The Principles of Clinical Cytogenetics - Extra Materials - Springer

506 Dana Crawford and Patricia Howard-Peebles
Re-evaluation of Positive Results
It is now apparent that the false-positive rate for cytogenetic testing was significant in both affected
and carrier individuals (130,138). The other three fragile sites in the Xq27-28 region, one common site
(FRAXD) and two rare sites (FRAXE and FRAXF), were the major contributors, because the rare sites
cannot be cytogenetically distinguished from FRAXA. However, technical and interpretative problems
in the laboratory were also factors. Any family with a cytogenetic diagnosis of fragile X syndrome
should have one family member (affected or obligate carrier) tested with DNA methodology, especially
prior to carrier testing in other family members via DNA technology. Normal females who were defined
as carriers based on low-level cytogenetic expression should be retested as well (138).
Molecular Testing
By the time DNA-based diagnosis of fraX became available, the problems with cytogenetic testing
had become apparent (139). First, fraX expression was variable (between 1% and 50%), with
females usually having fewer positive cells than males, and obligate carriers often tested negative.
Second, the presence of the other three fragile sites on Xq reduced the reliability of cytogenetic
scoring. Finally, lower expression in cell types other than lymphocytes compromised prenatal diagnosis.
DNA-based testing has solved all these problems and usually costs less as well. Thus, cytogenetic
fraX testing should be retired, as it is less accurate and more expensive. In fact, the
reimbursement (CPT) code for such testing has been deleted.
The objective of all DNA-based methods for fraX is to identify a piece of DNA containing the
CGG repeat and determine its length by electrophoresis in order to classify it as normal, premutation
or full mutation.
DNA-based Methods
The two DNA-based methods available for fraX testing are Southern blot, with or without methylation,
and PCR (polymerase chain reaction). PCR is more sensitive for premutations or carrier testing, and the
results are usually expressed as total repeat number. Southern blots are better for full mutations and, if double
digestion is utilized, the methylation status can be determined. The results are expressed as ∆ kb (delta kb
defined as the difference between the patient and a normal reference). According to a recent report by the
Quality Assurance Subcommittee of the American College of Medical Genetics Laboratory Practice Committee,
both DNA-based methods are considered diagnostic and are 99% sensitive and 100% specific (140).
Detailed descriptions of these techniques and illustrations are provided by Maddalena et al. (92).
An important caveat for DNA-based methods is the fact that a small percentage (