HLA Diagnostic Sequencing ± Conception, Application and Automation
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Transfusions- und Tranplantationsmedizin Redaktion: Mariam Klouche HLA Diagnostic Sequencing ± Conception, Application and Automation Diagnostische HLA-Sequenzierung ± Konzept, Anwendung und Automatisierung R. Blasczyk Summary: Sequencing gives the most reliable and ac- 2-digit HLA typing, a gene-specific amplification curate information of the DNA sequence of a gene and approach for sequencing of both alleles simultaneously is therefore of particular interest for fully characterising should be applied. Thus, the progress made in diagnos- the genetic complexity and allelic diversity of the HLA tic HLA sequencing now offers a real alternative to the genes. The full complexity of allelic diversity in HLA conventional molecular typing techniques, and will in- class Iand class IIgenes,as well as recent improve- creasingly replace them for the patients' benefit. ments in the convenience and quality of automated se- quencing, makes sequencing the method of choice for Keywords: HLA analysis; sequencing; automation. HLA low- and high-resolution typing. HLA typing by means of sequencing should be done whenever the Zusammenfassung: Die Sequenzierung liefert die HLA type of an individual is needed. The current devel- zuverlaÈssigste und genaueste Information uÈber die opments with regard to HLA-sequencing systems, se- DNA-Sequenz eines Gens und ist daher fuÈr die Charak- quencing instruments and software solutions for data in- terisierung der genetischen KomplexitaÈt und allelischen terpretation have made sequencing equally simple and Vielfalt der HLA-Gene von besonderem Interesse. Die robust, providing formats for each level of throughput hohe KomplexitaÈt der allelischen DiversitaÈt der HLA and automation in order to match each laboratory's in- Klasse Iund IIGeneund die aktuellen Verbesserungen dividual requirements. Modern sequencing is based on in der Anwendung und QualitaÈt der automatischen Se- direct sequencing of PCR products from genomic DNA quenzierung machen die Sequenzierung zur Methode with the help of fluorescent-labelling methods using der Wahl fuÈr niedrig- und hochaufloÈsende HLA-Typi- dye terminator cycle sequencing chemistries with spe- sierungen. Eine Sequenzierung sollte daher immer in cialised DNA polymerases and automated capillary se- Betracht gezogen werden, wenn eine HLA-Typisierung quencers. The presently available HLA sequencing sys- erforderlich ist. Die modernen Entwicklungen der HLA- tems differ mainly in regard to their PCR amplification Sequenziersysteme, SequenziergeraÈte und Software- strategies and software solutions for allele identifica- loÈsungen zur Dateninterpretation haben die Sequenzie- tion. rung gleichermaûen einfach und robust werden lassen, As the diversity of all HLA loci developed through ex- bieten jede gewuÈnschte Stufe fuÈr Hochdurchsatz und tensive sequence exchanges, resulting in chimeric struc- Automatisierung und koÈnnen die individuellen Anfor- tures of each HLA allele, the allele-specific sequencing derungen jedes einzelnen Labors erfuÈllen. Die moderne approach is the most successful for high-resolution typ- Sequenzierung beruht auf der direkten Sequenzierung ing (4 digits), as it is capable of determining the cis von PCR-Produkten aus genomischer DNA unter Ver- linkage of sequence motifs. This is achieved by apply- wendung von Fluoreszenzmarkierungen mit 4-Farben- ing multiple group-specific amplifications in parallel, Terminatoren, Zyklus-Sequenzierungen mit speziali- thus splitting the alleles into individual groups. This ap- sierten DNA-Polymerasen und automatischen Kapillar- proach increases the capacity for cis linkage definition sequenzierern. Die zur Zeit verfuÈgbaren HLA-Se- and in turn decreases the number of ambiguities. When quenziersysteme unterscheiden sich hauptsaÈchlich sequencing shall be established as the only method for hinsichtlich ihrer PCR-Amplifikationsstrategien und 4-digit HLA typing, these group-specific approaches der Software-LoÈsungen fuÈr die Allelidentifizierung. Da must be used. When sequencing shall be used also for die DiversitaÈt aller HLA-Gene durch intensive segmen- tale Rekombinationen entstanden ist, stellt jedes HLA- Allel eine ChimaÈre anderer HLA-Allele dar. Die Allel- Institut fuÈr Transfusionsmedizin, Medizinische Hochschule spezifische Sequenzierung ist daher fuÈr eine hochaufloÈ- Hannover Correspondence: Prof. Dr. Rainer Blasczyk, Institut fuÈr Transfu- sende (4-stellige) Typisierung am besten geeignet, da sionsmedizin, Medizinische Hochschule Hannover, Carl-Neuberg- sie die cis-Kopplung der Sequenzmotive erkennt. Eine Str. 1, 30625 Hannover, Germany. Allel-spezifische Sequenzierung wird erreicht, indem Tel.: +49 51 15 32 67 00 Fax: +49 51 15 32 20 79 