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Frataxin levels in peripheral tissue in Friedreich ataxia.

Lazaropoulos M, Dong Y, Clark E, Greeley NR, Seyer LA, Brigatti KW, Christie C, Perlman SL, Wilmot GR, Gomez CM, Mathews KD, Yoon G, Zesiewicz T, Hoyle C, Subramony SH, Brocht AF, Farmer JM, Wilson RB, Deutsch EC, Lynch DR - Ann Clin Transl Neurol (2015)

Bottom Line: Such mutations, usually expanded guanine-adenine-adenine (GAA) repeats, give rise to decreased levels of frataxin protein in both affected and unaffected tissues.Site-directed mutant frataxin was also transfected into human embryonic kidney cells to model results from specific point mutations.The G130V mutation led to decreased levels of frataxin in vitro as well as in vivo, while the R165C mutation produced normal immunoreactive levels of frataxin both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics and Neurology, Children's Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania Philadelphia, Pennsylvania, 19104.

ABSTRACT

Objective: Friedreich ataxia (FRDA) is an autosomal recessive ataxia resulting from mutations in the frataxin gene (FXN). Such mutations, usually expanded guanine-adenine-adenine (GAA) repeats, give rise to decreased levels of frataxin protein in both affected and unaffected tissues. The goal was to understand the relationship of frataxin levels in peripheral tissues to disease status.

Methods: Frataxin levels were measured in buccal cells and blood, and analyzed in relation to disease features. Site-directed mutant frataxin was also transfected into human embryonic kidney cells to model results from specific point mutations.

Results: There was no evidence for change in frataxin levels over time with repeated measures analysis, although linear regression analysis of cross-sectional data predicted a small increase over decades. GAA repeat length predicted frataxin levels in both tissues, and frataxin levels themselves predicted neurological ratings (accounting for age). Compound heterozygous patients for a GAA expansion and a point mutation in FXN generally had lower levels of frataxin than those homozygous for the presence of two GAA repeat expansions, though levels varied dramatically between tissues in some compound heterozygotes for point mutations. The G130V mutation led to decreased levels of frataxin in vitro as well as in vivo, while the R165C mutation produced normal immunoreactive levels of frataxin both in vitro and in vivo. Start codon mutations led to low levels of frataxin in buccal cells but preserved immunoreactive frataxin levels in blood.

Interpretation: The present data show that peripheral frataxin levels reflect disease features in FRDA, but emphasize the need for interpretation of such levels in the context of specific mutations.

No MeSH data available.


Related in: MedlinePlus

Relative levels of frataxin in blood of FRDA patients with selected point mutations. As three patients with protein truncating mutations in the first few amino acids had extremely low ratios of frataxin in buccal cells to blood, we addressed the localization in one such individual in greater detail (A). In this subject, frataxin levels were extremely low in buccal cells, while in the carrier range in blood. His father, who carried the start codon mutation c.2delT, had a similar pattern, being in the carrier range in buccal cells and in the control range in blood. When blood was fractionated, the high level in blood in the FRDA patient and the father reflected the RBC component. His mother, who did not carry this mutation, had carrier levels in all fractions. (B) This pattern of high levels in RBC was not found in subjects with 2 GAA repeats or in a subject with a frameshift mutation at amino acid 34 (c.100delG). (C) We assessed the molecular weight (MW) of frataxin in these subjects to insure that the atypical levels did to reflect processing issues. Frataxin had the same MW in buccal cells and blood in control individuals, c.100delG subject, and the subject with the start codon mutation (c.2delT). FRDA, Friedreich ataxia; RBC, red blood cell; GAA, guanine–adenine–adenine.
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fig06: Relative levels of frataxin in blood of FRDA patients with selected point mutations. As three patients with protein truncating mutations in the first few amino acids had extremely low ratios of frataxin in buccal cells to blood, we addressed the localization in one such individual in greater detail (A). In this subject, frataxin levels were extremely low in buccal cells, while in the carrier range in blood. His father, who carried the start codon mutation c.2delT, had a similar pattern, being in the carrier range in buccal cells and in the control range in blood. When blood was fractionated, the high level in blood in the FRDA patient and the father reflected the RBC component. His mother, who did not carry this mutation, had carrier levels in all fractions. (B) This pattern of high levels in RBC was not found in subjects with 2 GAA repeats or in a subject with a frameshift mutation at amino acid 34 (c.100delG). (C) We assessed the molecular weight (MW) of frataxin in these subjects to insure that the atypical levels did to reflect processing issues. Frataxin had the same MW in buccal cells and blood in control individuals, c.100delG subject, and the subject with the start codon mutation (c.2delT). FRDA, Friedreich ataxia; RBC, red blood cell; GAA, guanine–adenine–adenine.

