Ancestry informative markers

Have you guys seen the commercials about ancestry DNA from ancestry.com, my heritage, and 23andme? Ancestry informative markers (AIMs) have been around for a long time but it is only coming into light in the public eye because of these companies trying to make a profit. I've even fallen for the mystery of discovering my own ancestry, although I am pretty sure it will only tell me that I am full European (duh!). 

In missing persons or mass disaster situations, the biggest concern is identifying the remains either through visual examination, dental and/or medical records, or STR typing. DNA analysis is typically a last resort because it is time consuming and costly. When all methods have failed and there is low template and highly degraded DNA, alternative solutions are required such as DNA phenotyping (see previous blog) and lineage markers, to name a few. Mitochondrial DNA and Y chromosome DNA are examples of lineage markers that can provide useful information on the maternal and paternal lineage, respectively. However, because these are haplotypes (a group of genes passed down from a single parent) and non-recombinant genetic markers, individualization is limited especially when whole families are missing. Therefore, analysis of DNA markers from autosomal chromosomes that are small enough for degraded samples are preferred. Biallelic markers called insertions/deletions (INDELS) can be analyzed by targeting very short fragments of DNA less than 200 base pairs. One group of scientists selected 46 AIM-Indels considering allele frequencies from four human population groups: African, European, East Asian, and Native Americans. The developed a multiplex allowing all 46 AIM-Indels to be amplified in a singe PCR reaction. Later, another study evaluated the panel on bones displaying various degrees of DNA degradation. Anthropological examinations and other information regarding the individual was provided prior to testing. They found that samples from non-admixed individuals were successfully assigned to their continental group. Samples from admixed individuals were assigned with an estimate of the proportion of co-ancestry. Of course, there is ethical and confidentiality concerns with this type of analysis. The scientists conclude that although this type of information may be extremely useful in several scenarios (forensic or genetic populations studies), this type of information is sensitive and should only be used for the purpose of victim identification.

However, I found an interesting article about the use of AIMs as a tool to combat illegal trafficking of human kidneys. Human organs can extend and improve the lives of people, but the demand is so high and the supply so low. This has created the basis for a black market that is highly profitable at selling organs. Some people sell their organs when their families are in financial distress. Some countries have been known to donate organs of executed prisoners. Even worse, people have been murdered for their organs. Analysis of AIMS could be useful in cases when donor and recipient are from different origins or when the origin of the donor is unknown.




References:
Romanini C, et al. Ancestry informative markers: Inference of ancestry in aged bone samples using an autosomal AIM-Indel multiplex. FSI: Genetics 16 (2015) 58-63.

Pereira R, et al. Straightforward inference of ancestry and admixture proportions through ancestry-informative insertion deletion multiplexing. PLoS ONE (2012) 7(1)

Severini S et al. Use of ancestry-informative markers as a scientific tool to combat the illegal traffic in human kidneys. FSI: Genetics Supplement Series 5 (2015) e302-e304.

Freeze Tolerant Frogs!

Since a lot of our posts seem to be about cool organisms and how they can be beneficial to humans, I thought about one that I was interested in a few years ago. There's an amphibian that undergoes freeze-thaw cycles due to its surrounding arctic environment and lives through it! Normally, animals would migrate to warmer climates or hibernate for the winter, but the North American wood frog (Rana sylvatica) buries in the leaves and allows itself to freeze in suspended animation. They are able to survive weeks at a time without a beating heart and brain activity. When the temperature drops, urea concentrates in the tissues and their liver produces mass amounts of glucose and packs it into the cells to prevent intracellular ice formation. When water turns into ice, it forms ice crystals, which are harmful to cells causing shrinkage and cell death. Obviously, this must be avoided in order to survive freeze thaw cycles. Therefore, the urea and glucose are used as a cryoprotectant to lower the freezing temperature in the tissues. However, it has been suggested that enduring many freeze thaw cycles could result in significant energy depletion and be fatal to the frog. Applications for this freeze tolerant ability include aiding human organ transplants and cryopreservation of whole humans.

http://voices.nationalgeographic.com/2013/08/21/how-the-alaska-wood-frog-survives-being-frozen/

http://news.nationalgeographic.com/news/2007/02/070220-frog-antifreeze.html

https://www.youtube.com/watch?v=Fjr3A_kfspM


They are able to do this with special proteins in their blood called nucleating proteins.

