DNA Analysis of the HLA Gene Complex Focus: HLA gene complex - major functions of immune system - direct role in organ transplants Goal: Determine student HLA Genotypes using modern biotechnology instruments and technology.

Organ Transplants Pre-transplant: Determine who will accept a transplanted organ / tissue (bone marrow) and who will reject it. Post-transplant: Once a patient receives a transplant, how to identify those that may be rejecting their organ / tissue (bone marrow). A significant driver: mismatched organs and poor transplant outcomes cost the US Healthcare System $1.5 billion annually.

How do you match organs and patients? Patient and organ must have same HLA genotype HLA: Human Leukocyte Antigen HLA genes control our immune system responses If the patient’s HLA system sees the organ as a foreign invader, the body will attack it and cause organ rejection.

HLA: Self vs. Non-Self + = + = HLA proteins are the body’s “surveillance system” The “HLA System” discriminates between “self” and “non-self” “Self” is OKAY, but “Non-Self” is NOT OKAY and the body tries to eliminate it “Self” Immune System Cells Response Proteins on your own cells MHC + T cell or B cell = No Attack “Non-Self” Bacteria & Viruses MHC T cell or B cell + = Attack! Proteins on cells that are NOT your own (Example: transplanted tissue or organ)

HLA Gene Complex All HLA proteins are coded by genes located on short arm of chromosome 6 Spans about 4 megabases Contains more than 200 loci (genes)

HLA Gene Complex Highest polymorphism of any human genetic system (more than 2,400 variants/alleles of these genes) Associated with more diseases than any other genetic region Divided into Class I (loci A, B and C), Class II (DR, DQ..) and Class III

HLA Genes and Alleles Within a gene locus, there are different alleles creating different proteins on the surface of cells. For example, HLA-A2 and HLA-A68 class I molecules differ from one by 13 amino acids These differences give rise to dramatic differences in protein structure and shape. The alleles can be expressed co-dominantly

HLA Allele Discoveries

Current HLA Allele List

Allele Names and Numbers Indicates HLA the HLA region and prefix for an HLA gene HLA-DRB1 a particular HLA locus i.e. DRB1 HLA-DRB1*13 a group of alleles which encode the DR13 antigen HLA-DRB1*1301 a specific HLA allele HLA-DRB1*1301N a null allele HLA-DRB1*130102 an allele which differs by a synonymous mutation

How do you find the HLA genotype? SSO Probes Sequence Specific Oligonucleotide Probes: Small segments of single stranded DNA ready to bond or “hybridize” with a second matching strand of DNA. Only correctly matching DNA probes hybridize with a DNA sample, so we can see which probes are positive matches to find a genotype.

SSO Genotype Analysis SSO probes are short segments of single stranded DNA. Each probe has a unique DNA sequence. Matching DNA will hybridize on the probe. Then, chemicals markers are added so we can detect which DNA matches. Detection Hybridization We can see the detection chemicals on the probes, and we can find the genotype.

SSO Probe Sample Data Dark spots show positive matches on SSO probes. Weak or blank spots show no match.

SSO Systems: RELI vs. Prodigy Probes on membrane Strips Limited # of Probes More hands on time Larger quantities of reagents External Scanner for imaging Low to medium resolution Prodigy Probes on chips More than 500 Probes/chip Fully automated Smaller quantities of reagents Internal camera for imaging Medium to high resolution

Inside the Prodigy Instrument

RELI Manual Assay We can perform the procedure manually without the RELI or Prodigy instrument and get the exact same results!

SSO Manual Assay All of these molecules can be attached to the SSO probes by simply applying the correct chemical reagents. Detection Hybridization

RELI Strips The RELI system creates paper strips with dark bands showing positive DNA matches to the SSO probes. Each student will add their own DNA sample to their own RELI strip and read the positive matches to find the HLA genotype.

Weak positive = Matching DNA RELI Strip Analysis There are many probes on each RELI strip, but only certain ones will show a positive match to your DNA sample. Start Stop Control Probes Weak positive = Matching DNA Strong Positive = Matching DNA Negative = No matches

RELI Strip Analysis We will use an Overlay and a Scoresheet to analyze and record all positive and negative matches on your RELI strip.