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STAT

An MUSC blog
Keyword: gene

Dr. Betty Pei-tie Tsao and colleagues

Betty Pei-tie Tsao, Ph.D. (front, center), Richard M. Silver Endowed Chair for Inflammation Research at MUSC and senior author on the Nature Genetics article, with first author Jian Zhao, Ph.D. (to Dr. Tsao's left) and second author Yun Deng, M.D. (to Dr. Tsao's right).

Investigators at the Medical University of South Carolina (MUSC)  report pre-clinical research showing that a genetic variant encoded in neutrophil cytosolic factor 1 (NCF1)  is associated with increased risk for autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis, and Sjögren's syndrome, in the January 2017 issue of Nature Genetics. Data indicate that increased NCF1 protects against SLE while decreased NCF1 raises SLE risk and highlight the pathogenic role of reduced reactive oxygen species in autoimmune disease development.

Single-nucleotide polymorphisms (SNPs – pronounced 'snips') are the most common type of human genetic variation; each one represents a small difference in a nucleotide – the building blocks of our DNA. The Immunochip for fine-mapping is an important tool for conducting genome-wide association studies of the genetic components of disease. Researchers use the Immunochip to investigate DNA samples from people with a particular disease for linkage disequilibrium (LD) signals that illuminate associations between specific SNPs and the disease. Autoimmune diseases such as SLE are known to have a strong genetic component and, to date, dozens of SNPs associated with SLE have been identified and included on the Immunochip.

The Achilles heel is, of course, that the Immunochip cannot identify associations with SNPs that it does not include.

When MUSC researchers genotyped DNA samples from Chinese, European-American, and African-American SLE patients, they found a strong signal in the Chinese sample at the rs73366469 locus in the GTF2IRD1–GTF2I intergenic region at 7q11.23. This was puzzling because that locus was not consistent with SLE loci identified by other genome-wide association studies. Furthermore, the very strong signal in the Chinese sample appeared as a modest signal in the European-American sample and did not appear at all in the African-American sample.

"A true risk gene should be the same in all populations,” explained Betty Pei-tie Tsao, Ph.D., Richard M. Silver Endowed Chair for Inflammation Research at MUSC and senior author on the article. “And for such a strong signal, we wondered, 'why hasn't anyone else seen it?' We wanted to find out if what we were seeing was true and explain it." 

The team confirmed their finding using a different genotyping platform in an independent Asian sample provided by Nan Shen, M.D., Ph.D., professor of medicine and director of the Shanghai Institute of Rheumatology at Shanghai Jiao Tong University's School of Medicine. But, because rs73366469 did not show LD with any SNPs in the Immunochip, the researchers hypothesized that the SNP containing the true underlying risk factor was not included in it.

"We sort of came into the study from our Asian samples and then started looking for this signal in other populations,” said Tsao. “Every ethnic group has a different ancestral background and different LD patterns. We used the LD signal strength as a guide to find our way to the true risk gene – the particular variant that actually caused the increased risk for lupus."

Because the SNP they were looking for was most likely not included in the Immunochip, the team turned to the 1000 Genomes Project dataset, where they found two SNPs that were not only not on the Immunochip, but also produced stronger LD signals with rs73366469 in Asian patients than European or African patients. One of these two, rs117026326 located on intron 9 of GTF2I, showed a stronger association with SLE than either the original or the other locus from the 1000 Genomes Project.

As the researchers focused in on rs117026326, they saw that the NCF1 gene was nearby. This was important because NCF1, which encodes a subunit of NOX2, is thought to be related to SLE due to its role in activating the phagocytic complex NOX2.

Preclinical studies have shown that non-functional NOX2 exacerbates lupus in mice. Furthermore, NCF2, which encodes another subunit of NOX2, is associated with SLE risk in European Americans. The strong association of rs117026326 with SLE and the functional implications of nearby NCF1 took the team to their next hypothesis: that the rs117026326 SNP might tag causal variants of NCF1 that were not present in the 1000 Genomes Project database.

But unraveling this mystery was not going to be easy.

 "This is a very complex genomic region,” explained Tsao. “The NCF1 gene has two nearly identical twins – NCF1B and NCF1C – that are 98% the same. But they are non-functional pseudo-genes. This makes working in this region of the human genome very difficult. That's why the next-generation sequencing method that the 1000 Genomes Project has been doing doesn't pertain to this region."

