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Published: April 2014Print Record of Viewing
Friedreich ataxia is an inherited disorder causing progressive damage to the nervous system due to a deficiency in frataxin, a critical protein for iron metabolism, antioxidant protection, and overall energy production. An immunoassay recently developed at Mayo Clinic to measuring frataxin concentration in whole blood and in dried blood spots is applicable to the diagnosis, population and newborn screening, and therapeutic monitoring of this disorder. Early diagnosis in the form of newborn screening appears promising for early intervention to reduce and prevent morbidity and mortality of Friedreich ataxia.
Presenter: Devin Oglesbee, PhD
Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice. Our speaker for this program is Dr. Devin Oglesbee, Consultant in Laboratory Genetics and Medical Genetics at Mayo Clinic, Rochester, Minnesota. Dr. Oglesbee discusses Friedreich ataxia—clinical presentation, the natural history of the disorder, and a new laboratory test for diagnosis. Thank you Dr. Oglesbee for presenting with us today. Thank you for the introduction, I have nothing to disclose.
And also, thank you for taking the time to learn more about a new test offered by Mayo Medical Laboratories. Today, I will discuss a new diagnostic assay for an inherited neurological disorder, Friedreich ataxia, based on the analysis of a protein, frataxin. Besides informing you about the new opportunity to quickly detect this disease, I would also like to educate you about the clinical presentation, natural history, and potential therapies available for this progressive neurological condition. Moreover, I will touch on the utility of protein-based Friedreich ataxia diagnostic testing, covering the acceptable specimen types, the assay method, its performance, and provide an update on testing recommendations in light of the availability of this new assay.
As you are aware there are numerous hereditary ataxias, encompassing a large and diverse group of degenerative diseases of the nervous system. And it is difficult to distinguish hereditary ataxias from acquired or sporadic forms of this condition. While incoordination, or ataxia, are a collective presentation and a common manifestation, the occurrence of hereditary ataxia is not confined to an early disease onset. Indeed, there are also adult-presenting hereditary ataxias that are led by a long latent phase. Moreover, the severity of disease symptoms will depend on the age of onset, type of condition, and other poorly understood factors that might include environmental exposure and yet-to-be identified genetic modifiers.
The most common inherited ataxia is called Friedreich ataxia. It was originally described by the German professor, pathologist, and neurologist, Nicolaus Friedreich, at the prestigious University of Heidelberg in Germany. He published a series of publications recounting a family with numerous affected individuals. Professor Friedreich noted that the condition caused progressive sensory loss, muscle weakness, and ataxia. Further clinical research over the last 150 years, summarized on the table on the right, has recognized that Friedreich ataxia presents with a variety of different conditions and symptoms: progressive ataxia, dysarthria in the form of slurred speech, absent lower limb reflexes, scoliosis, and pes cavus. And in the majority of affected individuals, you will see that cardiomyopathy develops and is a high cause of mortality. Affected individuals are at an increased risk for vision loss, hearing impairment, diabetes, and swallowing difficulties as shown by the percentages of individuals with these symptoms from several different large case series published to date.
More recently, it has been observed that there is a demarcation in the clinical presentation of Friedreich ataxia. In particular, that there are different forms of the disease, often called classic or atypical Friedreich ataxia. For classic Friedreich ataxia, the onset of symptoms occurs in childhood or early teens with poor balance, progressive speech impairment, and upper-limb ataxia. The age of onset can be between 10 to 15 years of age.
In over 60% of classic Friedreich ataxia, hypertrophic cardiomyopathy will develop and lead to the risk of arrhythmias and congestive heart failure in early life. The progression of these symptoms is difficult to predict and may depend on a variety of modulators. In contrast, atypical Friedreich ataxia is often divided into 2 groups based on the age of presentation. Late-onset Friedreich ataxia frequently refers to individuals who become progressively ataxic by 25 to 39 years of age. While very-late onset Friedreich ataxia describes individuals who are symptomatic above the age of 40. For both late-onset and very-late onset Friedreich ataxia, the disease progression is slower than classic Friedreich ataxia and the co-morbidities may also be different or delayed.
The prevalence of Friedreich ataxia is rare in the general population, with an incidence around 1 in 50,000 individuals depending on ancestry. For individuals with European, Indian, and Middle-Eastern descent, it is the most common inherited ataxia. In contrast, the condition appears to be absent in populations with Southeast Asian, Sub-Saharan African, or Native American ancestry.
Friedreich ataxia is different from sporadic or acquired ataxias due to the very fact that it is inherited in the following manner, as diagramed on the right. Unaffected parents can pass copies of a defective Friedreich ataxia gene onto all future offspring, such that their children have an equal opportunity to present with Friedreich ataxia at a rate of 25%. In addition, 50% of offspring could be asymptomatic carriers, just like their parents. This type of inheritance is called autosomal recessive, as it requires 2 defective copies of a gene to dictate a clinical presentation. Thus, unless there is genetic intervention before each pregnancy, every child has the same risk (1 in 4) of being affected.
