Friedreich ataxia is commonly caused by large expansions of a GAA triplet-repeat (GAA-TR) sequence in the first intron of the FRDA gene. We used small-pool PCR to analyze somatic variability among 7190 individual FRDA molecules from peripheral blood DNA of subjects carrying 12 different expanded alleles, ranging in size from 241 to 1105 triplets. Expanded alleles showed a length-dependent increase in somatic variability, with mutation loads ranging from 47% to 78%. We noted a strong contraction bias among long alleles (>500 triplets), which showed a 4-fold higher frequency of large contractions versus expansions. Some contractions were very large; of all somatic mutations scored, approximately 5% involved contractions of >50% of the original allele length, and 0.29% involved complete reversion to the normal/premutation length (< or =60 triplets). These observations contrast sharply with the strong expansion bias seen in expanded CTG triplet repeats in myotonic dystrophy. No somatic variability was detected in >6000 individual FRDA molecules analyzed from 15 normal alleles (8-25 triplets). A premutation allele with 44 uninterrupted GAA repeats was found to be unstable, ranging in size from 6 to 113 triplets, thus establishing the threshold for somatic instability between 26 and 44 GAA triplets. Analysis of an additional 7850 FRDA molecules from serially passaged lymphoblastoid cell lines carrying nine expanded alleles (132-933 triplets) showed very low mutation loads, ranging from 0% to 6.2%. Our data indicate that expanded GAA-TR alleles in Friedreich ataxia are highly mutable and have a natural tendency to contract in vivo, and that these properties depend on multiple factors, including DNA sequence, triplet-repeat length and unknown cell-type-specific factors.

Friedreich ataxia patients are homozygous for expanded GAA triplet-repeats containing 66 to 1,700 triplets. We report two patients with delayed-onset, hyperreflexia and gradually progressive disease. Both were heterozygous for large expansions and also carried alleles with 44 and 66 triplet-repeats, respectively. Due to somatic instability, 15% (GAA-44) and 75% (GAA-66) of cells contained alleles with >/=66 triplet-repeats, constituting a plausible mechanism for their mild phenotype. A sibling with a stable GAA-37 allele and a large expansion was clinically normal. Instability of borderline alleles confers a risk for Friedreich ataxia, and the range of pathogenic alleles is broader than previously recognized.

ABSTRACT:The purpose of this study was to determine whether combining different testing modalities namely ␤-hexosaminidase A (HEXA) enzyme analysis, HEXA DNA common mutation assay, and HEXA gene sequencing could improve the sensitivity for carrier detection in non-Ashkenazi (AJ) individuals. We performed a HEXA gene sequencing assay, a HEXA DNA common mutation assay, and a HEXA enzyme assay on 34 self-reported Tay-Sachs disease (TSD) carriers, six late-onset patients with TSD, and one pseudodeficiency allele carrier. Sensitivity of TSD carrier detection was 91% for gene sequencing compared with 91% for the enzyme assay and 52% for the DNA mutation assay. Gene sequencing combined with enzyme testing had the highest sensitivity (100%) for carrier detection. Gene sequencing detected four novel mutations, three of which are predicted to be disease causing ͓118.delT, 965A3 T (D322V), and 775A3 G (T259A)͔. Gene sequencing is useful in identifying rare mutations in patients with TSD and their families, in evaluating spouses of known carriers for TSD who have indeterminate enzyme analysis and negative for common mutation analysis, and in resolving ambiguous enzyme testing results. (Pediatr Res 67: 217-220, 2010)

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