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Projects
Sequence based typing of HLA-DQA1:
A study to determine the level
of molecular heterogeneity of HLA-DQA1 and its haplotypes.
Coordinator: Dr C Voorter, Tissue Typing Laboratory, University
Hospital Maastricht, NL
Project proposal within the Sequence Based Typing workshop
The project will be a collaboration between different laboratories. All data will be submitted to the IHWG database.
Those laboratories already having experience with SBT of DQA1 are invited
to participate in the project. Also laboratories with experience in SBT
of other HLA genes, but not DQA1, are welcome to join. Laboratories with
interesting samples (i.e. peculiar SSP or SSO patterns for DQA1 or rare
DQB1 alleles) and not prepared to perform SBT of DQA1 are invited to
make these samples available for the project.
Quality control samples have to be tested to assure the quality of typing
results from the different participating laboratories. One of the possibilities
is the UCLA exchange.
The aims of the project comprises the following:
- Selection of the most efficient and validated protocol:
The available direct sequencing protocols for DQA1 exons 1-4 will be
exchanged and evaluated. The selected protocol will be validated by
the use of reference samples.
- Polymorphism: filling in gaps that exist in currently described
alleles.
To enable full length exon sequencing, primers for amplification
and sequencing will be developed and evaluated. It is the intention to
complete the gaps in the exon 2 sequences of DQA1 in the IMGT database
and to determine the additional polymorphism beyond exon 2.
- Sequence determination of HLA-DQA1 alleles in samples with:
3a. Unusual SSO or SSP profiles for DQA1 typing
3b. Unusual DRB1 - DQB1 associations
The intention is that these samples are gathered by sample exchange
with workshop participants. During this workshop high resolution
typing of DQA1 will be performed
of these samples. They can be made available by the participating labs,
but also by non-participating laboratories. Laboratories, not having
the possibility
to perform SBT of DQA1, are invited to send interesting samples (i.e. peculiar
SSP or SSO patterns for DQA1 or rare DQB1 alleles) to the project coordinator
in order to be typed by SBT. If necessary a selection of the samples will
be made.
- Comparison of the sequences of reported alleles from different ethnic
groups, to study the presence of intra-allelic polymorphism. Again these
samples will be gathered by exchange. Both participating and non-participating
laboratories are invited to send samples of different ethnic origin with
known HLA-DQA1 high resolution typing to the project coordinator. These
samples will be used to resolve the intron sequences.
Those laboratories interested in participation can send a lettre or
e-mail to the coordinator, giving their name, affiliation and information
on the SBT experience.
April, 2005, Update
Progress report on Workshop project
Typing of DQA1 by sequencing has been a challenge due to a 3 nucleotide deletion in exon 2 in half of the alleles. Furthermore, 19 of the 28 alleles cannot be identified on basis of exon 2 alone, but additional information is needed from the other exons. Most protocols for SBT of DQA1 described sofar are based on sequencing exon 2 or exons 2 and 3, using primers located in the exons. They do not reveal complete sequence information for all exons. By elucidating the intron sequences, the Tissue Typing Laboratory of Maastricht was able to develop a sequencing strategy for exons 1 to 4, obtaining the complete coding sequence. Since the participants in the project had no experience with SBT of DQA1, the protocol of Maastricht was used.
The protocol includes separate amplification of exons 1 to 4 with primers located in the 5' UT region and the introns. Standard heterozygous sequencing is performed in the forward and/or reverse direction. The combined sequences are used for automatic allele assignment.
In the framework of the workshop project the protocol was sent to the participants, together with the list of amplification and sequencing primers, located in the adjacent introns or 5' untranslated region. Also control DNA samples were sent to validate the method. Furthermore, the participants will type a control population from their country or any other interesting population to obtain HLA-DQA1 frequencies for these populations.
Contact:
Dr C Voorter
Tissue Typing Laboratory,
University Hospital Maastricht,
P.O. Box 5800,
6202 AZ Maastricht.
E-mail cvoo@lwee.azm.nl;
phone: +31 43 3874680,
fax: +31 43 3874678.
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