DNA Phenotyping

When I first started my education in forensic science, I wrote a paper about craniofacial reconstruction, which is the process of giving a face to unidentified skulls. I thought maybe I could do this because I also love to draw. My specialty is the human form,  and specifically the face because that's what people pay for. However, forensic artists can't determine the color of the skin, hair, or eyes based on features of the skull so the final product will be a black and white sketch or a beige and bald clay sculpture. Then I read about a rapidly developing field of research called DNA phenotyping and it has been one of my favorite topics since. 

DNA phenotyping uses single nucleotide polymorphisms (SNPs) found within or around genes linked to externally visible characteristics (EVCs). One gene that is commonly used as an EVC is amelogenin for sex determination of biological evidence. One of the coolest labs I got to do during graduate school was verify our own eye color via genotyping using IrisPlex, a DNA-based eye color prediction assay. IrisPlex uses six informative SNPs: MC1R, OCA2, SLC24A5, MATP (SLC45A2), ASIP, and TYR.  Then you imput the allele into a table and it gives you a predicted phenotype for blue, intermediate, and brown eye color. We were able to accurately predict everybody's eye color that did the lab so I was impressed. Now, there is a company that  does DNA phenotyping, ancestry and kinship analysis. The Snapshot Forensic DNA Phenotyping System by Parabon can generate a likely profile using DNA genotyping. This tool can be used for investigative leads when a DNA profile from an unknown sample (hopefully being from the suspect) generates no hits from the convicted offender database, as well as for missing persons, disaster victim identification, and repatriating war remains.

I strongly urge you guys to go look at the following websites. It's pretty neat!


References:

• http://hirisplex.erasmusmc.nl/ 
• https://snapshot.parabon-nanolabs.com/
• Dembinski G M, Picard C J. Evaluation of the IrisPlex DNA-based eye color prediction assay in a United States population. Forensic Science International: Genetics 9 (2014) 111-117.

Rapid DNA Analysis Systems

Rapid DNA analysis refers to a fully automated process of DNA typing, essentially a "swab in - profile out" system that only takes a couple of hours. Standard DNA typing methods take about 10 hours over several days for DNA profiling of just one sample. These traditional methods require experienced technicians and the use of several expensive instruments and commercial kits. Therefore, scientific advancements have been made to traditional protocols in order to reduce costs and the amount of time for testing. Rapid DNA systems perform a modified method of extraction, short tandem repeat (STR) amplification, and separation and detection of DNA fragments driven by active microfluidics. They also contain an on-board platform for allele calling and generating electropherograms. There are many applications for this type of system: police booking stations, border security, and human identification in mass disasters just to name a few. These systems could provide quick investigative leads, minimize laboratory backlogs, and reduce contamination issues.

There are two systems on the market right now. One is called DNAscan (GE Healthcare and NetBio) and the other is RapidHIT 200 (IntegenX). They are both able to process five samples in less than 90 minutes, but RapidHIT also includes positive and negative controls. Both systems require minimal training on account of fully automated technical capabilities. When I was doing my internship in the summer of 2013, I was able to use these instruments during a demonstration and I even got my own profile from the RapidHIT instrument. I compared it to the one that I produced in my lab and it matched!

Furthermore, there are two types of this type of analysis, rapid DNA analysis and modified rapid DNA analysis. Rapid DNA analysis generates a profile without any human intervention, whereas modified rapid DNA analysis requires human interpretation of the profile and technical review. The distinction is important because only the modified rapid DNA analysis profiles are able to be uploaded into CODIS. This database is maintained by the FBI and contains DNA profiles from convicted offenders, arrestees, and forensic unknowns. DNAscan is the first and only system approved by the FBI to upload profiles to CODIS after scientists have analyzed them.

For more information:

• https://www.fbi.gov/services/laboratory/biometric-analysis/codis/rapid-dna-analysis
• http://www.gelifesciences.com/webapp/wcs/stores/servlet/ProductDisplay?categoryId=364838&catalogId=10101&productId=71366&storeId=11787&langId=-1
• https://promo.gelifesciences.com/gl/DNASCAN-RAPID-DNA/
• https://integenx.com/products/rapid-dna/
• https://www.fbi.gov/services/laboratory/biometric-analysis/codis/codis-and-ndis-fact-sheet

X-Inactivation

If you've ever had a basic biology class then you know female humans have the sex chromosomes, XX, whereas males have XY. You inherit one X from your mother and either an X or a Y from your father and that determines your sex. However, females would have double the X-linked genes that males would have had. Therefore, a process called X chromosome inactivation (XCI) compensates for this dosage of X-linked genes in females. In humans, this process is random meaning that the maternal or paternal X can be silenced. Three articles were published this year in the same journal about this phenomenon. I just want to focus on one that I found the most interesting.