The researchers believed that mapping techniques commonly used by the larger projects, while efficient, limited their ability to find unique sequences among all the copies and duplications in this region. So, they decided to set up their own, novel PCR assay.

"You can't easily sequence this region using the next-generation techniques,” said Tsao. “So, we had to do it the old-fashioned way, which was very time consuming and labor intensive. To genotype the region correctly, we used PCR to selectively amplify the NCF1 copies and conduct copy number variation tests. Then we only used samples with no copy number variation to examine the NCF1 variant. This method ensured that what we identified as an NCF1 variant was truly a variant."

Using this strategy, the team identified 67 SNPs, four of which had a strong association with rs117026326. After conducting a long series of multiple tests in samples from various ethnic populations, they gradually eliminated three of the four SNPs and determined that the one called p.Arg90His was the likely genetic variant causing SLE susceptibility across all populations.

In addition, p.Arg90His was associated with increased risk for other autoimmune diseases, including rheumatoid arthritis and Sjögren's syndrome. The team also found that having only one copy of NCF1 was associated with a higher SLE risk, but having three or more  NCF1 copies was associated with reduced SLE risk. Finally, while the underlying mechanism is unclear, the team found that having reduced NOX2-derived reactive oxygen species also raised the risk for these autoimmune diseases.

Tsao notes that perseverance was a critical component of this work. This work was started years ago when the team was at the University of California Los Angeles  and was completed after moving to MUSC.

"We just stuck with it as a labor of love. Our lead author, Jian Zhao, devoted several years of his life to this project,” explained Tsao.” At the time we started, we didn't know it was going to be so complex. We just wanted to explain what we were seeing. It turned out to be quite a chase and very interesting and rewarding to finally bring this project to this point."

This work also points out an important unmet need in the field of genetic mapping.

"We need a more efficient platform to screen complex genome regions for variants. For a lot of diseases we've identified some, but not all, of the variants. There may be more variants hiding in these complex regions," said Tsao. "You have to sort it out like a puzzle. Autoimmune diseases share certain risk factors but also have unique genetic variants that drive the molecular pathogenesis of the disease. Each time you find a variant, you get more puzzle pieces and you can start to understand more about that disease and other autoimmune diseases as well."

Reb-1Researchers at the Medical University of South Carolina and elsewhere resolve the first protein structure in a family of proteins called transcription terminators that could provide insight into aging and cancer. The work reveals the protein Reb1 to be a traffic signal for coordinating transcription and gene replication, rather than a passive roadblock as previously thought.

Image Caption: Space-filling model of Reb1 bound to DNA. Reused with permission from PNAS.

 

In a study published on 28 March 2016 in the Proceedings of the National Academy of Sciences, researchers at the Medical University of South Carolina (MUSC) and Virginia Commonwealth University have resolved the first protein structure in a family of proteins called transcription terminators. The crystal structure of the protein, called Reb1, provides insight into aging and cancer, according to Deepak Bastia, Ph.D., Endowed Chair for Biomedical Research in the MUSC Department of Biochemistry and Molecular Biology and co-senior author of the study.

During transcription, large molecular machines read genes by traveling along double-stranded DNA. This machinery simultaneously reads out the gene code in continually lengthening chains of single-stranded RNA. The RNA code is then used to assemble proteins that cells use for growth and division. At certain times during the life of a cell, transcription must be stopped–in order to conserve cellular energy or prevent uncontrolled growth, for example. At other times, cells may be preparing to divide, during which period trouble can arise.

Before a cell can divide, the DNA must be exactly replicated for use in the new cell. During part of this process, two types of machinery are now moving along the DNA strand–transcriptional machinery and replication machinery. In regions where the two machines are moving in opposite directions, collisions can occur and DNA broken, causing mutations. Harmful gene mutations can be passed into the new cell. That’s where Reb1 comes in.

One way to prevent genome instability is to prevent replication from colliding with transcription,” says Bastia. That’s what these terminator proteins do.”

Bastia’s group knew that there are specific sites on the DNA strand called terminator regions to which Reb1 binds itself. Reb1 was thought of as a simple physical barrier that sits on DNA and blocks both the transcriptional and replication machinery from moving further along the DNA strand and colliding with each other. Then Bastia’s group did an experiment to cut the transcription terminator region (tail) off of Reb1. Intriguingly, Reb1 was no longer able to halt the transcription machinery without its tail but was still able to bind to DNA. Therefore, the simple roadblock theory couldn’t be correct.

