Download ASHI U Module Chapter II: DNA Based Testing Section: Application

ASHI U Module
Chapter II: DNA Based Testing
Section: Application Modules
Module (H): Killer-cell Immunoglobulin-like Receptor (KIR) Genotyping
Authors:
Joel Y. Sun, Stanford Hospital and Clinics, [email protected]
Laima Gaidulis, City of Hope National Medical Center: [email protected]
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David Senitzer, City of Hope National Medical Center: [email protected]
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Date Prepared: 08/23/2006
Date Updated: 07/04/2012
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OBJECTIVES
 Understand KIR components, gene location and standard nomenclature.
 Be familiar with currently available methodologies for KIR genotyping and
understand how to determine the genotype.Understand the steps involved in
identification of KIR genes using a simple multiplex PCR-SSP method as an
example.
 Understand and identify the ASHI standards that pertain to the compliance of the
assay, and the quality control measures incorporated in the test system.
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INTRODUCTION
Natural killer (NK) cells are increasingly recognized as an important immune
component involved in immune surveillance, anti-infection and alloimmune response.
Killer cell immunoglobulin-like receptors (KIRs) are the major receptor cluster
expressed on human NK cells and a subset of T cells. A KIR molecule consists of two or
three extracellular immunoglobulin-like domains, a transmembrane stem and a long (L)
or short (S) cytoplasmic tail (Fig. 1). Standard nomenclature describes these features
with a number followed by the letter D for the extracellular domains (2D or 3D), the letter
L or S in the third digit for the cytoplasmic tail and a number in the forth digit to
distinguish loci (in most cases). The letter P in the third digit refers to pseudogenes.
Generally, long tail KIRs transmit inhibitory signals and short tail KIRs transmit
stimulatory signals. Some inhibitory KIRs (iKIRs) specifically recognize HLA-A, B or -C
allotypes on target cells as iKIR ligands (iKIRLs, Fig.1). The interaction of iKIRs and HLA
class I iKIRLs controls NK cell function [Ref. 1-3]. The recognition of KIR and its ligands
is a complex issue. For example, it was a consensus that 3DL1 recognizes HLA-Bw4
allotypes but not those HLA-A sharing the same epitope [Ref. 4]. However, a recent
study using HLA-A tetramers demonstrated the binding of HLA-A and 3DL1, but the
functional implication of this binding was undetermined [Ref. 5]. A full presentation of
the knowledge is out of the scope of this module.
KIR is another highly polymorphic system, like the notorious HLA system. Its
diversity is generated by both gene content (a variation in the number of genes present
on individual haplotypes) and allelic variation. The KIR gene cluster is located on
chromosome 19q13.4 (Fig. 1). The most common KIR haplotype is haplotype A, defined
by the presence of only one short tail KIR 2DS4 and a fixed number of nine KIR genes
3DL3-2DL3-2DP1-2DL1-3DP1-2DL4-3DL1-2DS4-3DL2. Haplotype B harbors more than
one short tail KIR (except some very rare haplotypes) and varies greatly in gene content.
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Some KIR gene loci exist in almost every haplotype and are called framework genes.
They are 3DL3 in the centromeric end, 3DP1 and 2DL4 in the middle and 3DL2 in the
telomeric
end
of
the
KIR
gene
cluster.
The
IPD
database
(http://www.ebi.ac.uk/ipd/kir/index.html) named 614 KIR alleles in the 15 April 2011
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release, and that number is increasing quickly. Most of the allelic variants do not change
KIR function. However, some do affect either KIR protein expression or receptor-ligand
interaction. For example, the proteins of alleles KIR2DS4*003/4/6-10/12/13, differing
from KIR2DS4*001 by a 22bp deletion in exon 5, are transcribed but not expressed on
the cell surface due to the absence of a transmembrane and cytoplasmic domain. The
frequency of these alleles is very high [Ref. 1, 6].
There is a growing body of literature that has addressed the effects of KIR and its
ligand on transplantation (both bone marrow [Ref. 7-9, 24] and solid organ [Ref. 10, 11,
25]), infectious [12] and autoimmune diseases [Ref. 13]. KIR genotyping was widely
performed in these studies.