The first one was an overview of XCI and reactivation in X-linked diseases. Maternal and paternal X chromosomes have the same probability of inactivation, but the authors state that one particular X chromosome can be preferentially inactivated in most cells arising to a skewed or unbalanced XCI. It has been proposed that XCI skewing is heritable with possibly multiple X-linked genes driving this process. Some of these genes can escape XCI and have been suggested to play a role in Turner and Klinefelter syndromes. Turner syndrome occurs in females partially or completly missing an X chromosome (XO), whereas Klinefelter syndrome results in two X chromosomes in males (XXY). In addition, females who are carriers and heterozygous for X-linked dominant disorders can be phenotypically okay because the active X chromosome has the normal allele. Conversely, a skewed XCI can lead to an X-linked recessive trait transforming into a dominant trait if the normal allele has been silenced in the majority of cells. Another interesting finding is that some inactivated genes in the mouse model escape inactivation in humans and expression of these escapee genes is tissue dependent. Therefore, discrepancies were found between phenotypes of patients and the mouse model. Studies on humans have only recently been introduced and some of the results are interesting. It appears that human cells are more sensitive to environmental factors in culture conditions. Apparently, this could be attributed to humans having a more complex XIC process but this requires deeper exploration through research (1).

References:

1) Vacca M, Ragione F D, Scalabri F, D'Esposito M. X inactivation and reactivation in X-linked diseases. Seminars in Cell & Developmental Biology 56 (2016) 78-87.

For more on this topic:

2) Migeon B R. An overview of X inactivation based on species differences. Seminars in Cell & Developmental Biology 56 (2016) 111-116.

3) Goodrich L, Panning B, Leung K N. Activators and repressors: A balancing act for x-inactivation. Seminars in Cell & Developmental Biology 56 (2016) 3-8.

MicroRNAs and Body Fluid Identification

MicroRNAs (miRNAs) are small (18-25 nucleotides), non-coding, single-stranded RNA molecules found in plants, animals, and viruses. These molecules play a role in RNA silencing and post-transcriptional regulation of gene expression and, therefore, can be used as a biomarker for body fluid identifications (BFID). While messenger RNA (mRNA) and miRNA demonstrate tissue-specific expression, miRNAs are more stable in compromised samples due to their small size and highly abundant per cell. For these reasons, forensic scientists have looked into these biomolecules for determining the DNA source recovered from casework evidence. Forensic DNA analysis is often performed on many types of substrates such as saliva, semen, venous blood, menstrual blood, tissues, teeth and bone, etc. In some cases, it would be beneficial to know whether the DNA came from a sperm cell versus an epithelial cell, for example. Traditionally, this was done by cutting a vaginal swab in half and smearing one half on a slide to visualize if sperm was present under a microscope. The other half would go onto DNA analysis. This is not ideal because the foreign male DNA could be present in low amounts and half of the material has now been consumed. Therefore, one study looked at co-extraction and co-analysis of DNA and miRNA to obtain an STR profile (for human identification, HID) and determine the origin of that DNA (BFID). They specifically looked at 4 miRNA markers for menstrual blood, venous blood, and semen in a multiplex reaction. Then they amplified the co-extracted DNA with a commercial STR kit. Both amplified products from DNA and miRNA were separated and detected on the same capillary electrophoresis (CE) platform. They were successfully able to determine the source of DNA and the origin of where it came from. Likewise, another study looked at 8 miRNA markers for blood, saliva, semen, and vaginal material using quantitative PCR (qPCR) compared to CE. The results of the study showed that blood and semen were highly distinguishable from the other fluids, but saliva and vaginal fluid have many markers in common. In addition, the CE analysis was able to detect absence or presence of miRNAs like qPCR; however, CE analysis also offers an indication of expression level. In conclusion these studies show the possibility for confirmed BFID with co-analysis of DNA with the potential for an extended panel of markers.

References:

1)     Li Y, Zhang J, Wei W, Wang Z, Prinz M, Hou Y. A strategy for co-analysis of microRNAs and DNA. FSI:Genetics 12 (2014) 24-29.

