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Johns Hopkins flowFISH Cytometry Assay Predicts Risk of Genetic Diseases

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NEW YORK (360Dx) – Researchers at Johns Hopkins University are using a more-than-decade-old technology to potentially predict the risk of individuals with shortened telomeres to develop certain genetic diseases.

In a study published last week in Proceedings of the National Academy of Sciences, the JHU team described using the technology called flowFISH to examine telomere length (TL) in patients with a family history of telomere-related genetic disorders in a diagnostic setting. They reported that TL measurement had prognostic value for patients with shortened telomeres, which correlated with a patient's genetic disease and age of diagnosis.

In the study, Mary Armanios, a professor at JHU and the corresponding author for the PNAS study, and her team used flowFISH to test the value of TL measurement in patients with mutations in telomerase and telomere maintenance genes. The study focused on four degenerative diseases, including bone marrow failure, liver cancer, pulmonary fibrosis, and emphysema.

Originally invented by University of British Columbia researcher Peter Lansdorp in 2006, flowFISH is a method that combines fluorescence in situ hybridization with flow cytometry to quantify the copy number of specific repetitive elements in DNA of whole cell populations. Available through Johns Hopkins Pathology Labs for $400, the test is commonly used to determine the length of telomeres, and JHU has developed and commercialized it as a diagnostic tool.

Since McMaster University Carol Greider and her colleagues discovered that telomere shortening was associated with chromosome instability in 1990, people have associated TL with the processes of aging. In addition, researchers at the University of Washington have found the flowFISH assay to be useful for analyses such as measuring the rate of telomere attrition length during the cell cycle.

In an interview Armanios said "[W]e know that on the cellular level, telomeres need to be at least 500 base pairs in order to provoke the DNA damage response that is sufficient to cause cells to die or senesce."

For their study, Armanios and her team used the flowFISH assay to measure TL in lymphocytes and granular cells using 10 ml of a patient's blood sample. They poked holes into the nucleus of each cell and inserted fluorescently-labeled DNA that attached to telomeres.

"We take live cells from blood, and we need them to be alive so that we can distinguish the different subsets of leukocytes," in addition to distinguishing "lymphocyte length from granular telomere length, as each of those have their normal range," Armanios explained.

Researchers then performed flow cytometry to identify the types of white blood cells in each blood sample and quantified their fluorescence, averaging the number of lymphocyte and granular cells to give the average TL. Using, the flowFISH test, they then determined a range for normal telomere length among 192 healthy subjects, recruited from The Johns Hopkins Hospital. The control group included an age range from newborns to patients in their 80s.

In terms of clinical specificity, the team found that an arbitrary threshold would miss a subset of children with short telomere syndromes and nearly-adult mutation carriers. While TL above the 50th age-adjusted percentile had a 100 percent negative predictive value for clinically relevant mutations, the lower threshold in mutation carriers was age-dependent, and adult mutation carriers often overlapped with the lowest decile of controls.

However, Armanios found that the extent of telomere shortening correlated with a patient's age of diagnosis, as well as the short telomere syndrome phenotype: extremely short TL was linked to bone marrow failure and immunodeficiency in children and young adults, while milder defects often manifested as pulmonary fibrosis and emphysema in adults.

Importantly, the team saw that the the flowFISH assay maintained consistency and reproducibility with a 5 percent difference in TL. Comparing data published by the University of British Columbia Cancer Agency, which used flowFISH to measure TL, the researchers found they had a coefficient of variation of 5 percent. The normal TL range, which is between 8 and 13 kilobases of DNA, were also similar for both labs.

The researchers then examined whether TL can be used to identify patients with germline defects in telomere maintenance. Measuring the TLs of 100 people from 60 families carrying mutations in genes linked to telomere and telomerase, the team found that 73 individuals had symptoms of diseases associated with shortened telomeres. Armanios and her colleagues found 21 patients with bone marrow failure, for example, as well as 41 with mild pulmonary fibrosis or emphysema. The 21 with bone marrow failure, they added, were on average three decades younger than the 41 with mild pulmonary fibrosis/emphysema.

"We think there is an attrition in people with telomerase mutations who have very severe shortening," Armanios explained. "You hardly ever see adults with a high delta telomere length, as most who have different diseases earlier in life don't survive."

The team, therefore, believes that flowFISH-derived normal TL range can potentially be defined and standardized as a gold standard, depending on the patient's age and severity of disease.

"Physicians [therefore] can be sure that this diagnosis of short telomere syndromes is excluded at that threshold," Armanios explained. "The interpretation of telomere length should then consider the [patient's] clinical history, the deviation from the median for age, and genetic information."

flowFISH is meant for clinical use but a handful of companies have also set up shop, claiming they can predict a person's cellular age and health based on telomere lengths, but with qPCR-based methods. California-based TeloYears, for example, offers its qPCR blood-based test kit for $99 with a turnaround time of three to four weeks.

"Using qPCR, we amplify and measure three different DNA sequences, the telomeric DNA and two single copy genes," TeloYears VP of Marketing Greg Tomita explained. Averaging the values of two single copy genes, the company performs a ratio of the telomeric sequence value to the single-copy gene ratio, which determines the relative average telomere length per cell.

TeloYears runs its test in its CLIA-certified lab. It works by comparing a consumer's telomere length to its own representative reference population of over 10,000 individuals across a broad age range between 20 and 80 years old. TeloYears' aging test measure total leucocyte telomeric DNA from whole-blood samples, including lymphocytes and granulocytes.

Armanios, however, pointed out that PCR-based testing does not provide a person's biological age accurately or help make an accurate clinical diagnosis.

"If you use PCR to diagnose short telomeres, you miss one-third of the patients with certain issues because it's not precise enough of a measurement," she explained.

While the PCR tests are cheaper than the flowFISH assay, which costs $400, Armanios noted that recent studies have found that different environmental factors and methods of DNA preparation can cause different and questionable answers for PCR-based tests. She also highlighted that in contrast to PCR, TL measurement using flowFISH can be standardized across different days and laboratories.

Armanios plans to define the role of flowFISH TL testing beyond the current study and hopes to "set rigorous standards for reproducibility and clinical interpretation of other clinical research and diagnostics in this area."

Importantly, the researchers believe the flowFISH assay can be used to make diagnosis and treatment decisions in certain cases. In addition, they believe the test's availability will potentially help provide molecular information to hematologists and lung doctors to better confirm certain risks for their patients.

Armanios believes that new research will uncover several other indicators and applications of TL testing by flowFISH in the next few years. While research labs across the US could potentially develop their own versions of the assay, Armanios does not envision developing test kits that clinicians can use in hospitals.

"The protocols are published, so theoretically others could test if they can replicate the data we and other [labs] have obtained, but I suspect that this assay will more likely be a send out at least in the short-term," she said.

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