A Model System to Study the Instability of the GAA Triplet Repeat Expansion in Friedreich Ataxia

Sanjay I. Bidichandani, M.B.B.S., Ph.D.
Assistant Professor, Biochemistry & Molecular Biology, and Pediatrics
Oklahoma University Health Sciences Center

The following is an update on research funded by NAF in 1999.

Friedreich ataxia, the most common inherited ataxia, is caused by a deficiency of frataxin. Frataxin is essential for the maintenance of appropriate levels of iron in the mitochondria, the powerhouse of the cell. In 1996, a pivotal study showed that this deficiency of frataxin was caused by a large GAA triplet repeat expansion (100 - 1700 triplets) inboth copies of the gene that codes for frataxin (Ref. 1).Unaffected individuals also have GAA triplet sequences in theirfrataxin genes, but these are typically very short (< 40triplets). Expansion of the GAA triplet repeat sequence beyond 100 triplets results in the shutting off of the frataxin gene (Ref.2), and the consequent deficiency of frataxin protein. Severalparameters of gene function and disease severity have shown acorrelation with repeat length: longer repeats are associated withan increasingly severe deficiency of frataxin (and its function),and clinical severity. Understanding the factors that determinethe length of the GAA expansion, possibly different in differenttissues, is clearly important to understand disease pathogenesis -and perhaps in the future could yield clues to strategies toreverse this pathologic process.

Nearly 1% of the generalpopulation carries a large GAA triplet repeat expansion in onecopy of their frataxin gene (with a notable exception of theJapanese population), making this a fairly common human genemutation. These individuals, termed "carriers", have approximatelyhalf the normal level of frataxin, which is compatible with anormal, disease-free state. Clearly, to maintain such a highprevalence of this mutation in the population, expansions ofotherwise normal repeat tracts would have to expand withremarkably high frequency.

Many questions need to beanswered. What causes the otherwise normal GAA triplet repeat toexpand into the disease range? How does a repeat tract with about40 triplets (premutation) expand to contain more than ten timesits original length (mutation) in only one generationaltransmission? Why does it happen as frequently as is expected fromthe population studies? Why do patients have varying repeatlengths, and therefore a wide range of clinical severity? Why dodifferent tissues from the same patient have varying repeatlengths? Can the process of expansion or contraction of the GAAtriplet repeat be controlled?

It was decided that a simplemodel system to mimic the instability of the GAA repeat in the labwas needed, which could possibly help answer some of the abovequestions.
Development of a modelsystem to study the behavior of the GAA triplet repeatexpansion Lymphocytes, cells circulatingin the blood, can be "transformed" into immortal "lymphoblastoid"cells in the lab using the Epstein Barr virus.
These cells areuseful for analyzing DNA, RNA, proteins, and certainpathophysiological processes in patients with genetic diseases,and since they are immortal it also ensures a permanent supply ofcellular material. Although these cells may not be ideal for thestudy of some genes, they offer a unique setting to investigatethe behavior of "dynamic" mutations such as triplet repeatexpansions in the context of a human cellular environment.Furthermore, their immortal nature allows us to track changes asthese cells divide over a prolonged period of time.

Our experimental model systemwas as follows (Ref. 3):
Determinethe size of the GAA expansion(s) using DNA isolated from bloodsamples of patients and carriers Transformlymphocytes from these individuals into lymphoblastoid cells anddetermine the size of the GAA expansion(s) using DNA from thesetransformed cells Grow(culture) these cells under specified conditions in an incubatorand analyze the behavior of the GAA repeat in DNA extracted fromvarious generations (passages) of these cultured lymphoblastoidcells.

A total of 16 lymphoblastoidcell lines were established (7 patients and 9 carriers) comprisinga total of 23 GAA expansion bearing chromosomes. These wereselected in order to study a wide distribution of expansion sizes:the chromosomes analyzed contained between 97 and 1250 (average =744) GAA triplet repeats. Repeat sizes were calculated using areproducible and robust method involving "Southern blot" analysisof DNA. These cell lines were passaged over 20 to 39 generations(a single passage involved one week of growing time).

The GAA triplet repeatexpansion contracts when lymphocytes are transformed intolymphoblastoid cells.
Twenty chromosomes (6 patientsand 8 carriers) were analyzed prior to lymphoblastoidtransformation. The expansions ranged in size from 111 to 1105triplets (average = 750). Following transformation, as many as 12of these 20 chromosomes (i.e. 60%) showed contractions rangingfrom 67 to 236 triplets (average size of contraction = 134). Theremaining 8 chromosomes did not show any change in size.Significantly, none of the 20 chromosomes thus studied showed anexpansion. Since only a small proportion of lymphocytes areselected during the process of transformation, this may mean thateither the cells containing short repeats have a selectiveadvantage, or that during transformation the repeats do indeed getshorter, perhaps due to specific changes in the cellularphysiology.

The GAA triplet repeatexpansion is highly unstable in passaged lymphoblastoidcells
Perhaps the most significantfinding of this project was the discovery that expanded GAAtriplet repeats alter significantly in size as the cells arepassaged in the lab (Ref. 3). Basically, as the cells multiply inculture flasks the size of the GAA repeat does not remain static.This is potentially an important finding, since this system can beused as a model to further investigate this "instability" of theGAA triplet repeat within the context of human cells.

The extent of the changesobserved in the various GAA expansions is depicted graphically infigure 1A (cells from patients) and 1B (cells from carriers). Notethat only those chromosomes that showed length changes aredepicted in the two figures. Four of the seven patient cell lines,and five of the nine carrier cell lines showed these changes. Asseen in the two graphs, significant length changes are seen in GAAexpansions of all sizes, and at all stages of the experiment.
Nosignificant difference is seen in the behavior of the expansionsin cells from patients and carriers. While contraction was mostlikely during transformation of lymphocytes, expansions andcontractions were equally frequent during the passaging oflymphoblastoid cells. Some GAA expansions seem to be more unstablethan others, with six of them changing in size more than onceduring the experiment.

Given that the GAA tripletrepeat expansion is capable of significant length variation duringthe division of somatic cells, it is conceivable that there mayarise similar length variation during fetal development, such thatdifferent organs may harbor widely varying GAA expansion lengths.It is likely that this is the underlying basis for the discrepancybetween the observed repeat length in DNA isolated from blood (asused in most patients) and the overall clinical severity of somepatients, the latter obviously governed largely by the repeatlength in pathologically affected tissues.
We have recentlyreported one such patient where large repeat lengths in blood DNA(and in several other tissues) were surprisingly associated withvery late onset (symptoms started after age 40) and exceptionallymild Friedreich ataxia (Ref. 4).