Recently, a team of researchers and physicians at Mayo Clinic in Rochester identified a new type of renal amyloidosis derived from the protein apolipoprotien C-II (Apo-CII). The type is so rare that it was met with skepticism from other health care providers, but the group’s teamwork and indisputable data persevered and has also brought an end to one woman’s long journey for answers.
Amyloidosis is a rare disease in which misfolded proteins, called amyloid, become insoluble and abnormally accumulate in various tissue sites. It also causes damage to major organs, the digestive tract, or even the nervous system. There are about 30 known types of amyloid and, as yet, there is no cure for this disease, which can be life-threatening. Diagnosing the type is critical because proper treatment can help patients manage their symptoms and limit the production of amyloid.
Where It All Began
The case study, recently published in the Journal of the American Society of Nephrology, started when a 61-year-old white female’s kidney biopsy sample was sent to Mayo Medical Laboratories for amyloid typing. The woman had been hospitalized at another health care institution for peripheral edema and had been diagnosed with renal amyloidosis.
“It was pretty clear, from the evaluation we did in our clinic, that this woman did not have the type of amyloid she was alleged to have had.”
- Morie Gertz, M.D., Chair of Internal Medicine at Mayo Clinic
The woman’s referring physician diagnosed her type as amyloid light chain (AL) amyloidosis, the most common form of systemic amyloidosis in the U.S., and wanted confirmation from Mayo Clinic. The doctor also recommended a bone marrow transplant—typical treatment for a patient with AL amyloidosis. For the initial consult, he referred the patient to Morie Gertz, M.D., Chair of Internal Medicine at Mayo Clinic, who also heads the Amyloidosis Therapy Group, regarded as one of the best clinics in the world.
“It was pretty clear, from the evaluation we did in our clinic, that this woman did not have the type of amyloid she was alleged to have had,” says Dr. Gertz.
The Mayo Clinic Approach to Typing Amyloid
Amyloid is associated with diseases as diverse as Alzheimer’s disease and plasma cell neoplasms. Some amyloidosis cases are hereditary, and others are due to longstanding chronic inflammation. So in order to know which type the 61-year-old patient actually had, Dr. Gertz needed her test results from his close colleague Paul Kurtin, M.D., a hematopathologist who directs the Clinical Tissue Proteomics Laboratory (CTPL), a premiere mass spectrometry facility at Mayo Clinic.
“The pathology features of these many amyloid types are basically the same in that they all cause protein deposits in the tissues and show up via Congo red (CR) staining,” says Dr. Kurtin, senior author on the study paper. “But CR analysis isn’t enough to distinguish among proteins. So the next step is typing of the amyloid to figure out which of these abnormal proteins is deposited in the tissue. For this test method, we cut out the amyloid deposits from the tissues using microdissection and then introduced them into a mass spectrometer. This analysis allows us to identify the specific protein types present in the amyloids.”
This “shotgun proteomic testing” for amyloidosis was developed at Mayo Clinic in 2008 and was the first such method used in a clinical laboratory. Since then, CTPL has typed more than 10,000 amyloid cases using this proteomic method—an amazing feat considering the rarity of the disease. For example, it’s estimated that between 1,500 and 2,500 people are diagnosed with AL amyloid annually in the U.S. (6 to 10 patients per million people).
Since 2008, the Clinical Tissue Proteomics Laboratory has typed more than 10,000 amyloid cases using this proteomic method—an amazing feat considering the rarity of the disease.
Teamwork Transforms Test Method
Because this form of testing is much more accurate and reproducible than prior methods like immunohistochemistry, immunofluorescence (particularly with kidneys), and immunoelectron microscopy, it has been hugely successful at Mayo Clinic and has transformed the way amyloidosis is approached in the clinic.
“And we really owe this to my predecessor, Dr. Ahmet Dogan and his collaborators, the visionaries behind this test method,” says Dr. Kurtin.
Given CTPL’s extensive experience in typing amyloidosis, the laboratory team was able to identify something different in the woman’s protein sample. “We recognized that this woman had a novel kind of amyloidosis that we thought was due to apolipoprotein CII. We called the new form AApoCII,” he says. “That was our hypothesis.”
His findings were surprising—even to Dr. Gertz.
“When Paul told me the amyloid type, I had never heard of it and wondered, ‘Does it really exist?’” he says. “But I had to believe it because the diagnostic accuracy of his laboratory is absolutely unparalleled. He also found an abnormality in the amyloid protein, and that abnormality suggested it could be an inherited mutation.”
Bringing in Bioinformatics
To further demonstrate this finding, the doctors reached out to Surendra Dasari, Ph.D., a bioinformatician in Mayo Clinic’s Department of Health Sciences Research. Dr. Dasari has designed many novel proteomics software programs, one of which is an informatics pipeline called DirecTag-TagRecon that he developed as a post-doc at Vanderbuilt University School of Medicine.
“The pipeline uses a concept called sequence tagging to interrogate the tandem mass spectra for unanticipated mutant peptides,” says Dr. Dasari. “I brought it to Mayo Clinic and validated it for clinical use so that we can identify yet unknown pathogenic mutations.”
Hence, DirecTag-TagRecon was able to find the Apo-CII mutation in the female patient’s amyloid deposits with a specific amino acid change that had never before been described, consistent with the CTPL findings.
Confirming the Hereditary Link
Meanwhile, the patient had arrived at Mayo Clinic to consult with Dr. Gertz, as well as receive genetic counseling for her and her two children (a son, 22, and daughter, 23).
Given the possible hereditary nature of the disease because of the amino acid change, both children were interested in knowing if they had the mutation identified in their mother’s kidney tissue.