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Figure 1. Killer Ig-like Receptors (KIRs), their Ligands and Genes
Ligand
KIR
D0,
Gene arrangement
D1,
D2
Haplotypes: A,
-S-S-
-S-S-
2DL1
-S-S-
2DL2 &
2DL3
HLA-Clys80,
Cw*02,04,etc.
B1, B2, B3, …
3DL3
2DS2
HLA-Casn80,
Cw*01,03,etc.
-S-S-
2DL2/3
HLA-Clys80,
3
2
3
2
2DL5B
Cw*02,04,etc.
-S-S-
-S-S-
2DS1
2DS3/5
Unknown
-S-S-
-S-S-
2DS2
2DP1
Unknown
-S-S-
-S-S-
2DS3
2DL1
Unknown
-S-S-
-S-S-
2DS4
Unknown
-S-S-
-S-S-
2DS5
3DP1
2DL4
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HLA-G
-S-S-
-S-S-
2DL4
Unknown
-S-S-
-S-S-
2DL5
HLA-Bw4
-S-S- -S-S-
-S-S-
3DL1
HLA-A*03,*11
-S-S- -S-S-
-S-S-
3DL2
Unknown
-S-S- -S-S-
-S-S-
3DL3
Unknown
-S-S- -S-S-
-S-S-
3DS1
3
3DL/S1
L
L
S
S
2DL5A
2DS3
2DS5
2DS1
NK cell
2DS4
3DL2
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METHODOLOGIES
KIR genotyping methods rely on molecular biology techniques, such as PCR
(polymerase chain reaction). The currently developed methods include SSP (sequencespecific primers) [Ref. 15, 16], direct sequencing [Ref. 26] or SSOP (sequence-specific
oligonucleotide probes) [Ref. 17]. Another direction of methodologies is based on single
nucleotide extension and the mass spectrometry. A research group has reported a
method that uses a matrix-assisted laser desorption/ionization time-of-flight mass
spectrometer (MALDI-TOF) [Ref. 18] to detect single nucleotide extension products of
KIR templates. This method holds promise for high-throughput testing, but needs
expensive instruments. The real time PCR platform has also been utilized to do KIR
genotyping [Ref. 23]. Real time PCR needs more expensive reagents, but could be
easily automated.
Although commercial kits are supplied with detailed protocols, most of them
require reagents and technical support. Laboratories are required to validate the assay
prior to clinical use. Alternately, the assays for KIR genotypes can be developed in the
laboratory; however, this requires extensive quality control and expertise. In the research
area, especially disease association and large-scale gene frequency studies, most
laboratories [Ref. 11-14, 19-22] used in-house-developed methods due to budget limits.
Commercial kits may be suitable for clinical laboratories when KIR genotyping becomes
a clinical practice.
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Section : Click here to link to the web based ASHI U Procedure Manual
FREQUENTLY ASKED QUESTIONS
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Question:
Why do we need mineral oil in a modern PCR machine with a heated lid?
Answer:
Even with commonly used machines, such as those listed in the protocol,
evaporation still occurs with a 10l volume in a 0.3/0.5 ml PCR tube/well. Mineral oil may
be omitted for samples of 20l or more. However, mineral oil is necessary for this
protocol.
Question:
Does any other hot start DNA polymerase work in this protocol?
Answer:
No. DNA polymerase must be AmpliTaq Gold LD because all the
experimental conditions were optimized with this enzyme. One of the hot start enzymes
was tried in the conditions established with AmpliTaq Gold, but it did not work. Other
laboratories may optimize different hot-start enzymes.
Question:
Is template DNA concentration flexible?
Answer:
Yes. We have tested the template range, and the 15ng human genomic
DNA per 10l PCR volume is at the relative low end. Usually, when the template DNA is
increased up to 3 times, the specific bands will be stronger without increasing the nonspecific bands.
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ASHI STANDARDS
The ASHI standards sited in this document are from the version noted at the top
of the page. Revisions to the ASHI standards may have taken place since the
development of this document. Laboratories and individuals should always
reference the most current ASHI approved standards.
D.2.2.4
Laboratories performing amplification of nucleic acids must:
D.2.2.4.1
Use physical and/or biochemical barriers to prevent nucleic acid
contamination (carry-over).
D.2.2.4.2
Perform pre-amplification procedures in a work area that excludes
amplified nucleic acid that has the potential to serve as a template in any other
amplification assays performed in the laboratory (e.g., PCR product, plasmids containing
HLA genes or relevant STR/VNTR sequences). Restricted traffic flow is recommended.
