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Somatic Mutations in HLA Genes
Anajane Smith
Clinical Immunogenetics Laboratory
Seattle Cancer Care Alliance
What are somatic mutations?
Genome wide studies detect somatic mutations
Seattle experience in HSCT patients (2000-2008)
Implications for HLA typing of patients with a malignant
disease or a myelodysplastic syndrome
Somatic Mutations
Extensive publications document clonal gene mutations resulting
from chromosome rearrangements among patients with malignant
hematopoietic cell diseases.
Cytogenetic analysis is employed to analyze chromosome
rearrangements that can be of prognostic significance in potential
recipients of a hematopoietic stem cell transplant.
Minimal residual disease testing targets clonal somatic mutations in
disease specific genes.
BCR-ABL fusion gene in CML.
FLT3 tyrosine kinase gene in AML.
Clinical chimerism testing in HSCT patients may detect mutations
that result in “missing” or “extra” VNTR/STR alleles in disease
cells.
Tumor specific changes in HLA expression have been documented
extensively in solid tumors.
Genome Wide Studies of Somatic
Mutations in Hematopoietic Diseases
Studies of patients with various hematopoietic stem cell diseases have
revealed previously undetected somatic mutations that may be of
prognostic significance.
The recent availability of microarray technology has allowed high
resolution genome-wide genotyping through the analysis of singlenucleotide polymorphisms (SNPs) in panels of patients with
hematologic diseases.
Whole genome re-sequencing of disease cells in a cytogenetically
normal AML genome revealed 8 previously unknown somatic
mutations as well as identifying two well-known AML-associated
mutations.
A study reported in an EFI 2009 abstract described LOH of HLA loci
detected in 29% of AML patients in relapse post HSCT.
Terminology
Somatic mutation is a genetic change in a subset of cells (disease population)
that is not present in germline cells.
SNPs: Single Nucleotide Polymorphisms are located throughout the human
chromosome and are usually bi-allelic polymorphisms.
Indel: An insertion or deletion polymorphism.
CN: Copy Number change is the result of a deletion or amplification (such as a
duplication) of a genetic locus.
UPD: UniParental Disomy is the result of duplication of one parental
chromosome during mitosis and loss of the other parental chromosome,
resulting in homozygosity without CN change.
LOH: Loss of Heterozygosity is the result of a genetic change that results in
homozygosity at a genetic locus and/or loss of gene expression.
Hemizygous: Genotype that has lost heterozygosity.
Seattle Experience with Somatic
Mutations in HLA Genes
The growing number of anecdotal case studies reported by
different hematopoietic stem cell transplant centers, including
ours, suggests that somatic mutations in HLA genes in
hematopoietic disease cell clones are infrequent, but not rare
events.
We present here a collection of 15 cases of somatic mutations in
HLA genes detected in patients diagnosed with a variety of
hematologic diseases (AML, ALL, MDS, NHL and CLL) in a
clinical HLA laboratory supporting a hematopoietic stem cell
transplant program.
Materials and Methods
Samples tested:
Among 14 patients, mutations in HLA genes were detected in
DNA isolated from peripheral blood mononuclear cells (PBMC)
during routine clinical HLA typing prior to HSCT.
In one patient, HLA typing was hemizygous in DNA isolated from
post-transplant tumor cells with extensive loss of heterozygosity in
VNTR/STR loci.
DNA was isolated from buccal swabs for germline typing in most
cases.
HLA typing methods:
Intermediate resolution typing by reverse SSOP.
Allele level typing by DNA sequencing.
Novel HLA Sequences in Disease Cells
AML: A*0201 with a single base insertion in exon 3.
ALL: A*0301 with a SNP TGG>TGA at codon 147.
Mutation generated a stop codon.
Loss of A3 expression demonstrated by flow cytometry in a tumor cell population.
AML: B*1501 with a SNP GCG>GTG at codon 182.
CLL: B*0702 with a SNP GCC>GTC at codon 117.
Initially typed by rSSOP as B*0717.
DNA sequencing found novel C>T at N422.
HLA matched sibling and patient buccal DNA typed as B*0702.
NHL: B*3501/42 allele with a 28N insertion in exon 3.
HLA-B locus specific
sequencing exhibited
the pattern of an
Indel polymorphism.
HLA-B*35 group
specific primers also
exhibited the Indel
pattern.
Buccal Swab DNA with B*35 Primers
Hemizygous HLA Typing
10 patients had molecular HLA typing consistent with loss of
one full HLA haplotype (n=9) or at least one HLA locus (n=1)
in disease cell populations.
HLA typing by rSSOP
Strong probe reactions for the HLA alleles on one haplotype.
Weak or absent probe reactions for the alleles of the other haplotype.
Sequencing of HLA-A, B, C and/or DRB1 genes revealed
sequences that were virtually homozygous, with very low or
absent heterozygous peaks.
Buccal swab samples provided accurate HLA typing of patient
germline DNA.
Haplotype Loss Case Examples
Patient diagnosed with ALL.
Post-transplant (d72) CD45/19 tumor cells exhibited hemizygous patterns
in over 50% of the VNTR/STR loci analyzed.
HLA-A-DQB1 loci were hemizygous in tumor cells.
Patient diagnosed with AML.
Pre-transplant DNA from peripheral blood mononuclear cells (PBMC) had
predominantly hemizygous rSSOP patterns, HLA-A through DQB1.
Potential twin donor gave clear heterozygous typing at all HLA loci.
15 STR loci showed identical patterns in both patient and twin with no
evidence of Loss of Heterozygosity in the STR profile of the patient.
Buccal swab DNA from the patient gave HLA typing concordant with the
twin.