2)     van der Meer D J, Williams G A. Performing body fluid identification with microRNAs using capillary electrophoresis. FSI: Genetics Supplement Series 5 (2015) e592-e594.

 

Increased DNA damage after alcohol exposure

I had a good friend in undergrad that refused to drink alcohol because of its negative affects on the brain. More power to ya... but there's nothing I love more than a margarita with my beloved mexican food! Yum! I read an article by Suman et al. that was recently published about DNA damage and loss of repair mechanisms in rat hippocampus after alcohol exposure. Alcohol is a toxic substance to the brain, which leads to structural and functional damage. The hippocampus is found in the brain and plays a role in flexible cognition and complex activities including navigation, decision-making, and social behavior. Research by Rubin et al. suggests that damage to the hippocampus is associated with failure to adapt to social situations. Damage is also linked to medical conditions such as schizophrenia, autism, Alzheimer's disease, and depression. DNA damage occurs through oxidative stress-induced damage resulting in increased double strand break and decreased repair proteins in the hippocampus. The scientists induced binge drinking on rats and observed the effects on the hippocampus. They specifically looked at alcohol-induced oxidative stress using immunofluorescence staining of 4-Hydroxynonenal (4-HNE), which is a chemical found in higher quantities in animal tissues after oxidative stress due to increased activity in the lipid peroxidation chain reaction. DNA damage was also assessed using immunofluorescence staining of gamma-H2AX, which is a protein that recruits DNA repair proteins and can be used as a biomarker for equal parts of double strand breaks. Furthermore, they looked at downregulation of DNA double strand break repair mechanisms using immunoblots and immunohistochemistry 24 hours after a 4 day alcohol binge. Image quantification was performed by measuring staining intensity using the color deconvolution portion of an open source image processing program called ImageJ. They observed more intense staining of 4-HNE and gamma-H2AX cells in the alcohol treated groups in comparison with the control groups, suggestive of oxidative damage and increased double strand breaks. Results of the signaling pathways connected to DNA damage showed decreased levels of proteins after the alcohol binge. The authors suggested that the damage, reduced repair mechanisms, and/or misrepaired DNA may lead to cell cycle arrest, apoptosis, and even senescence. This could adversely affect the structure and function of the hippocampus and neuron formation.

So why is this research beneficial? What are its potential applications?

The location of the hippocampus in the rat (left) and human brain (right).

References:

Suman S, Kumar S, N'Gouemo P, Datta K. Increased DNA double-strand break was associated with downregulation of repair and upregulation of apoptotic factors in rat hippocampus after alcohol exposure. Alcohol 54 (2016) 45-50.

Rubin R D, Watson P D, Duff M C, Cohen N J. The role of the hippocampus in flexible cognition and social behavior. Frontiers in Human Neuroscience (2014) 8:742

DNA Degradation and Linear Amplification

DNA degradation is a major concern for forensic evidence and human identification. Analysis of DNA in the forensic community utilizes short tandem repeats (STRs) that range from 100 to 500 base pairs (bp) in size. The more extensive the degradation, the smaller the fragments of DNA, and the less likelihood to obtain a full profile for identification due to drop-out of the larger loci. I've been wanting to try this pre-PCR enhancement method that increases DNA typing success for low template DNA and I'm curious about its application for degraded samples. If degradation is observed via drop-out of larger loci or a ski-slope effect, the sample usually gets re-amplified with additional PCR cycles. However, this can lead to stochastic effects.

The method I'd like to try, linear amplification, increases starting template DNA by producing a single copy of the template for every cycle number as opposed to exponential amplification. This occurs in separate forward and reverse primer reactions that are then pooled together for downstream analysis. The thinking behind this was that the linear (and non-exponential) fashion of amplification would be more representative of the DNA in a sample without the stochastic effects of low template DNA. However, if a degraded samples can be amplified with specifically the larger loci in mind then perhaps we could see more balanced peaks and additional true alleles getting called. The drawback to this is that you would have to get permission to use commercial kit's proprietary primers and it would be difficult to customize a linear amplification multiplex specific to a sample's observed locus drop-out. In addition, most scientists prefer first pass success for increased productivity and lower costs.

References:

Grisedale, K., et al. Linear amplification of target prior to PCR for improved low template DNA results. Biotechniques 56(3) (2014) 145-7.