Development of a PCR-SSOP approach capable of defining the natural killer cell inhibitory receptor (KIR) gene sequence repertoires
Development of a PCR-SSOP approach capable of defining the natural killer cell inhibitory receptor (KIR) gene sequence repertoires
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DOI：
10.1034/j.1399-0039.2000.560403.x
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发表时间：
2000-10-01
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影响因子：
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通讯作者：
Middleton, D
中科院分区：
文献类型：
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作者：
Crum, KA;Logue, SE;Middleton, D
建立了一种多态杀伤抑制受体（KIR）基因的分子分型方法，该方法基于聚合酶链反应（PCR）扩增3个不同的目标结构域（免疫球蛋白结构域1和3，以及跨膜-细胞质结构域），然后与26个地高辛标记的序列特异性寡核苷酸探针（SSOP）杂交。除了鉴定12个KIR亚家族外，我们的PCR-SSOP分型方法还可以区分构成KIR3DL1亚家族的推定等位基因NKB1和NKAT3。90名无血缘关系的献血者和13个家庭（52人），包括父母双方，接受了我们的KIR PCR-SSOP分型方法。包括2DS5变异序列在内的所有12个KIR亚家族均存在于90个个体中，并且表现出不同的表型频率：2DL1（0.96）、2DL2（0.31）、2DL3（0.95）、2DS1（0.56）、2DS2（0.51）、2DS3（0.27）、2DS4（0.96）、2DS5v（0.35）、3DS1（0.47）、3DL1（0.96）、3DL2（1.0）和2DL4（1.0）。本研究共定义了23种不同的KIR表型，其中10种仅在一个个体中发现一次，表明爱尔兰人群中KIR表型谱存在相当大的多样性。大多数个体（93%）对三种明确定义的HLA-B和c配体具有抑制性KIR特异性。在3个个体中观察到不寻常的3DS1探针模式，表明胞质结构域内核苷酸位置1185-1186的3DS1基因序列发生了变化。测序结果显示，3DL1 NKB1基因外显子3有一个新的单核苷酸多态性（195,G-A）， 2DS4基因外显子5有一个22 bp的缺失多态性（核苷酸777 ~ 798缺失）。许多强KIR关联被观察到，即2DL1与2DL3, 2DS4与3DL1, 2DL2与2DS1/2DS2/2DS3, 2DS1与2DS3/2DS5v/3DS1, 2DS2与2DS3和2DS5v与3DS1。在13个家族中观察到的KIR分离分析证实了这些强关联，并允许定义一些部分KIR单倍型，例如2DL2-2DS1-2DS2-2DS3-3DL1。分离分析表明，至少有3个不同的基因位点编码2DL1-4，至少有4个基因位点编码无抑制性KIR2DS1-2DS5。在3DL1-2和3DS1的情况下，我们的数据表明3个基因位点，每个亚家族一个。
A molecular typing method based on polymerase chain reaction (PCR) amplification of three different target domains (immunoglobulin domains 1 and 3, and the transmembrane-cytoplasmic domain), followed by hybridisation with 26 digoxigenin-labelled sequence-specific oligonucleotide probes (SSOP) has been established for the polymorphic killer inhibitory receptor (KIR) genes. In addition to identifying the 12 KIR subfamilies, our PCR-SSOP typing approach could also distinguish the putative alleles, NKB1 and NKAT3, that comprise the KIR3DL1 subfamily. Ninety unrelated blood donors and 13 families (52 individuals), including both parents, were subjected to our KIR PCR-SSOP typing approach. All 12 KIR subfamilies, including a 2DS5 variant sequence, were present in the 90 individuals and displayed varied phenotype frequencies: 2DL1 (0.96), 2DL2 (0.31), 2DL3 (0.95), 2DS1 (0.56) 2DS2 (0.51), 2DS3 (0.27), 2DS4 (0.96), 2DS5v (0.35), 3DS1 (0.47), 3DL1 (0.96), 3DL2 (1.0) and 2DL4 (1.0). A total of 23 different KIR phenotypes were defined in this study, and 10 of these were only found on one occasion in one individual, indicating considerable diversity in the KIR phenotype profiles within the Irish population. Most individuals (93%) possessed the complement of inhibitory KIR specificities for the three well-defined HLA-B and -C ligands. An unusual probe pattern for 3DS1 was observed in 3 individuals indicating a variant 3DS1 gene sequence with changes at nucleotide positions 1185-1186, within the cytoplasmic domain. Sequencing analysis revealed a new single nucleotide polymorphism in exon 3 of 3DL1 NKB1(195, G-A) and a 22-bp deletion polymorphism in exon 5 of 2DS4 (nucleotides 777-798 deleted). A number of strong KIR associations were observed, namely 2DL1 with 2DL3, 2DS4 with 3DL1, 2DL2 with 2DS1/2DS2/2DS3, 2DS1 with 2DS3/2DS5v/3DS1, 2DS2 with 2DS3 and 2DS5v with 3DS1. Analysis of the KIR segregation observed in the 13 families confirmed these strong associations and permitted the definition of a number of partial KIR haplotypes, e.g 2DL2-2DS1-2DS2-2DS3-3DL1. The segregation analysis concluded that at least 3 distinct gene loci encode 2DL1-4 and at least 4 gene loci encode the non-inhibitory KIR2DS1-2DS5. In the case of 3DL1-2 and 3DS1, our data suggests 3 gene loci, one for each subfamily.