Simple method for haplotyping the poly(TG) repeat in individuals carrying the IVS8 5T allele in the CFTR gene.
BACKGROUND
The 5T allele of the polyT tract located within intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is a variant that in trans with a severe CFTR mutation can result in normal phenotype, congenital bilateral absence of vas deferens (CBAVD), or mild cystic fibrosis. The 5T allele has been associated with the skipping of exon 9, a process that seems to be influenced by an adjacent 9-13TG tandem repeat. The 12- or 13TG repeats are often associated with an abnormal phenotype. We present here a single-step method for direct haplotyping of the TG repeats in 5T carriers.


METHODS
The method is based on a single-step PCR, using a fluorescently labeled forward primer and a reverse allele-specific primer matching the 5T allele. We validated the test in 30 control samples of known 5T-poly(TG) haplotype and then used this method to evaluate 57 clinical samples.


RESULTS
The expected TG genotypes were obtained for all 5T control samples, and no nonspecific amplification of either the 7T or 9T alleles was detected. In our 5T-positive collection 9 of 9 (100%) CBAVD patients, 6 of 12 (50.0%) chronic pancreatitis patients, and 12 of 36 (33.3%) individuals undergoing assisted reproduction showed 5T-12TG haplotype.


CONCLUSIONS
Our method is an accurate, specific, and simple tool to characterize the 5T poly(TG) haplotype. Our results confirm the high frequency of 5T-12TG in CBAVD patients and do not preclude a potential effect also in pancreatitis. This assay can be useful in assessment of the disease risk in 5T carriers.
Mantovani
Vilma
V
Medical Genetics Unit, S.Orsola-Malpighi University Hospital, Bologna, Italy. mantovan@med.unibo.it
Garagnani
Paolo
P
Selva
Paola
P
Rossi
Cesare
C
Ferrari
Simona
S
Cenci
Marinella
M
Calza
Nilla
N
Cerreta
Vincenzo
V
Luiselli
Donata
D
Romeo
Giovanni
G
D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000097:blood / Q000235:genetics*; D005307:Fertilization in Vitro; D005838:Genotype; D006239:Haplotypes; D006579:Heterozygote; D006801:Humans; D008297:Male; D010195:Pancreatitis / Q000235:genetics; D011119:Polynucleotides / Q000235:genetics*; D012680:Sensitivity and Specificity; D020080:Tandem Repeat Sequences; D014649:Vas Deferens / Q000002:abnormalities
C505032:CFTR protein, human; D011119:Polynucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator