In contrast, transcripts were easily obtainable from an individual found to be homozygous for KIR1D.
A new model for KIR haplotypes
In this study, we describe extended family studies with very large sibships to define specific haplotypes, following them through three generations within the families. Analysis of the resolved haplotypes invites a reassessment of the initial two haplotype groups, first proposed by Uhrberg et al. (29) and further supported by the genomic sequencing of the complete KIR region for a representative of the A and B haplotypes (24). From our studies, it is evident that what was traditionally viewed as haplotype A is actually a combination of two different genotype groups, one containing KIR2DS4 and the other containing KIR1D in association with pseudogene 3DP1. The latter subtype is the more common, comprising 73% of haplotype A cases, and observed in 39% of unrelated Caucasian individuals. In contrast, the 2DS4-positive haplotype A has a haplotype frequency of 12%. The combined frequency of both haplotype A subtypes of 51% is consistent with previously published estimates (29, 36). It is interesting to note that because the most common genotype in the Caucasian population reflects homozygosity for the haplotype A containing KIR1D (14%), these individuals are lacking all activating KIR receptors, with the exception of KIR2DL4, a ubiquitously found receptor characterized by a long cytoplasmic tail, but exhibiting activating function (48, 49).
Although our studies are consistent with the broad classification of
KIR regions into A and B haplotypes,
the inclusion of typing in families for the KIR pseudogenes
and the discovery of the human KIR1D as a likely allele of
the 2DS4 locus has provided a more detailed view of the
KIR genomic region, as also recently described by Shilling
et al. (36). Our data can best be accommodated by an
alternative model for KIR haplotypes (Fig. 7). The KIR haplotype can be
considered as two halves, the centromeric half bordered upstream by
KIR3DL3 and comprised of those KIR genes upstream
of anchor gene KIR2DL4 and the telomeric half bordered
downstream by 3DL2 and comprised of those KIR
genes downstream of 2DL4. The centromeric half may be
characterized by the presence of KIR2DL3 or 2DL2,
but not both, and rarely, neither. In the rare case where neither
KIR2DL3 nor 2DL2 was identified, the possibility
exists for the presence of a new KIR gene (S. Chida and D.
Geraghty, personal communication). In our studies,
KIR2DL2 was found always to segregate with 2DS2,
the latter occupying the more upstream position of the two. Alteration
of our model to reflect the gene position of KIR2DS2 is
consistent with a reinterpretation of the RP5 chromosomal KIR genomic
sequence (accession no. AL133414) and with gene order studies performed
by S. Chida and D. Geraghty (personal communication). Following
the locus occupied by either KIR2DL3 or 2DL2 is
either pseudogene 3DP1v or the trio of pseudogene
2DP1-KIR2DL1-pseudogene 3DP1. In our studies,
KIR2DL3 was exclusively associated with the trio
2DP1-2DL1-3DP1 to define a common partial haplotype (Fig. 7, haplotype models 1 and 4). Although this trio could also be seen to
associate with KIR2DL2 and 2DS2 (Fig. 7, haplotype models 2 and 5), 2DL2 and 2DS2 are more
commonly found to associate with the pseudogene 3DP1v, in a
partial haplotype defined by the en bloc absence of
2DP1-2DL1-3DP1 (Fig. 7, haplotype models 3 and 6). This
partial haplotype is consistent with an available genomic sequence of
this region of the 2DL2-positive chromosome
(24).
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, haplotype models 1–3). In the "long" haplotypes, a number
of activating KIR genes may be seen, almost always
accompanied by KIR2DL5. KIR2DL5 has four known
variants, two of which (2DL5.1 and 2DL5.3) are
expressed and, in this paper, collectively labeled 2DL5.1. The two
nonexpressed variants (2DL5.2 and 2DL5.4) are
collectively labeled 2DL5.2. Previous gene order studies
(44) and LD analysis performed in this report support the
likelihood that the nonexpressed 2DL5 variants are located
in the centromeric half adjacent to 2DL2 and are most often
accompanied by 2DS3, while the expressed 2DL5
variants remain in the telomeric half accompanied by 2DS3 or
2DS5 (Fig. 7, haplotype models 5 and 6). In our studies,
haplotypes containing both expressed and nonexpressed
KIR2DL5 variants have been identified. Finally, in the long
haplotypes, the locus adjacent to KIR3DL2 can be occupied by
KIR1D, 2DS4, or 2DS1 (Fig. 7, haplotype models 4–6). Accordingly, the vast majority of our data fits
the model presented in Fig. 7, which classifies KIR
haplotypes into one of six major groups. Haplotype 1 in Fig. 7 is
synonymous with the canonical haplotype A, with the
remainder comprising the classical B haplotype. The
documentation of the three short haplotypes can readily be seen in the
family studies. The high frequency of these haplotypes with their small
gene content in the telomeric KIR region is evident also in
the sibling analysis and in the unrelated panel studies.
Not all 30 permutations within the three major subtypes of the long
B haplotypes (Fig. 7, haplotypes 4–6) were identified in
the family studies, in part due to the limitations of the family
materials, but also likely in part due to differences in prevalence for
each permutation. For instance, haplotype 5 (Fig. 5) identified in the
pedigree studies was the most common long haplotype found in the
population analysis, occurring in 21 of 85 individuals (24.7%) with a
haplotype frequency of 12.4%. Because KIR2DL5 was observed
to pair with either 2DS3 or 2DS5, with one rare
exception (Fig. 5B, haplotype 12), it is therefore very
possible that KIR2DS3 and 2DS5 are alleles of the
same locus, but our studies cannot resolve this. Although we did not
observe KIR2DL5, 2DS3, and 2DS5 segregating
together, it is theoretically possible for such a haplotype to exist if
both 2DL5 subtypes are present. Similarly, we did not
observe KIR1D, 2DS1, and 2DS4 together
either as pairs or as triplets, which in combination with their
negative LD, suggests their relationship as alleles. Although the
nucleotide sequencing data would support KIR1D and
2DS4 being alleles, we cannot unequivocally claim
2DS1 as an allele of the same locus. Identification of
possible recombinant haplotypes or direct sequencing of appropriate
KIR genomic regions will be needed to resolve this issue.
The finding that the pairing of 2DL5/2DS5 most frequently
segregated with 2DS1 was also very characteristic for
well-defined haplotypes in the families. We have no explanation for the
mechanisms underlying these phenomena, but some functional interactions
between the gene products may have preserved this relationship during
evolution.
It is to be expected that "new" unusual "recombinant" haplotypes or haplotypes with gene duplication of fragments of the KIR region will be identified which cannot be accommodated by our model. This would be expected for a genomic region with multiple highly homologous genes, as has been observed for the HLA chromosomal region (50). The emerging picture of the gene content within the KIR region is becoming very similar to the findings made for the HLA class II region. In this study, the HLA-DR region demonstrates a variable gene content with functional class II genes and pseudogenes. Recently, several other genomic regions within the human genome have displayed similar variable gene compositions (50).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bo Dupont, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. E-mail address: b-dupont{at}ski.mskcc.org
3 Abbreviations used in this paper: KIR, killer Ig-like receptor; ILT, Ig-like transcript; BLCL, B-lymphoblastoid cell line; SSP, sequence-specific primer; UTR, untranslated region; LD, linkage disequilibrium.
Received for publication May 30, 2002. Accepted for publication August 30, 2002.
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