“So I called Ed Highsmith and asked him if he could design a test to screen this mother and her kids, who were all here at Mayo together,” says Dr. Gertz. “And he said he could, so we collected blood samples and sent them to him.”
Edward Highsmith, Jr., Ph.D., directs the Molecular Genetics Laboratory at Mayo Clinic. With the invaluable help of his then molecular genetics fellow Linda Hasadsri, M.D., Ph.D., he was able to design a specific DNA sequencing assay. With the new test, they found a DNA sequencing base-pair change in the mother that corresponded to the amino acid change in the protein Dr. Dasari had discovered via mass spectrometry.
Discovery Met with Doubt
Once certain of the patient’s Apo-CII protein abnormality, Dr. Gertz informed the woman of the diagnoses. He also contacted her health care provider who had sent the original tissue samples to Mayo Medical Laboratories for analyses.
“The referring physician didn’t believe this new amyloid type was the correct diagnosis . . . . The difference is, I know Paul Kurtin. And if he says this is what it is, there’s no mistake."
- Dr. Morie Gertz
“The referring physician didn’t believe this new amyloid type was the correct diagnosis,” says Dr. Gertz, who co-authored the study paper. “He didn’t believe the mass spec data because he had never heard of it either. The difference is, I know Paul Kurtin. And if he says this is what it is, there’s no mistake.”
After further discussions with the health care provider, all physicians were eventually in agreement that this was indeed a new type of amyloid.
A Path Forged with New Information
Although there are treatments for other amyloid types, a specific targeted therapy for AApoCII does not exist. So, given the mother’s advanced renal damage, she is currently being evaluated for a kidney transplant—her only option at this point, according to Dr. Gertz.
As for her children? Screening confirmed the woman’s daughter does not share the same mutation as her mother. Unfortunately, the son does carry it, although he remains asymptomatic for renal disease. And unlike his mother, he will at least not have to endure a similar medical journey to derive a correct diagnosis.
“Knowing the son is at risk, he can be monitored very simply with blood and urine tests,” says Dr. Gertz. “If he starts developing changes in his samples, physicians will know the reason right away, without futzing around for a year trying to figure out what disease he has. This will short circuit a lot of unnecessary, invasive, and risky testing—maybe even avoid a kidney biopsy. And now that he knows he’s a carrier, it may affect his decision on whether or not to bring children into the world.”
Dr. Gertz estimates it could be several decades before the son shows symptoms, which would support strong evidence this mutation is hereditary. And perhaps by then, he’ll have more treatment options than his mother.
“It’s hard to imagine this is not a hereditary form,” Dr. Gertz says. “We found the abnormality in the mother’s amyloid, then we found the mutation in her blood, and now we find the mutation in her son. And since she didn’t become symptomatic until her late 50s/early 60s, we would expect the course of the disease to be very similar for him.”
Applying New Knowledge to Previous Cases
Having solidified this novel amyloid type, the Mayo Clinic team now wanted to reexamine those 10,000-plus amyloid cases that CTPL had done using mass spectrometry. Dr. Dasari, after doing a deep dive into Mayo Medical Laboratories’ database, was able to retrospectively identify seven other patients who showed the same pattern of the Apo-CII abnormality.
“These cases were still undiagnosed because, at the time of the original testing, we weren’t sure what type of amyloid these seven patients had because amyloidosis due to Apo-CII had not yet been described."
- Paul Kurtin, M.D., Mayo Clinic hematopathologist
“These cases were still undiagnosed because, at the time of the original testing, we weren’t sure what type of amyloid these seven patients had because amyloidosis due to Apo-CII had not yet been described,” says Dr. Kurtin. “Surendra brought those cases to me, and we verified that the data indicated this abnormality.”
The collaboration at Mayo Clinic doesn’t end here. Enter Samih Nasr, M.D., anatomic pathologist in Mayo Clinic’s Division of Anatomic Pathology, the final critical link in this group effort. An expert in renal pathology, Dr. Nasr looked at the kidney biopsies from the seven patients and verified that their pathology features were all similar. He also obtained important clinical data from the institutions where these patients were originally seen. Dr. Nasr is now in the process of contacting the treating nephrologists, or hematologists, of these seven individuals to let them know the new findings.
“These health care providers will then contact their patients to inform them that they have a novel, hereditary form of amyloidosis,” Dr. Nasr says. “The patients and their families will then be offered genetic testing to see what further information we can learn in helping them with this disease.”
International Cooperation and Recognition
AApoCII has since been recognized by the International Society of Amyloidosis as a new amyloid type. This is the result of the Mayo Clinic team’s study paper, as well as another paper by co-author Giampaolo Merlini, M.D, who has studied this mutation in Pavia, Italy. Dr. Merlini directs the Center for Research and Treatment of Systemic Amyloidosis.
“Before we encountered the 61-year-old female patient from our study, we did an analysis on a couple of cases Dr. Merlini sent to us that he thought were [this new type of] amyloid,” says Dr. Kurtin. “So we were clued in that AApoCII was a potential new amyloid type, by virtue of this earlier interaction with Dr. Merlini. These research collaborations just make you smarter about what’s going on in the field—and can accelerate potential discoveries that will change patient care.”
The collaborators agree, this discovery could not have happened without a strong culture of collaboration at Mayo Clinic. “We’re used to working together as a team without competitive pressures and we all get along well,” says Dr. Kurtin. “Mayo really promotes an environment in which we can collaborate more freely and readily. Everyone who contributed to this paper works together day-to-day to take care of our patients.”
Christoph Bahn covers emerging research and discovery for Mayo Medical Laboratories. His writing has also appeared in The New York Times, Los Angeles Times, and Smithsonian Air & Space. He divides his time between Southern California and Northwest Ohio.