HLA sequencing - the barcode on your cells

HLA Typing:Understanding the

BARCODE on your cells.

Swati Kadam, Ph.D.

Barcodes help identify cells

What is HLA?• HLA = Human Leukocyte Antigen• Immune cells read HLA-barcode on cells to help identify

self vs. non self cells or uninfected vs. infected cells.– An immune response is triggered if a infected cell is identified.

Y

Y YY

Y Y

Y Y

Y

Y YYY Y

Y Y

Y

Y YY

Y Y

Y Y

Y

Y YY

Y Y

Y Y

Infected cell

T-cell

Uninfected cell

Uninfected cell

Uninfected cell

• Any cell displaying some other HLA type is "non-self" and is seen as an invader by the body's immune system, resulting in the rejection of the tissue bearing those cells.

Y

Y YY

Y Y

Y Y

Y

Y YYY Y

Y Y

Y

Y YY

Y Y

Y Y

YY YY

Y Y

Y Y

Non-self cell : INVADER

T-cell

Self cell

Self cell

Self cell

What is HLA?

MHC Region

MHC Class II genes MHC Class III genes MHC Class I genes

HLA-AHLA-C

TNF C4

HLA-DQHLA-DP HLA-DR

HLA-B

MHC houses HLA genesChromosome 6

MHC Region

MHC Class II genes MHC Class III genes MHC Class I genes

HLA disease associationsChromosome 6

- Leprosy- Multiple Sclerosis- Lymphoid Leukemia- Rh(D) isoimmunization- Psoriasis- Ankylosing spondylitis- Hemophilia with synovitis- Malaria- Susceptibility or Resistance to HIV-1- Type1 autoimmune hepatitis- ANCA-positive autoimmune disease

- Multiple Sclerosis- Psoriasis- Systemic Lupus- Asthma- Childhood Acute Lymphoblastic Leukemia (ALL)- HIV-related disease- Thyroid Carcinoma- Nephropathy- Kawasaki disease- Celiac Disease

http://ccg.murdoch.edu.au/private/yurek_kulski/mhc/Table4.html

HLA disease associations

HLA status and control over HIV infection

- HIV controllers = HIV positive individuals that do not develop a clinical disease and do not progress to AIDS.

- HLA Class I SNPs can determine whether you are a HIV controller or progressor http://aids.gov/hiv-aids-basics/hiv-aids-101/what-is-hiv-aids/

X

X

HLA drug hypersensitivity testing

Allergy to Abacavir is strongly associated with the presence of the HLA-B*5701 gene

Gluten intolerance

• Celiac disease is an autoimmune disorder of the small intestine triggered by gluten consumption.

• Over 95% of people with celiac have the isoform of DQ2 or DQ8, on Class II MHC genes.

• Organ transplantation• Hematopoietic stem cell transplant• HLA types associated with specific immune

diseases.• Parental testing• Reduce instance of GVHD– Non-related donor– Related donor

Importance of HLA typing

Did you know?• In the United States:

– Nearly 120,000 need lifesaving organ transplants.

– Every 10 minutes another name is added to the national organ transplant waiting list.

– An average of 6000 people die each year from the lack of available organs for transplant.

– In 2012, there were 14,013 Organ Donors resulting in 28,052 organ transplants.

– More than 1 million tissue transplants are done each year and the surgical need for tissue has been steadily rising.

• $230M worldwide transplant test market.

1, 8, 103, 14, 17

10, 16, 82, 7, 11

1, 8, 10 1, 8, 102, 7, 11 10, 16, 8

3, 14, 172, 7, 11

3, 14, 1710, 16, 8

Mother Father

Daughter 1 Daughter 2Son 1 Son 2

10, 16, 8

Son 3

1, 8, 10

Variation in HLA alleles even between siblings.Random and varied distribution of Haplotypes from each parent.

High resolution typing provides a more accurate match

Finding a match is not easy!

Match

Savior Siblings

• Serotyping– Detection of antibodies is serum (non

sequencing based method).

• Sanger sequencing of amplified regions– Amplicon sequencing of specific exon regions.

• Sequence Specific Oligonucleotide Hybridization (SSO)– Oligo hybridization based detection of allele

status.

• (NGS) MHC sequencing for donor matching– Sequencing of entire MHC region.– Sequencing of amplicons for target regions.

Typing Methods

Method About method Pros Cons

SerotypingNon-sequencing based typing method where antibodies specific to HLA proteins are used to identify the proteins on the cell surface.

- Low Cost- Rapid- Traditional

- Crude Method- Protein based detection- Inaccurate typing- Protein binding to more

than one serotype

Sequence Specific Oligonucleotide

Hybridization (SSO)

Typing method where specific oligos are first designed for genes of interest and then hybridized to patient or donor DNA to check for hybridization.

- Checking of specific target

- Efficient

- Cannot account for unrecorded alleles

- Hybridization errors- Need to know target

sequence- Cannot phase

Sanger SequencingSanger sequencing or Sequencing by Termination (SBT) is a classical method used for sequencing specific regions of the MHC.