Patient Diagnosed With MDS Typed in 2000
Patient typing 2000:
Outside lab:
A*01,11-B*51,52-Cw*03,1202-DRB1*0701,1501-DQB1*02,06.
CIL rSSOP:
A*01, (+ weak A*11 probes)-B*51,52-DRB1*07,15-DQB1*02,06.
CIL SSP:
A*01,11-Cw*03, 1202.
CIL SBT:
A*0101 homozygous with 3 different sets of sequencing reagents.
Patient buccal DNA sequencing clearly identified A*0101, 1101, with no evidence of A*11
allele dropout.
Patient typing 2008 (new DNA isolated from cryopreserved PBMC).
rSSOP: A*01, (+ weak A*11 probes)-B*51, 52-DRB1*07, 15-DQB1*02,06.
SBT: A*0101, with very weak or absent peaks at expected positions for A*11.
HLA-B and HLA-C sequencing had no evidence of hemizygosity.
Probes for A*11 were weak.
Cutoff values were adjusted
to give the expected A*01,
11 result.
Probes for A*01 gave strong
clear reactions.
Heterozygous
peaks for A*11
were weak or
absent.
HLA-B and HLAC (right panel)
sequences
exhibited no
evidence of allele
loss.
2008 Case: Patient Diagnosed with MDS
Typing discrepancy between DRB1 rSSOP and SBT detected a
the recent case (2008) of HLA haplotype loss.
rSSOP: DRB1*01, 13
SBT: DRB1*13 only (very weak or absent peaks for DR1).
Review of rSSOP revealed weak probe reactions for the alleles of
one haplotype and strong reactions for probes consistent with the
other haplotype.
SBT of HLA-A, B, C showed a virtually hemizygous pattern at all
three loci.
DNA from a buccal swab provided accurate typing consistent with
a family study and an HLA identical sibling.
Detection of Somatic HLA Mutations
Year
2000
2000
2001
2001
2003
2004
2004
2004
2005
2006
2006
2007
2007
2008
2008
Allele
A*02 indel
Hemizygous
HLA-A
A*03 SNP
Haplotype
Haplotype
Haplotype
Haplotype
B*15 SNP
Haplotype
Haplotype
B*07 SNP
Haplotype
Haplotype
B*35 indel
Haplotype
Disease
AML
MDS
ALL
ALL
MDS
NHL
ALL
AML
AML
AML
CLL
AML
AML
NHL
MDS
Summary
Mutation
AML
ALL
MDS
NHL
CLL
Total
Allele Sequence
2
1
0
1
1
5
Haplotype/locus loss
4
2
3
1
0
10
Total
6
3
3
2
1
15
Frequency estimate of somatic HLA gene mutations
15/5000 = approximately 0.3%.
This is a minimal frequency estimate due to variability in the presence
of disease cells in patient peripheral blood specimens submitted for
HLA typing prior to transplant.
Somatic mutations in HLA genes may not be detected due to the
typing method and/or the absence of tumor cells in the specimen.
HLA Somatic Mutations
10 patients had loss of at least one HLA locus due to either:
Deletions with copy number changes (CN).
Uniparental disomy (UPD) with no CN change.
5 patients had HLA allele polymorphism
2 with SNPs resulting in novel HLA antigen expressed on tumor cells.
3 with HLA allele polymorphisms known or highly likely to have resulted in
loss of cell surface expression.
SNP resulting in a stop codon in an A*0301 allele.
Indel polymorphisms in A*0201 and B*3501 alleles.
Overall, 13 of 15 patient showed loss of heterozygosity (LOH).
UPD or deletion of an HLA locus or full haplotype.
Allele polymorphisms consistent with loss of HLA antigen expression.
Significance of HLA Somatic Mutations?
Detection of mutations that result in LOH in HLA genes
across multiple hematopoietic stem cell diseases might
suggest a prognostic significance?
Mechanism for tumor escape from immune surveillance.
Alternatively, these might be random mutations in “fragile”
cancer genomes.
SNP genome wide analysis in 119 patients with “low-risk
myelodysplastic syndromes” (Ref: Mohamedali).
Chromosomes 4, 1 and 6 had the most frequent regions of
UPD.
Chromosomes 6, 19, 1, 12 had the most frequent CN changes.
Implications for Clinical HLA Typing
Novel HLA sequences in patients with a hematologic diseases
or a myelodysplastic syndrome should be confirmed in DNA
from disease-free cells.
HLA homozygosity not confirmed by family study in patients
should be carefully evaluated to assure accuracy, especially
when starting an unrelated donor search.
References
A mutated HLA-A2 molecule recognized by autologous cytotoxic T lymphocytes on a human renal
cell carcinoma. Brandle D et al. J.Exp. Med. 1996: 183: 2501.
DNA sequencing-based HLA typing detects a B-cell ALL blast-specific mutation in HLA-A*2402
resulting in loss of HLA allele expression. Sayer DC et al. Leukemia 2004: 18: 174.
DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Ley TJ et al.
Nature 2008: 456: 66.
Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP
arrays. Pfeifer D et al. Blood 2007: 109: 1202.
Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Mulligan CG et al.
Nature 2007: 446: 758.
Prevalence and prognostic significance of allelic imbalance by single-nucleotide polymorphism
analysis in low-risk myelodysplastic syndromes. Mohamedali A et al. Blood 2007: 110: 3365.
HLA class I expression, tumor escape and cancer progression. Current Cancer Therapy Reviews
2008: 4: 105.
Genomic loss of mismatched HLA in leukemia is a major mechanism of in vivo escape from T cell
immunosurveillance following haploidentical hematopoietic stem cell transplantation. Luca Vago
et al. Tissue Antigens 2009: 73:385.