D.2.2.4.3
Use dedicated lab coats, gloves and disposable supplies in the
preamplification area.
D.4.1
Laboratories performing nucleic acid testing must have written criteria
or protocols for:
D.4.1.1
Accepting the validity of each molecular assay.
D.4.1.2
Preventing DNA contamination using physical and/or biochemical barriers
for assays involving amplification of templates.
D.4.6
Test systems, equipment, instruments, reagents, materials,
supplies, nucleic acid sequence databases, and clinical requirements (systems,
applications).
D.4.6.1
Test systems must be selected by the laboratory. The testing must be
performed following the manufacturer's instructions or as modified and validated by the
laboratory and in a manner that provides test results within the laboratory's stated
performance specifications for each test system.
D.4.6.2
The laboratory must define and follow criteria for those conditions that are
essential for proper storage of reagents and specimens, accurate and reliable test
system operation, and test result reporting. The criteria must be consistent with the
manufacturer's instructions, if provided. These conditions must be monitored and
documented and, if applicable, include the following:
D.4.6.2.1
Water quality
D.4.6.2.2
Temperature
D.4.6.2.2.2
Ambient temperatures must be monitored every working day.
D.4.6.2.2.3
Refrigerator and freezer temperatures must be monitored continuously
and the temperature recorded each day of testing.
D.4.6.2.2.4
Refrigerators and freezers must be monitored to ensure maintenance of
optimal temperatures for storage of each type of sample or reagent. The laboratory’s
storage and maintenance of both critical reagents and relevant transplant candidate
specimens must use an audible or centrally monitored temperature alarm system and
have an emergency plan for alternative storage.
D.4.6.2.3
Humidity
D.4.6.2.4
Protection of equipment and instruments from fluctuations and
interruptions in electrical current that adversely affect patient test results and test
reports.
D.4.6.4
Reagents, solutions, culture media, control materials, calibration
materials, and other supplies whether commercially purchased or prepared in-house
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must not be used when they have exceeded their expiration date, have deteriorated, or
are of substandard quality.
D.4.6.4.1
There must be a documented system in place for identifying which lots
and shipments of reagents were used for each assay.
D.4.6.4.2
Prior to reporting results obtained with new lots or shipments of reagents,
satisfactory performance must be verified and documented.
D.4.6.11
Laboratories performing nucleic acid testing must:
D.4.6.11.2.
Ensure that the laboratory has criteria for accepting each lot and
shipment of primers or probes.
D.4.6.11.3
Have acceptable limits of signal intensity for positive and negative results.
If these are not achieved, acceptance of the results must be justified and documented.
D.4.6.11.4
Have an independent review of the data and its interpretation.
D.4.6.11.5
Include in each electrophoretic process, controls that verify that the
specific targets can be detected.
D.4.6.11.6
If the size of a nucleic acid is a critical factor in the analysis of the data:
D.4.6.11.6.1 In each gel, include size markers that produce discrete electrophoretic
bands spanning and flanking the entire range of expected fragment sizes.
D.4.6.11.6.2 The amount of DNA loaded in each lane must be within a range that
ensures equivalent migration of DNA in all samples, including size markers.
D.4.6.11.7
Ensure that each lot and shipment of primers or probes is monitored to
confirm stability and performance of the primers or probes.
D.4.6.11.11 Ensure and document acceptable electrophoretic conditions used for
each gel electrophoresis.
D.4.6.11.13 Laboratories performing amplification-based methods must:
D.4.6.11.13.4 Ensure that thermal cycling instruments achieve the appropriate target
temperatures during cycling.
D.4.6.11.13.5 Ensure that all batches of aliquotted reagents (solutions containing one or
multiple components) utilized in the amplification assay are demonstrated to be free of
contamination.
D.4.6.11.13.6 Ensure that reagents used for primary amplification are not exposed to
post- amplification work areas.
D.4.6.11.13.8 Verify that the conditions for primer extension (e.g. polymerase type,
polymerase concentration, primer concentration, concentration of nucleotide
triphosphates) are appropriate for the template (e.g. length of sequence, GC content).
