Trinucleotide Repeat Measurements

Basic research and technological advances in human genetics, biochemistry, and model systems have brought much progress toward the understanding of human infectious, hereditary, and somatically acquired diseases. In fact, whereas in 1991, triplet repeat expansion diseases could be described in a single article, now the remarkable developments within each disease has created volumes of work (4,5). One factor contributing to triplet repeat diseases is the adoption of unusual non-B DNA structures by the repetitive elements. These structures are associated with disease progression and severity. Despite all of the advances, an effective therapy is yet unrealized. However, the defining element of these diseases, the unstable DNA mutation, can accurately be measured. Some diseases already have diagnostics methods available [i.e., Huntington's disease and Fragile X syndrome (6,7)]. Yet, in the case of Fragile X syndrome, accurate size determination of the triplet repeat region is not easily determined because of the nature of the repeat elements associated with this disease; consisting entirely of the C and G nucleotides. This sequence content increases the error rate of polymerase chain reaction (PCR) amplification and is commonly methylated in the disease state.

2.1. MEASUREMENT TECHNOLOGIES FOR ACCURATE SIZING: FRAGILE X SYNDROME Current measurement technologies for repetitive elements and their adjacent flanking sequences are Southern blot hybridization, PCR amplification and electrophoretic separation, and DNA sequencing (7). The need for reference materials for Fragile X syndrome was discussed at a workshop held at NIST in 1998 entitled "Standards for Nucleic Acid Diagnostic Applications" (8). Reference materials for this syndrome are needed to provide accurate repeat size measurements across technology platforms and interlaboratory diagnostic and prognostic agreement. The specific size range for such a standard reference material was based on the likelihood of full mutation transmission (9).

The Biotechnology Division established a measurement program in this area, focusing on accurate size measurements after PCR amplification and sequencing. The accuracy of an optimized PCR amplification protocol to correctly measure the number of (CGG)n repeats from normal, gray zone, and premutation length alleles was determined (4). The DNAs used in this study were reported to contain CGG repeat elements ranging from 29 to 110 repeats. Both slab-PAGE (polyacrylamide gel elec-trophoresis) and capillary measurements were conducted, and the factors impacting sensitivity, accuracy, and reproducibility of

Table 1

Measurement of (CGG) Repeats

Table 1

Measurement of (CGG) Repeats

Coriell No.

Allele size(s):

Peak

Allele size(s):

Peak

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