- Used to sequence regions of interest

- Fast- Base pair resolution- Coverage only 2x

- Different HLA alleles share similar sequences, difficulty aligning.

- Cannot phase

Next-Gen SeqPerforming long range PCR to amplify HLA genes in MHC region, fragmenting the amplified genes and then preforming deep sequencing.

- Deep coverage (1000x)- Total MHC coverage- Rapid high throughput- Accurate and efficient- Phasing

- Data Analysis

Typing Methods

HLA nomenclature

http://hla.alleles.org/

We have just scratched the surface

Table S1: PCR primers and estimated amplicon size for the 6 HLA genes

Locus Forward primer Seq   Reverse primer Seq Length (bp)

HLA-A HLA-A_F7 ATCCTGGATACTCACGACGCGGAC HLA-A_R8 CATCAACCTCTCATGGCAAGAATTT 3398

HLA-C HLA-C_F4 GGCCGCCTGTACTTTTCTCAGCAG HLA-C_R3 CCATGGTGAGTTTCCCTGTACAAGAG 4440HLA-B HLA-B_F3 AGGTGAATGGCTCTGAAAATTTGTCTC HLA-B_R3 AGAGTTTAATTGTAATGCTGTTTTGACACA 4296HLA-DRB1 HLA-DRB1_F1 TGATTGACTTGCTGGCTGGTTTCTCATC HLA-DRB1_R2 GCATCCACAGAATCACATTTTCTAGTGTT 11899HLA-DQB1 HLA-DQB1_F6 TCATGTGCTTCTCTTGAGCAGTCTGA HLA-DQB1_R7 TGTGACAGCAATTTTCTCTCCCCT 7118HLA-DPB1 HLA-DPB1_F1 TGGTCCAACAGGATCACATTTATAAGTGT   HLA-DPB1_R1 CCCAGTTTGGATGGTCTCTCAGCTCTT 13605

13,605bp 7,118bp 11,899bp

4,296bp 4,440bp 3,398bp

Long range PCR

Nextera

MiSeq

• Nextera/MiSeq– Coverage of coding and non-coding HLA

regions.– Nextera tagmentation protocol provides easier

faster processing of samples– Greater depth delivers accuracy exceeding

existing technologies.

Sequencing

1, 8, 103, 14, 17

10, 16, 82, 7, 11

1, 8, 10 1, 8, 102, 7, 11 10, 16, 8

3, 14, 172, 7, 11

3, 14, 1710, 16, 8

Mother Father

Daughter 1 Daughter 2Son 1 Son 2

10, 16, 8

Son 3

1, 8, 10

Variation in HLA alleles even between siblings.Random and varied distribution of Haplotypes from each parent.

Finding a match is not an easy task.

Unique HLA fingerprint

Match

• MiSeq install at HistoGenetics and DKMS Life Science Lab in Germany.

High resolution HLA typing because: – Read length (2x250, 2x300) allows phasing– Highest accuracy and speed– 1000x Coverage

• (consensus accuracy 99.999%)

– No Homo polymer errors– Easier workflow

MiSeq leads the way

• Analyze SNPs, allelic differences, status of microsatellites, and HLA typing all in one.

• Basic SNP or genotyping analysis tool

• IMGT-HLA database

Data Analysis

LET’S START DECIPHERING THE HLA-BARCODE.

Questions : Swati kadam, Ph.D. [email protected]

Questions : Abizar [email protected]

Appendix

• Currently at about 50 HLA related MiSeq installs in the last few months– DKMS. From Martin Allgaier’s install report: “DKMS Life Science Lab, a subsidiary of

DKMS, is a state-of-the-art HLA tissue typing laboratory in Dresden, Germany performing all sequencing activities to MiSeq. This was the 11th (!!!) Miseq for this customer and they are still planning for more. Currently, there are no signs of switching to another platform as the MiSeq gives them the output and flexibility they need for their service.”

– HistoGenetics. From Peter Kraus’ install report: “MiSeq Install Report for the first 10 of 20 total MiSeqs to be installed at HistoGenetics in Ossining, NY. HistoGenetics is a company that performs HLA typing as a service using sequencing assays” • Press release."We anticipate building on our leadership position and established

success by leveraging the MiSeq platform," said Dr. Nezih Cereb, Chief Executive Officer and Co-founder of HistoGenetics. "Based on our experience with other technologies, we think the MiSeq's quality data output and simple workflow, combined with Illumina's commitment to work collaboratively, is the ideal solution to enable us to provide our customers with the superior results and turnaround time they require."

Example: MiSeq installs in HLA labs

• Research customers– Association of HLA variants

with disease• Inflammatory, infection

susceptibility, cancer, etc.• More accurate cataloging of

HLA types

• Translational customers• Tissue registries• Marker validation for diseases

• Clinical customers• Transplantation match

– Registry– Confirmatory

HLA sequencing: Business Opportunity

$230M worldwide

Software companies

Conexio • SW market leader. Sanger legacy

Omixon • FDA-compliance

GenDx • mature SW, nice UI