1. HLA Allelic and Genotypic Ambiguity Reduction in ImmPort
Paula Guidry, Ph.D.
BISC Team – UT Southwestern
4/22/2017

2. HLA Typing Platforms and Ambiguity
SSOP – single-stranded oligonucleotide probe
SSP – sequence-specific priming
SBT – sequence-based typing
SSCP – single-stranded conformation polymorphism
Allelic Ambiguity
Typing methodology cannot distinguish between a set of alleles
Polymorphisms that distinguish these alleles are not interrogated by the method
Outside of exon 2 (class II) or exons 2 and 3 (class I)
Sections of these exons not covered by the probes/primers
Genotypic Ambiguity
Inability to determine phase in heterozygous samples
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3. Ambiguity in HLA Typing Data
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4. Ambiguity in HLA Typing Data
Allelic Ambiguity Reduction
Genotypic Ambiguity Reduction
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5. Ambiguity Reduction Principles
CWD Alleles
Common and Well-Documented (CWD) HLA Alleles
Allele has an estimated frequency of at least in any reference population
Accurate frequency estimates require an allele to be observed 3+ times
Studies with 1500 unrelated individuals (2n = 3000 chromosomes) can only accurately detect allele frequencies ≥ 0.001 OR
Allele has been detected unambiguously in at least three unrelated individuals but gene frequencies have not been estimated accurately
Rare HLA Alleles
Alleles that are not CWD
Cano et al (2007). Common and Well-Documented HLA Alleles. Human Immunology 68:
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6. Ambiguity Reduction Principles
Regional CWD
Meta-Analysis to Determine Worldwide HLA Allele Frequencies
Data sources
13th IHW Anthropology/Human Genetic Diversity component
12th IHW Anthropology component
Literature
8 journals
Every issue from 1990 – 2007
4 studies from other journals
AlleleFrequencies.net
≥ 20 individuals per population sample
497 population samples from around the world
Total of 66,800 individuals
Solberg et al (2008). Balancing Selection and Heterogeneity Across the Classical Human Leukocyte Antigen Loci: A Meta-Analytic Review of 497 Population Studies. Human Immunology 69:
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7. Ambiguity Reduction Principles
Regional CWD
Reg-CWD HLA Alleles
Allele is common and well-documented in a particular world region*
OTH – significantly admixed populations
* Ignoring the past 1000 years of human migration
Reg-Rare HLA Alleles
Not reg-CWD
Not detected in other world regions
Neither Reg-CWD nor Reg-Rare
Not reg-CWD
Detected in other world regions
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8. HLA Allelic Ambiguity Reduction Heuristic
Steve Mack, Glenys Thomson,
Rich Single

9. Allelic Ambiguity Reduction

10. Allelic Ambiguity Reduction
Phase 1
Elimination of less-probable alleles by CWD status
Are any of the alleles reg-CWD?
Are any of the alleles CWD?
Are any of the alleles reg-Rare? no no
yes
yes
yes
Eliminate others
Eliminate others
Eliminate others
Possible results: single allele call or multiple allele possibilities

11. Allelic Ambiguity Reduction
Phase 2
Reduction of multiple alleles to G- or P-codes
Do any alleles belong to a common G-code?
Do any alleles or G-codes belong to a common P-code?
Do the alleles contain any G- or P-codes? no
yes
yes
yes
Combine to G-code
Combine to P-code
Eliminate rare alleles
G-code: Identical nucleotides across ex2 or ex2/3
P-code: Identical proteins encoded by ex2 or ex2/3
Possible results: single G- or P- call, multiple G- or P- calls or multiple alleles not related by G- or P- code
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12. Allelic Ambiguity Reduction
Phase 3
Reduction of alleles in the same family by frequency
Are the alleles part of the same allele family?
Is the ratio of MRWAFs* for any pair of these alleles > ?
yes no
Keep the allele/code with the lowest 2nd field (third and fourth digits)
yes no
yes
Are there still multiple alleles in the set?
User determines most likely allele
Eliminate the lower frequency alleles
MRWAF – is there a big difference in the regional frequencies of any of the possible alleles?; if so, eliminate those that are 9.5 times less frequent than any of the other alleles
*MRWAF is the mean regional weighted allele frequency:
mean frequency for an allele in a given region, weighted by sample sizes (2n)
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13. Outcome of HLA Allelic Ambiguity Reduction

14. HLA Genotypic Ambiguity Reduction Heuristic
Steve Mack, Glenys Thomson,
Rich Single

15. Genotypic Ambiguity Reduction

16. Genotypic Ambiguity Reduction
Phase 1
Reduction of sub-genotypes by CWD status
Are any sub-genotypes reg-CWD/reg-CWD?
Are any sub-genotypes reg-CWD/CWD or CWD/CWD?
Are any G/G, P/P, G/P, G/reg-CWD, G/CWD, P/reg-CWD, P/CWD? no no no
yes
yes
yes
Eliminate others
Eliminate others
Eliminate others
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17. Genotypic Ambiguity Reduction
Phase 1, continued
Reduction of sub-genotypes by CWD status
Are any reg-CWD/reg-Rare, CWD/reg-Rare, G/reg-Rare or P/reg-Rare?
Are any sub-genotypes reg-Rare/reg-Rare? no
yes
yes
Eliminate others
Eliminate others
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18. Genotypic Ambiguity Reduction
Phase 2
Reduction of sub-genotypes by zygosity and allele numbers
Are any of the sub-genotypes homozygous?
Keep the sub-genotype with the lowest sum of the 2nd field (third and fourth digits) no
yes
Keep homozygous sub-genotypes and sub-genotypes containing these alleles
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19. Outcome of HLA Allelic and Genotypic Ambiguity Reduction
Allelic Ambiguity Reduction
Genotypic Ambiguity Reduction
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20. ImmPort HLA Ambiguity Reduction Tool
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21. 4/22/2017

24. Example HLA Typing Results File Ready for Ambiguity Reduction in ImmPort
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25. 4/22/2017

27. 4/22/2017

28. Ambiguity Reduction Input File
Allelic Ambiguity Reduction Output
Genotypic Ambiguity Reduction Output

29. Acknowledgements Steven J. Mack , Ph.D.(CHORI)
Glenys Thomson, Ph.D. (Berkeley)
Richard Single, Ph.D. (UVM)
Nishanth Marthandan
Thomas Smith, Ph.D.
Patrick Dunn, Ph.D.
John Campbell
Jeff Wiser
Richard Scheuermann, Ph.D.
David Karp, M.D., Ph.D.
4/22/2017

31. Allelic Ambiguity Reduction
MRWAF Analysis
MRWAF is the mean regional weighted allele frequency:
mean frequency for an allele in a given region, weighted by sample sizes (2n)
If all alleles have MRWAF = 0 or all pairs have MRWAF ratio ≤ , then these are passed to the next step
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