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Genetic Testing Methodologies Used in Determining Relatedness of Survivors

It was initially anticipated that genetic testing would be the major hurdle in matching children of the Disappeared with their surviving grandparents. Strides in genetic technology development have allowed for relatively simple identification, and the calculations used in determining probability of relatedness have been straightforward. The major hurdles have been legal, with the government of Argentina showing variable levels of support and a strong tendency to develop roadblocks which prolong the process of reuniting children with their blood relatives. Ethical issues certainly arise, with the Grandmothers considering the best interests of the child in each individual case.

Four genetic methods have been used in efforts involving the children of the Disappeared. Those methodologies are: 1) HLA Typing, 2) Mitochondrial DNA Analysis, 3) Y Chromosome Short Tandem Repeat (STR) Analysis, and 4) Autosomal Short Tandem Repeat (STR) Analysis. Each is briefly introduced below, with a link to a more detailed explanation and student activities involving that particular methodology.

In the early 1980s, the preferred method of paternity and maternity testing used histocompatibility (HLA) typing. HLA antigens are antigens initially on the surface of white blood cells (leukocytes). The HLA system of humans consists of four closely linked genes, found on chromosome 6. Most people's familiarity with this system comes from publicity regarding tissue transplantation and the difficulties in finding "a match." Each of the genes has a number of different alleles, making this one of the most highly polymorphic gene systems in the human genome. Each of the genes is listed below with its number of alleles:

Each allele encodes a distinct antigen identified by a letter and number. For example B3 is allele 3 at the B locus (position), while B46 is allele 46 at the B locus. Because of the close positioning of the four genes, the HLA tends to be inherited as a distinct unit. To make this even more complex, all of the alleles are codominant, meaning that the information from each is expressed equally. An example should make this more clear. The typical person has 46 chromosomes - two sex chromosomes and forty-four non-sex chromosomes (autosomes) - per cell. Therefore, each cell has a pair of chromosome 6s.The array of HLA chromosomes on each chromosome 6 is called a haplotype, and each individual will have two HLA haplotypes.

Let's assume that an individual inherits A7, B14, C2, DR3 from one parent and A5, B36, C7, DR11 from the other parent, that persons genotype (genetic constitution) for HLA can be represented as:

In the case of organ and tissue transplants, successful transplantation of organs depends on matching of donor and recipient HLA types. Because there are so many variations, the likelihood a match with another person is slight. Often close relatives will undergo HLA testing to determine whether they are a likely match for an individual in need of a tissue or organ transplant. The ability to be able to test one's HLA types has led to a capability to link specific diseases with certain HLA combinations. Examples include narcolepsy, ankylosing spondylosis, psoriasis, autoimmune hepatitis, celiac disease, myasthenia gravis,, rheumatoid arthritis and systemic lupus erythmatosus.

HLA typing was suggested as a way to match children of the Disappeared with their surviving grandparents. In July, 1984 HLA typing of hundreds of possible grandparents began, with the goal of stockpiling data for future use. In 1987, a genetic database was developed. That same year, an Argentine law was enacted, stipulating that judges were to order genetic testing of families as well as children whose identities were in doubt, or when there was sufficient evidence that a child could be the son or daughter of one of the disappeared. In at least two dozen cases, the probability of a given set of grandparents were sufficiently high that further legal steps were taken. The best interests of the children were always a primary consideration. In some cases, children continued to live with their adoptive families while incorporating their biological families into their lives.

HLA Typing - Frequently Asked Questions - http://www.ibmtindy.com/faq/hla-typing.htm

Well written, concise discussion of likelihood of finding a bone/tissue transplantation match within family and ethnic group.

National Marrow Donor Program, Step 1: HLA Typing - http://www.marrow.org/PATIENT/HLA_typing.html

Brief description of HLA typing in the donor-recipient matching process.

DNA-Based HLA Molecular Typing - http://www.cbrlabs.com/hla.html

Brief, more scientifically oriented introduction to the HLA system.

What is HLA?
- http://www.innogenetics.com/Website/Website.nsf/7df3b6bb9c0862e8c12567380052687f/69a6ff86644d314ac12569a700578cec?OpenDocument

Well written discussion to HLA, good scientific detail. Commercial site selling a product.

In the late 1980s, Dr. Mary Claire King began using mitochondrial DNA testing to solve several of the unknown parentage cases. In the case of nuclear DNA, including the HLA genes, each individual gets half their DNA from each parent. Both the sperm and egg possess mitochondrial DNA. However, the sperm's mitochondria are located at the base of the flagellum, and these are left with the flagellum outside the egg during the fertilization process.Therefore the mitochondria, small sausage-shaped organelles that play an essential role in the breakdown of sugar for energy, are inherited from the mother.

Mitochondrial DNA offers an advantage not found in nuclear DNA. In the case of nuclear DNA, a given DNA sequence has only a 50% chance of being passed from parent to child, so that all 4 grandparents would have to be checked for a match to the DNA sequence of a particular child. Where one or two of the four grandparents were missing or deceased, definitive identification was often impossible. King found a 600-base pair, highly variable region of the mitochondrial DNA. The degree of variation is such that matches are unambiguous. Because mtDNA is passed directly from grandmother to mother to child, it serves as a perfect recognition marker to establish identity in the Argentina Disappeared situation. Even if the maternal grandmother is dead, maternal uncles and aunts should have identical DNA to that of the child's disappeared mother and the child.

Mitochondrial DNA analysis has been used regularly in solving forensic cases including determination of the identity of the Viet Nam conflict's Unknown Soldier, and more recently has been used in tracing ancestry and migration of many populations, including human populations.

About Mitochondrial DNA - http://www.mitotyping.com/aboutmt.html

An excellent introduction to the use of mitochondrial DNA analysis in forensic cases.

