Development of a novel, accurate, automated, rapid, high-throughput technique suitable for population-based carrier screening for Fragile X syndrome.
OBJECTIVE
To develop a high-throughput, automated, accurate method suitable for population-based carrier detection of fragile X syndrome.


METHODS
We developed a new method called capillary Southern analysis that allows automated high-throughput screening for expanded fragile X mental retardation 1 (FMR1) alleles. Initially samples are analyzed by a multiplex polymerase chain reaction that contains an internal control to establish gender. All females heterozygous for two normal alleles are reported as normal without further analysis. All females homozygous at the FMR1 locus (24% of all analysis) are then analyzed by capillary Southern analysis. Theoretically this method can detect expansion as high as 2000 CGG repeats, although in our series the largest nonmosaic FMR1 present was 950 CGG repeats. After assay development, we performed capillary Southern analysis on 995 female and 557 male samples submitted for fragile X syndrome testing by polymerase chain reaction and Southern blot.


RESULTS
The polymerase chain reaction/capillary Southern analysis technique identified 100% of six female premutation carriers, seven full mutation carrier females, one premutation male, and five affected males. There was only one discrepancy between analysis by polymerase chain reaction/Southern blot and analysis by polymerase chain reaction/capillary Southern analysis. A single female sample appeared to be heterozygous for a premutation allele by polymerase chain reaction/capillary Southern analysis but was negative by Southern blot. It is possible this patient is a mosaic for the premutation allele, but because samples were deidentified, we were unable to determine whether this was a true false positive.


CONCLUSIONS
We have developed an automated, high-throughput technique capable of detecting carriers of fragile X syndrome with 100% sensitivity and at least 99.5% specificity. This should allow population-based carrier detection for the most commonly inherited form of mental retardation.
Strom
Charles M
CM
Quest Diagnostics, Nichols Institute, San Juan Capistrano, California 92690, USA. charles.m.strom@questdiagnostics.com
Huang
Donghui
D
Li
Yuanyin
Y
Hantash
Feras M
FM
Rooke
Jenny
J
Potts
Steven J
SJ
Sun
Weimin
W
D015139:Blotting, Southern; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000032:analysis; D005600:Fragile X Syndrome / Q000175:diagnosis*; D006580:Genetic Carrier Screening / Q000379:methods*; D005820:Genetic Testing; D006801:Humans; D008297:Male; D008957:Models, Genetic; D012680:Sensitivity and Specificity
D051860:Fragile X Messenger Ribonucleoprotein 1