multiple Gruppen-spezifische Amplifikationen parallel E-mail: [email protected] durchgefuÈhrt werden und dadurch die beiden Allele ã 2003 Blackwell Verlag, Berlin J Lab Med 2003; 27 (9/10): 359±368 359 R. Blasczyk: Diagnostic Sequencing ± Conception, Application and Automation einer Probe in zwei Gruppen getrennt werden. Dieser are routinely used for clinical purposes [6±8]. With the Ansatz steigert die FaÈhigkeit eines Sequenziersystems discovery of the polymerase chain reaction (PCR), the die cis-Kopplungen von Sequenzmotiven zu definieren polymorphism of the MHC genes could be studied di- erheblich und minimiert auf diese Weise die Anzahl un- rectly on the nucleotide level. With the increasing avail- eindeutiger Sequenzierergebnisse. Wenn die Sequenzie- ability of sequenced alleles, the development of PCR- rung als alleinige Methode fuÈr 4-stellige HLA-Typisie- based typing techniques in the late 1980s and the 1990s rungen etabliert werden soll, muÈssen Gruppen- increasingly replaced conventional typing methods. spezifische Amplifikationen verwendet werden. Wenn The PCR-based methods for HLA class II genes have die Sequenzierung auch fuÈr 2-stellige HLA-Typisierun- meanwhile completely superseded serological typing gen eingesetzt werden soll, koÈnnen Genort-spezifische and MLC. For HLA class I, a few laboratories are still Amplifikationen zur simultanen Sequenzierung beider content with the continuing use of serology, but as the Allele verwendet werden. Die Fortschritte der diagno- beneficial effects of PCR-based typing approaches have stischen Sequenzierung bieten daher bereits jetzt eine been clearly demonstrated, the latter are now also con- wirkliche Alternative zu konventionellen molekularen sidered as the standard typing technique for HLA Typisierungstechniken und werden diese zum Wohle class I. des Patienten zunehmend ersetzen. SchluÈsselwoÈrter: HLA-Analyse; Sequenzierung; Nature of HLA diversity Automatisierung. The PCR technology has made it possible to investigate fragments of genes and has emerged as one of the most HLA-typing methods important molecular techniques for HLA typing. As the polymorphism of HLA genes is mainly restricted to ith respect to the clinical relevance and the only a few exons, the PCR-based methods for HLA- Wextensive polymorphism of HLA antigens, ad- typing purposes focus on only small fragments of HLA equate typing methods are extremely important. Early genes. These are mainly exon 2 in HLA class II genes work on the definition of HLA antigens was aggravated and exons 2 and 3 in HLA class Igenes. by the poor reproducibility of the leukocyte agglutina- The PCR-based diagnostics of the polymorphic HLA tion test. The replacement of leukocyte agglutination by system requires the consideration of some general rules lymphocytotoxicity tests in 1964 [1] and the systematic concerning the polymorphic structure of HLA genes. analysis of antibodies formed in pregnancy have The diversity of HLA genes at the antigen-presenting brought substantial progress in clinically applied HLA sites has evolved on the one hand by the gradual accu- typing. In the 1970s, homozygous typing cells were mulation of point mutations during successive ancestral used in the mixed lymphocyte culture (MLC) to define species, and on the other hand by gene conversion the polymorphism at the HLA-D locus, which enabled events or recombinations [9, 10]. The last mechanism the discrimination of 26 antigens named HLA-Dw1- appears to be the most important factor for the enor- Dw26 [2]. In the late 1970s, sera obtained from multi- mous diversification of HLA genes. This has conse- parous women, containing HLA antibodies, showed quences for their polymorphic structure. When scruti- similar specificities as the MLC for HLA-D typing. The nising the sequences of the different alleles per locus, it use of these sera permitted the definition of HLA-DR becomes apparent that the allelic diversity is not char- (D related) and later also the identification of a second acterised by allele-specific point mutations but by dif- HLA class II locus, which was named HLA-DQ [3]. ferent combinations of sequence motifs from a common The third HLA class II locus, HLA-DP, was detected in pool of different motifs. This patchwork structure of al- the late 1970s as a weak MLC locus, the polymorphism lelic sequences is reconciled with the polymorphism of which was defined by a secondary MLC or primed being generated by segmental sequence exchanges. lymphocyte test (PLT). The application of the PLT Thus, most of the alleles can be considered as chimeras allowed the discrimination of further six antigens: formed from various segments of other alleles. In most HLA-DPw1-DPw6 [4, 5]. Of note, HLA-DP could not cases, newly identified alleles are characterised by a be defined by means of the serological