D.4.6.11.13.9 Ensure that for each set of primers, conditions that influence the
specificity or quantity of amplified product have been demonstrated to be satisfactory for
the range of samples routinely tested.
D.4.6.11.13.10 Ensure that template quantity and quality are sufficient to provide
interpretable data for a locus (or loci) or allele(s).
D.4.6.11.13.11 Ensure that the amount of amplification template in each amplification
reaction is in an acceptable range.
D.4.6.11.13.12 Define and document the specificity and sequence of primer targets. The
genetic designation (e.g. locus) of the target amplified by each set of primers must be
defined and documented. For each locus analyzed, the laboratory must have
documentation that includes the chromosome location, the approximate number of
alleles, and the distinguishing characteristics (e.g. sizes, sequences) of the alleles that
are amplified.
D.4.6.11.15 Laboratories performing SSP methods must:
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D.4.6.11.15.1 Ensure that an internal control, that produces a product distinguishable
from the specific typing product, is included for each primer mixture.
D.4.6.11.15.2 Ensure that the amplification conditions are acceptable for the primers
used.
D.4.6.11.15.3 Include a negative (no nucleic acid) or contamination control in each
assay.
D.4.6.11.15.4 Ensure that primers used produce adequate amounts of amplification
products to be visualized.
D.4.9.2
For thermal cycling instruments, the appropriate target temperatures
must be achieved. Accuracy of these temperatures must be verified and documented at
least every six months.
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References:
1. Marsh SG, Parham P, Dupont B,et al. Killer-cell immunoglobulin-like receptor
(KIR) nomenclature report, 2002. Hum Immunol 2003: 64: 648-54.
2. Norman PJ, Parham P. Complex interactions: the immunogenetics of human
leukocyte antigen and killer cell immunoglobulin-like receptors. Semin Hematol.
2005;42:65-75.
3. Middleton D, Williams F, Halfpenny IA. KIR genes. Transpl Immunol.
2005;14:135-42.
4. Vilches C, Parham P. KIR: diverse, rapidly evolving receptors of innate and
adaptive immunity. Annu Rev Immunol. 2002;20:217-51.
5. Thananchai H, Gillespie G, Martin MP, et al., Allele-specific and peptidedependent interactions between KIR3DL1 and HLA-A and HLA-B. J Immunol.
2007;178:33-7.
6. Hsu KC, Chida S, Geraghty DE, Dupont B. The killer cell immunoglobulin-like
receptor (KIR) genomic region: gene-order, haplotypes and allelic polymorphism.
Immunol Rev. 2002; 190:40-52.
7. Ruggeri L, Mancusi A, Capanni M, et al. Donor natural killer cell allorecognition of
missing self in haploidentical hematopoietic transplantation for acute myeloid
leukemia: challenging its predictive value. Blood. 2007;110:433-40.
8. Sun JY, Dagis A, Gaidulis L, et al. Detrimental effect of natural killer cell
alloreactivity in T-replete hematopoietic cell transplantation (HCT) for leukemia
patients. Biol Blood Marrow Transplant. 2007;13:197-205.
9. Morishima Y, Yabe T, Matsuo K, et al.; Japan Marrow Donor Program. Effects of
HLA allele and killer immunoglobulin-like receptor ligand matching on clinical
outcome in leukemia patients undergoing transplantation with T-cell-replete
marrow from an unrelated donor. Biol Blood Marrow Transplant. 2007;13:315-28.
10. Cirocco RE, Mathew JM, Burke GW 3rd, Esquenazi V, Miller J. Killer cell
immunoglobulin-like receptor polymorphisms in HLA-identical kidney transplant
recipients: lack of 2DL2 and 2DS2 may be associated with poor graft function.
Tissue Antigens. 2007;69 Suppl 1:123-4.
11. Leung W, Handgretinger R, Iyengar R, et al. Inhibitory KIR-HLA receptor-ligand
mismatch in autologous haematopoietic stem cell transplantation for solid tumour
and lymphoma. Br J Cancer. 2007;97:539-42.
12. Martin MP, Qi Y, Gao X, et al. Innate partnership of HLA-B and KIR3DL1
subtypes against HIV-1. Nat Genet. 2007 Jun;39(6):733-40.
13. Karlsen TH, Boberg KM, Olsson M, et al. Particular genetic variants of ligands for
natural killer cell receptors may contribute to the HLA associated risk of primary
sclerosing cholangitis. J Hepatol. 2007;46:899-906.