The mutations that cause Friedreich ataxia are found in the FXN gene on chromosome 9q21.11. This gene encodes for a mitochondrial protein, frataxin, which has several unique functions in the mitochondria and cell. Specifically, it is involved in the biosynthesis of iron-sulfur clusters in enzymes involved in metabolism. It is also important for iron localization and impacts iron distribution in the cell. Also, when frataxin is present, it reduces iron-mediated oxidative damage. Mutations that cause Friedreich ataxia lead to reduced frataxin levels in all tissues. The most common cause of Friedreich ataxia is GAA-trinucleotide repeat expansions that are found in intron 1 of FXN.
In most individuals, intron 1 of FXN contains a small number of GAA repeats, often less than 33-trinucleotide repeats. There are some people, perhaps as many as 1 in 100, where intron 1 of FXN contains an expanded GAA-trinucleotide repeat, more than 66 repeats and as many as 1700 repeats. The outcome of GAA-repeats greater than 66 is the loss of FXN expression. When this occurs, RNA is not synthesized due to the fact that the FXN gene becomes more tightly bound to acetylated histones when a large GAA-repeat expansion is present. This turns off the gene, and in turn, leads to the loss of frataxin protein levels.
While GAA-repeats greater than 66 lead to reduced frataxin and disease, classic Friedreich ataxia is associated with even longer GAA-repeat lengths, commonly greater than 500 repeats, or more, on both alleles. In 2 to 5% of patients, Friedreich ataxia is caused by a point mutation inherited opposite a GAA-repeat expansion allele. These point mutations are often exonic and lead to amino acid changes that reduce frataxin levels. In rare circumstances, Friedreich ataxia is caused by a full or partial gene deletion that is inherited opposite another mutation. Deletions also lead to lower frataxin levels.
Laboratory testing is very important for the detection and diagnosis of Friedreich ataxia. Traditionally, DNA testing is performed to measure GAA-repeat length sizes. The laboratory analytical time for the analysis of GAA-repeat lengths in whole blood DNA can be as long as 14 days. Moreover, a big pitfall to the DNA GAA-repeat length measurement method is the fact that is misses point mutations and deletions unless it is coupled with other DNA analytical methods. To address this shortfall, molecular genetic laboratories often include DNA gene sequencing alongside GAA-repeat length determination. This DNA panel testing has increased the sensitivity of DNA-based methods but also is unable to detect exonic deletions unless it incorporates additional analytical methods, like deletion/duplication arrays or a method called MLPA. Deletion testing is not broadly used by laboratories at this time.
In order to side-step the shortcomings of DNA testing, we developed and validated an analytical method to diagnose Friedreich ataxia by measuring frataxin levels from whole blood or dried blood spots. The protein-based assay is specific and sensitive for Friedreich ataxia, just like the DNA panel test. However, it is also applicable to therapeutically monitor blood levels of frataxin, it is multiplexible with other analytes, and useful for high-throughput screening, or HTS screening, and is a cost-effective method to diagnose Friedreich ataxia. And, as with other protein assays, it is more easily reimbursable than DNA-based genetic testing. Lastly, it is not recommended to be used to determine carrier status, which is one of the applications of DNA panel testing.
The protein-based assay uses immunocapture methods to measure frataxin levels. As shown here in step 1, fluorescent microbeads are coated with frataxin-specific capture antibodies. Dried blood spot or whole blood eluents are incubated with coated microbeads and the antibodies bind available frataxin. In step 3, a detection antibody is added to the frataxin-coated beads, which sandwiches the frataxin between 2 antibodies and permits the bead to be labeled by a fluorescent reporter dye, shown in step 4. The fluorescence of each bead is counted and correlated with a standard recombinant protein curve in order to determine endogenous frataxin levels in a sample. As with all microbead assays, there is the capability to mix microbeads specific to different analytes together. In our assay, we used a second protein, as an internal control alongside the target protein, frataxin.
The frataxin assay can be performed on 2 different types of specimens, whole blood or dried blood spots. Whole blood collected in EDTA, sodium or lithium heparin. Blood is acceptable up to 70 days if kept frozen. In contrast, dried blood spots can be collected onto 1 of 3 different types of paper, commonly used for newborn screening, such as Whatman 903 paper or the Mayo Clinic Supplemental newborn screening card. Dried blood spots are stable at room temperature for up to 30 days.
