INVESTIGADORES
RADIC Claudia Pamela
congresos y reuniones científicas
Título:
Identification of a highly polymorphic tetranucleotide repeat locus (DXpS) at Xp and development of a DXpS/HUMARA biplex methylation-based PCR assay that enhances detection of X-chromosome inactivation
Autor/es:
MACHADO FELIPE; RAMOS ESTER S. ; RADIC CLAUDIA PAMELA ; DE BRASI CARLOS DANIEL; MEDINA-ACOSTA ENRIQUE
Reunión:
Workshop; 50 Years of Xinactivation Research; 2011
Resumen:
The methylation-based PCR assay at the polymorphic
(CAG)n repeat in exon 1 of the human androgen receptor AR gene (HUMARA)
is a standard method for determination of the methylation state of alleles in
active X (Xa) and inactive X (Xi) chromosomes. HUMARA assay is endowed with
heterozygosity rates ~85% worldwide. This means that in a proportion of females
it is uninformative. The HUMARA genotype is not neutral, being linked to
Kennedy´s disease. Moreover, allele designation and quantification from
trinucleotide repeats demand normalizing for minor (stutter) products differing
from the original template by multiples of the repeat unit. Here, we report on
the identification of a highly polymorphic tetranucleotide repeat (named DXpS)
mapping to within a CpG island on Xp. This island is 191 bp downstream from the
start of the repeat element, and contains sites for the HhaI, HpaII and BstUI
methyl-sensitive restriction enzymes. We developed the DXpS and the HUMARA
markers into a biplex methylation-based quantitative fluorescent PCR assay. For
DXpS we observed twelve alleles with negligible stuttering. DXpS exhibited a
heterozygosity rate of 87% (n = 60), matching that of HUMARA. The combined
informativeness of the biplex assay was 98%. Random and nonrandom
X-inactivation patterns inferred with DXpS in phenotypically normal females and
haemophiliac females carrying a defective F8 gene were highly concordant (r2
= 0.96) with HUMARA patterns. DXpS represents a notable advancement in
detecting X-chromosome inactivation due to the observed high rate of
heterozygosity, negligible stuttering, concordance with HUMARA, and the
apparent neutrality of allelic variants. Financial support: FAPESP
(09/10615-7), FAPERJ, CNPq.