14. Schellekens J, Rozemuller EH, Petersen EJ, et al. Patients benefit from the
addition of KIR repertoire data to the donor selection procedure for unrelated
haematopoietic stem cell transplantation. Mol Immunol. 2008;45:981-9.
15. Gomez-Lozano N, Vilches C. Genotyping of human killer-cell immunoglobulinlike receptor genes by polymerase chain reaction with sequence-specific primers:
an update. Tissue Antigens 2002: 59: 184-93.
16. Sun JY, Gaidulis L, Miller MM et al. Development of a multiplex PCR-SSP
method for Killer-cell immunoglobulin-like receptor genotyping. Tissue Antigens
2004; 64: 462-468.
17. Halfpenny IA, Middleton D, Barnett YA, Williams F. Investigation of killer cell
immunoglobulin-like receptor gene diversity: IV. KIR3DL1/S1. Hum Immunol.
2004;65:602-12.
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18. Houtchens KA, Nichols RJ, Ladner MB, et al. High-throughput killer cell
immunoglobulin-like receptor genotyping by MALDI-TOF mass spectrometry with
discovery of novel alleles. Immunogenetics. 2007;59:525-37.
19. Poggi A, Negrini S, Zocchi MR, et al. Patients with paroxysmal nocturnal
hemoglobinuria have a high frequency of peripheral-blood T cells expressing
activating isoforms of inhibiting superfamily receptors. Blood. 2005;106:2399408.
20. Norman PJ, Abi-Rached L, Gendzekhadze K, et al. Unusual selection on the
KIR3DL1/S1 natural killer cell receptor in Africans. Nat Genet. 2007;39:1092-9.
21. Yawata M, Yawata N, Draghi M, Little AM, Partheniou F, Parham P. Roles for
HLA and KIR polymorphisms in natural killer cell repertoire selection and
modulation of effector function. J Exp Med. 2006;203:633-45.
22. Hou LH, Steiner NK, Chen M, Belle I, Ng J, Hurley CK. KIR2DL1 allelic diversity:
four new alleles characterized in a bone marrow transplant population and three
families. Tissue Antigens. 2007;69:250-4.
23. Hong HA, Loubser AS, de Assis Rosa D, et al. Killer-cell immunoglobulin-like
receptor genotyping and HLA killer-cell immunoglobulin-like receptor-ligand
identification by real-time polymerase chain reaction. Tissue Antigens. 2011
78:185-94.
24. Cooley S, Weisdorf DJ, Guethlein LA, et al. 2010, Donor selection for natural
killer cell receptor genes leads to superior survival after unrelated transplantation
for acute myelogenous leukemia, Blood. 2010 116: 2411-9.
25. van Bergen J, Thompson A, Haasnoot GW, et al. 2011, KIR-ligand mismatches
are associated with reduced long-term graft survival in HLA-compatible kidney
transplantation, Am J Transplant 11, 1959-1964.
26. Hou L, Chen M, Steiner N, et al. Killer cell immunoglobulin-like receptors (KIR)
typing by DNA sequencing. Methods Mol Biol. 2012 882: 431-68.
27. Kulkarni S, Martin MP, Carrington M. KIR genotyping by multiplex PCR-SSP.
Methods Mol Biol. 2010 612: 365-75.
28. Abalos AT, Eggers R, Hogan M, et al. Design and validation of a multiplex
specific primer-directed polymerase chain reaction assay for killer-cell
immunoglobulin-like receptor genetic profiling. Tissue Antigens. 2011 77:143-8.
Recommended Reading:
1. Carrington, M. and Norman, P. The KIR Gene Cluster. Bethesda (MD): National
Library of Medicine (US), National Center for Biotechnology Information; 2003.
(Available on www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowTOC&rid=
mono_003.TOC&depth=2).
2. IPD-KIR Database: http://www.ebi.ac.uk/ipd/kir/introduction.html
3. Parham P, Norman PJ, Abi-Rached L, Guethlein LA. Human-specific evolution of
killer cell immunoglobulin-like receptor recognition of major histocompatibility
complex class I molecules. Philos Trans R Soc Lond B Biol Sci. 2012 19
367(1590): 800-11.
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