Mentions: In addition, there were substantial discrepancies in frataxin levels between blood and buccal cells in multiple subjects with point mutations and a single GAA repeat (not shown). These individuals had blood frataxin levels that were in the expected range for carriers or controls, while having buccal cell frataxin levels in the lower portion of the patient range. While the buccal to blood ratio varied among most subjects slightly, only five individuals had ratios of less than 0.10. Two of these individuals carried truncation mutations in the first 5 amino acids (c.2delT and c.11-12delTC). A third subject who carried a c.2delT mutation in conjunction with a pathologic but short (90) GAA repeat had disease levels in buccal cell (22%) with high control levels (157%) in blood. To understand this, we analyzed the distribution of frataxin in blood in more detail in one subject with the c.2delT mutation (subject PA 002 in reference 7). The proband had very low levels of frataxin in buccal cells (4.5% of control), consistent with his clinical phenotype of FRDA, but higher amounts of frataxin in whole blood (62.8% of control). To determine the source of the frataxin in blood, whole blood was fractionated from this subject and his father (who also carries the c.2delT mutation), and frataxin was measured from buccal cells, whole blood, platelets, and the RBC pellet (Fig.6A). The RBC pellet, which contains low levels of mitochondria but the highest absolute amounts of frataxin,14 was responsible for the relatively elevated frataxin level seen in whole blood in the proband and for an elevated amount of frataxin in blood in his c.2delT carrier father. This suggests that this start codon mutation in FXN leads to retained immunoreactive frataxin in RBC through an unknown mechanism. Fractionated blood from a subject with a frameshift mutation at amino acid 34 (c.100delG) and in subjects who are homozygous for GAA repeat expansions did not show this pattern (Fig.6B). Although there was variability in the amount of frataxin in different fractions in blood in the c.100delG subject, the variability was less than in the subject heterozygous for a c.2delT mutation.


Frataxin levels in peripheral tissue in Friedreich ataxia.

Lazaropoulos M, Dong Y, Clark E, Greeley NR, Seyer LA, Brigatti KW, Christie C, Perlman SL, Wilmot GR, Gomez CM, Mathews KD, Yoon G, Zesiewicz T, Hoyle C, Subramony SH, Brocht AF, Farmer JM, Wilson RB, Deutsch EC, Lynch DR - Ann Clin Transl Neurol (2015)

Relative levels of frataxin in blood of FRDA patients with selected point mutations. As three patients with protein truncating mutations in the first few amino acids had extremely low ratios of frataxin in buccal cells to blood, we addressed the localization in one such individual in greater detail (A). In this subject, frataxin levels were extremely low in buccal cells, while in the carrier range in blood. His father, who carried the start codon mutation c.2delT, had a similar pattern, being in the carrier range in buccal cells and in the control range in blood. When blood was fractionated, the high level in blood in the FRDA patient and the father reflected the RBC component. His mother, who did not carry this mutation, had carrier levels in all fractions. (B) This pattern of high levels in RBC was not found in subjects with 2 GAA repeats or in a subject with a frameshift mutation at amino acid 34 (c.100delG). (C) We assessed the molecular weight (MW) of frataxin in these subjects to insure that the atypical levels did to reflect processing issues. Frataxin had the same MW in buccal cells and blood in control individuals, c.100delG subject, and the subject with the start codon mutation (c.2delT). FRDA, Friedreich ataxia; RBC, red blood cell; GAA, guanine–adenine–adenine.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4554444&req=5