The Fire Within: The Unfolding Story of Human Mitochondrial DNA - http://biocrs.biomed.brown.edu/Books/Essays/MitochondrialDNA.html

Concise, well written introduction to mitochondrial DNA and its applications.

Mitochondrial DNA Analysis at the FBI Laboratory - http://www.fbi.gov/hq/lab/fsc/backissu/july1999/dnatext.htm#Back%20to%20text,%20Figure%201

Excellent detailed discussion of mitochondrial DNA, evidence collection, analysis, interpretation of data and legal aspects of working with mitochondrial DNA. Extensive bibliography at the end.

The Other DNA: Research on Mitochondrial Diseases - http://www.columbia.edu/cu/21stC/issue-1.3/dna-mitoch.html

Good overview of mitochondrial disorders and resulting diseases. Many links to diseases presented in this paper.

DNA & Native American origins - http://biocrs.biomed.brown.edu/Books/Essays/MitochondrialDNA.html

Much more expansive than one would expect - lots of articles, references, etc,, all dealing with mitochondrial DNA and human evolution.

Cohen Expected to Identify Unknown Soldier from Vietnam War - http://www.cnn.com/US/9806/30/unknowns.01/

CNN article announcing the identity of the Unknown Soldier. Includes video footage.

Human beings typically possess two sex chromosomes. Females have two X chromosomes, while males possess one X chromosome and one Y chromosome. The mother's eggs each receive an X chromosome, while sperm produced by the father possess either an X or a Y chromosome, thus determining the gender of the child (XX resulting from fusion with an X-bearing sperm, XY resulting from fusion with a Y-bearing sperm). Just as mitochondrial DNA is passed on maternally, Y chromosomes are passed on paternally, from father to son. The larger X chromosome is now known to have more than 1000 genes, while the Y chromosome has had 330 genes identified thus far. Though the Y chromosome is associated with maleness, a single gene, the SRY gene, is actually the gene that when activated causes the fetus to develop male reproductive characteristics.

Tandemly repeated DNA sequences are sets of base pairs (A-T, C-G) which are repeated without break along the DNA. These sequences are widespread throughout the human genome, yet show sufficient variability among individuals in a population that they have become important in human identity testing, including forensic testing. These tandemly repeated regions of DNA are typically classified into several groups depending on the size of the repeat region. Microsatellites (short tandem repeats, STRs) contain 2-5 bp repeats. Minisatellites (variable number of tandem repeats, VNTRs) have core repeats with 9-80 bp,

The forensic DNA community has moved primarily toward working with tetranucleotide repeats, which may be amplified using the polymerase chain reaction (PCR) with greater fidelity than dinucleotide repeats. The variety of alleles present in a population is such that a high degree of discrimination among individuals in the population may be obtained when multiple STR loci are examined. In the Argentina situation, if a father became one of the disappeared, his sons would all have the same Y chromosome sequence, which would in turn be identical to that of their father (missing), paternal grandfather, and paternal uncles. Dr. Daniel Korach of Argentina has been successful in using eight different Y chromosome-specific short tandem repeats: DYS19, DYS385, DYS389 I, DYS389 II, DYS 392 and DYS 393, to substantiate relatedness conclusions based on mitochondrial DNA data.

Y-Chromosome STRs - http://www.cstl.nist.gov/biotech/strbase/y_strs.htm

A more technical treatment of Y chromosome STRs.

Y-Chromosome Links - http://john.hynes.net/y.html

An excellent list of resources including images, general articles, Thomas Jefferson and his slave descendants, Jewish relationships to an African tribe, etc. This site begins with links to biometric analysis of Y chromosome data.

Jefferson-Hemmings DNA Testing: An On-Line Resource - http://www.monticello.org/plantation/hemings_resource.html

Numerous references regarding the most highly publicized use of Y chromosome testing in the U.S.

Using Genetic Variation on the Y Chromosome and Mitochondrial DNA to Study Populations and Their Migrations - http://www.ich.ucl.ac.uk/cmgs/ymito.htm

Brief discussion of each technology. Includes mutation rates.

Y Chromosome /mt DNA Analysis - http://store.yahoo.com/genetree/ycan.html

Well written commercial site that deals with the various tests they use to determine paternity, maternity, sibling relationships, grandpaternity and grandmaternity.

The Y Chromosome in the Study of Human Evolution, Migration and Prehistory - http://www.ucl.ac.uk/tcga/ScienceSpectra-pages/SciSpect-14-98.html

An excellent overview of the Y chromosome by itself, this article includes multiple cases of the use of the Y chromosome in early human migration.

Autosomal STRs are short tandem repeats found in the genetic material (DNA) that makes up the human genome. Several different STRs, regions that are highly variable in length from individual to individual, have been located and examined. Unlike, Y Chromosome Short Tandem Repeats or mitochondrial DNA analysis which indicates either a match or no match, autosomal short tandem repeats can be used to indicate degree of relatedness. The size of the autosomal STR is what scientists examine to help answer questions regarding relatedness. The different sizes of DNA found at these STR regions correspond to alleles. It is the frequency of these frequencies that researchers use to calculate the probabilities of relatedness.

Additional Resources Dealing with Autosomal and STRs

Y Chromosome /mt DNA Analysis - http://store.yahoo.com/genetree/ycan.html

Well written commercial site that includes a brief introduction to autosomal tandem repeat analysis.

Short Tandem Repeat DNA Internet DataBase - http://www.cstl.nist.gov/biotech/strbase/

Includes an introduction to short tandem repeats, fact sheets, sequence information, chromosomal location, mutation rates, etc..

Go to Student Activity #2: Alternative Applications of Selected Genetic Testing Methodologies

Go to Student Activity #3: Introduction to Online Scientific Journals

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