Reference values are slightly different between pediatric and adult individuals. For individuals less than 18 years of age, normal frataxin levels are greater or equal to 19 ng/mL, while adults have frataxin levels that are greater than 21 ng/mL. A normal result is reported for any individual whose frataxin levels are measured above 19 or 21 ng/mL, depending on age. For every test, an interpretation about the clinical significance of the result is also provided with each report. Moreover, as mentioned previously, frataxin blood levels are not recommended for determining carrier status for Friedreich ataxia.
The performance of the assay for measuring blood frataxin levels is excellent. As seen on the top panel on the right-hand side, a simple regression analysis of titrated frataxin levels results in a coefficient of determination, or R-squared, of 0.99985. The linearity has been determined out to 200 ng/mL, and the limit of detection is less than 1 ng/mL. On the lower panel, a box-and-whisker plot shows normal and disease ranges. In green, you can observe that the normal range starts at 19 or 21 ng/mL and extends up to greater than 80 ng/mL. In contrast, the affected range for Friedreich ataxia, seen labeled as in red as FA on the far right hand side, ranges from 1 to12 ng/mL, and is easily distinguishable from normal individuals. Also observed, are frataxin levels measured for very-late onset Friedreich ataxia patients, labeled here as VLO-FA. These individuals have frataxin levels slightly below the normal range, but can also overlap frataxin levels measured in a cohort of Friedreich ataxia carriers, making the protein assay ineffective for the determination of carrier status as it cannot exclude the possibility of a diagnosis of very-late onset Friedreich ataxia.
The imprecision of the frataxin assay is shown on the following slide. On the top table, intraassay imprecision coefficient of variation, or CV, is characterized. As seen for blood or dried blood spot, frataxin levels, or FXN, is less than 15% for protein levels at 3 different levels. For interassay imprecision, the percent coefficient of variation is a little larger but ranges from 9.8 to 15.8%.
As shown by the details of assay performance, the protein-based test for frataxin is useful for obtaining a diagnosis and excluding Friedreich ataxia from a list of potential differential diagnoses. In contrast, DNA panel testing can be used for detecting Friedreich ataxia but it is especially helpful to determine carrier status in asymptomatic individuals, measuring GAA-repeat lengths, and confirming point mutations or deletions.
The availability of an assay for frataxin can expedite a diagnosis of Friedreich ataxia if the testing strategy that is shown in this diagram is followed. First, if there is a clinical suspicion of Friedreich ataxia, or listed here as FRDA, the best assay is a quantitative frataxin level in dried blood spot or blood. This single assay will allow you to obtain a diagnosis, if frataxin levels are low, or refute the possibility of Friedreich ataxia, if measured levels are within the normal range. In the rare circumstance that a result for frataxin is close, but not within the normal range, for instance, a blood level of 19 ng/mL for an adult whose normal range begins at 21 ng/mL; carrier status cannot be excluded. In this rare circumstance, we recommended considering DNA panel testing, followed by deletion testing, if needed. For all normal results, there are other inherited ataxias that should be part of a differential diagnosis for individuals presenting with symptoms similar to Friedreich ataxia.
When faced with normal frataxin results, consider the following conditions listed on this slide that should be part of every Friedreich ataxia differential diagnosis.
While most treatment for Friedreich ataxia has not been shown to alter its natural history, and encompasses standard management of heart failure, arrhythmias, diabetes, hearing loss, and the like, there is a growing pipeline of new experimental therapies that may show promise from further study. There are clinical trials for compounds that reduce oxidative stress and increase mitochondrial function, such as coenzyme-Q and vitamin E. There are also other compounds under review that aim to modulate frataxin-mediated metabolic pathways through nutritional approaches or by specifically activating a protein, Nrf2, a transcription factor important for antioxidant pathways. Moreover, there is research on erythropoietin, or EPO, that demonstrate that this growth factor can enhance or stabilize frataxin in blood specimens. Although, additional research is needed to determine whether this induction is blood-specific or more widespread. Additional compounds that show therapeutic promise are specific histone-deacetylase inhibitors that can apparently relax the tight GAA-repeat length-mediated silencing of the FXN gene, and increase frataxin expression. But further work is needed to ensure that unique HDAC inhibitors, such as these, are capable to specifically target frataxin protein expression without toxic side effects. Lastly, gene therapy, through the introduction of copies of normal FXN genes, is also a pipeline of potential therapy.
For more information about Friedreich ataxia, I recommend a couple of resources that are helpful for both healthcare practitioners and patients. First, the Friedreich Ataxia Research Alliance, and its website, www.curefa.org, is very informative. In addition, the National Ataxia Foundation, the Muscular Dystrophy Association, the Office of Rare Diseases Research, and for contact information on current clinical trials, look at www.clinicaltrials.gov
This ends my presentation on frataxin protein testing, and I thank you for your attention and I hope that you found this presentation to be educational.