fig06: Relative levels of frataxin in blood of FRDA patients with selected point mutations. As three patients with protein truncating mutations in the first few amino acids had extremely low ratios of frataxin in buccal cells to blood, we addressed the localization in one such individual in greater detail (A). In this subject, frataxin levels were extremely low in buccal cells, while in the carrier range in blood. His father, who carried the start codon mutation c.2delT, had a similar pattern, being in the carrier range in buccal cells and in the control range in blood. When blood was fractionated, the high level in blood in the FRDA patient and the father reflected the RBC component. His mother, who did not carry this mutation, had carrier levels in all fractions. (B) This pattern of high levels in RBC was not found in subjects with 2 GAA repeats or in a subject with a frameshift mutation at amino acid 34 (c.100delG). (C) We assessed the molecular weight (MW) of frataxin in these subjects to insure that the atypical levels did to reflect processing issues. Frataxin had the same MW in buccal cells and blood in control individuals, c.100delG subject, and the subject with the start codon mutation (c.2delT). FRDA, Friedreich ataxia; RBC, red blood cell; GAA, guanine–adenine–adenine.
Mentions: In addition, there were substantial discrepancies in frataxin levels between blood and buccal cells in multiple subjects with point mutations and a single GAA repeat (not shown). These individuals had blood frataxin levels that were in the expected range for carriers or controls, while having buccal cell frataxin levels in the lower portion of the patient range. While the buccal to blood ratio varied among most subjects slightly, only five individuals had ratios of less than 0.10. Two of these individuals carried truncation mutations in the first 5 amino acids (c.2delT and c.11-12delTC). A third subject who carried a c.2delT mutation in conjunction with a pathologic but short (90) GAA repeat had disease levels in buccal cell (22%) with high control levels (157%) in blood. To understand this, we analyzed the distribution of frataxin in blood in more detail in one subject with the c.2delT mutation (subject PA 002 in reference 7). The proband had very low levels of frataxin in buccal cells (4.5% of control), consistent with his clinical phenotype of FRDA, but higher amounts of frataxin in whole blood (62.8% of control). To determine the source of the frataxin in blood, whole blood was fractionated from this subject and his father (who also carries the c.2delT mutation), and frataxin was measured from buccal cells, whole blood, platelets, and the RBC pellet (Fig.6A). The RBC pellet, which contains low levels of mitochondria but the highest absolute amounts of frataxin,14 was responsible for the relatively elevated frataxin level seen in whole blood in the proband and for an elevated amount of frataxin in blood in his c.2delT carrier father. This suggests that this start codon mutation in FXN leads to retained immunoreactive frataxin in RBC through an unknown mechanism. Fractionated blood from a subject with a frameshift mutation at amino acid 34 (c.100delG) and in subjects who are homozygous for GAA repeat expansions did not show this pattern (Fig.6B). Although there was variability in the amount of frataxin in different fractions in blood in the c.100delG subject, the variability was less than in the subject heterozygous for a c.2delT mutation.

Bottom Line: Such mutations, usually expanded guanine-adenine-adenine (GAA) repeats, give rise to decreased levels of frataxin protein in both affected and unaffected tissues.Site-directed mutant frataxin was also transfected into human embryonic kidney cells to model results from specific point mutations.The G130V mutation led to decreased levels of frataxin in vitro as well as in vivo, while the R165C mutation produced normal immunoreactive levels of frataxin both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics and Neurology, Children's Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania Philadelphia, Pennsylvania, 19104.

ABSTRACT

Objective: Friedreich ataxia (FRDA) is an autosomal recessive ataxia resulting from mutations in the frataxin gene (FXN). Such mutations, usually expanded guanine-adenine-adenine (GAA) repeats, give rise to decreased levels of frataxin protein in both affected and unaffected tissues. The goal was to understand the relationship of frataxin levels in peripheral tissues to disease status.

Methods: Frataxin levels were measured in buccal cells and blood, and analyzed in relation to disease features. Site-directed mutant frataxin was also transfected into human embryonic kidney cells to model results from specific point mutations.

Results: There was no evidence for change in frataxin levels over time with repeated measures analysis, although linear regression analysis of cross-sectional data predicted a small increase over decades. GAA repeat length predicted frataxin levels in both tissues, and frataxin levels themselves predicted neurological ratings (accounting for age). Compound heterozygous patients for a GAA expansion and a point mutation in FXN generally had lower levels of frataxin than those homozygous for the presence of two GAA repeat expansions, though levels varied dramatically between tissues in some compound heterozygotes for point mutations. The G130V mutation led to decreased levels of frataxin in vitro as well as in vivo, while the R165C mutation produced normal immunoreactive levels of frataxin both in vitro and in vivo. Start codon mutations led to low levels of frataxin in buccal cells but preserved immunoreactive frataxin levels in blood.

Interpretation: The present data show that peripheral frataxin levels reflect disease features in FRDA, but emphasize the need for interpretation of such levels in the context of specific mutations.

No MeSH data available.


Related in: MedlinePlus