The insight came when they solved the crystal structure–a laborious process during which Carlos R. Escalante, Ph.D., Bastia’s co-senior author from Virginia Commonwealth University, made monthly drives transporting freshly made crystals from MUSC to the X-ray crystallography facility at Brookhaven National Laboratory in New York. The crystal structure showed that, when bound to DNA, the transcription terminator tail of Reb1 can interact with a specific part of the transcriptional machinery, acting as a tether between the two.

The work illuminates Reb1 as a traffic signal for coordinating transcription and gene replication, rather than as a simple roadblock as previously thought.

Though the tether between Reb1 and the transcriptional machine is clear, the team is still not sure exactly how terminator proteins stop transcription, a question which drives their current work. And the connection between terminator proteins and colorectal cancer has been made, but work in other cancers and in aging has yet to be undertaken.

Still, Bastia suspects that this coordination prevents the type of gene errors that lead to many types of cellular aging and tumor growth, both of which are processes that result from uncontrolled transcription and replication. The group is currently researching another type of terminator protein, work which Bastia hopes will lend further knowledge to the diseases of aging.

 

Genetic Origin of Mitral Valve ProlapseAs part of a multi-center investigation recently reported in the journals Nature1 and Nature Genetics,2 researchers at the Medical University of South Carolina (MUSC) and Harvard/Massachusetts General Hospital as well as other international institutes have discovered genetic and biological causes for MVP. The investigators identify that MVP can be a result of heritable genetic errors that occur during embryonic cardiac development and progress over the lifespan of affected individuals.

Mitral valve prolapse (MVP) affects 1 in 40 individuals making it one of the most prevalent human diseases. Many individuals with MVP develop potentially life-threatening cardiac arrhythmia and heart failure.

In MVP, one or both flaps of the mitral valve bulge backward into the left atrium causing it to close improperly upon termination of atrial systole. Mitral valve prolapse is often detected as a heart murmur and is usually asymptomatic, but in roughly 10% of cases mitral valve regurgitation intensifies to a clinically severe stage. In severe cases, arrhythmic heartbeats develop, which increases the risk of stroke, heart failure and sudden cardiac death. In fact, the risks are high enough in MVP to make it the leading indication for mitral valve surgery.

In the Nature article,1 investigators used linkage analyses and capture sequencing technology to examine protein-coding genes on chromosome 11 in four members of a large family segregating non-syndromic MVP. They discovered a missense mutation in the DCHS1 gene, which codes for the protein dachsous homolog 1, a member of the calcium-dependent cell-cell adhesion family of cadherins. Another DCHS1 mutation was found in additional families segregating deleterious MVP. Both mutations reduce DCHS1 protein stability in mitral valve interstitial cells (MVICs), a finding corroborated with the discovery of the original mutation in MVICs in a human patient with MVP that underwent mitral valve repair surgery. Dchs1 mutant mice displayed similar pathology, along with scattered migration of MVICs during growth, suggesting that protein stability is essential to maintaining cues for cell polarity during mitral valve development.

In a subsequent manuscript published in Nature Genetics,2 the investigators performed a genome-wide association study (GWAS) to identify genetic variants in a population of  more than 10,000 subjects.  Single nucleotide variants (SNPs) with genome-wide significance were identified in the patient cohorts and genes surrounding these SNPs were functionally evaluated in multiple in vivo models. 

The results from both studies highlight a potential unifying biological cause for MVP in the population.

“We have found a genetic and biological reason for one of the most common diseases affecting the human population," says MUSC researcher Russell A. (Chip) Norris, Ph.D., who was a co-senior author on the studies. "This is a critical initial step as we transform this discovery into new remedial therapies to treat the disease.”  Roger R. Markwald, Ph.D.,  and Andy Wessels, Ph.D. both of the Department of Regenerative Medicine and Cell Biology at MUSC, were also co-authors.

If you are interested in supporting medical research, visit donorscure.org, an MUSC-affiliated 501(c)(3) nonprofit organization that allows you to fund biomedical research projects led by researchers across the United States.

References

1 Durst, et al. Mutations in DCHS1 cause mitral valve prolapse. Nature. 2015 Aug 10 [Epub ahead of print]. Available at http://dx.doi.org/10.1038/nature14670

2 Dina C, et al. Genetic association analyses highlight biological pathways underlying mitral valve prolapse.
Nat Genet. 2015 Aug 24.  [Epub ahead of print] Available at http://dx.doi.org/10.1038/ng.3383.

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