Left ventricular outflow tract obstructions (LVOTO) constitute a subgroup of congenital heart defects genetically determined.Significant male-gender bias is observed,pointing to involvement of X-chromosome.Skewed X-chromosome inactivation (XCI) is observed in heterozygous females carrying gene mutations involved in X-linked syndromes.We hypothesized that this non-random XCI may contribute to LVOTO in women.
METHODS:We accessed provincial patient and DNA database to who echocardiography was performed.To assess XCI in females,we determined methylation status at human androgen receptor locus on Xchromosome.Briefly,we assayed methylation of the different alleles by digestion of the DNA with a methylation sensitive enzyme (HpaII),followed by PCR amplification with fluoro-primers.Skewing was calculated as ratio of amplification of methylated versus unmethylated alleles.Individual data was analyzed in pedigree context.Affected status was defined as aortic stenosis,bicuspid aorta,coarctation or aortic root filation.Skewing was analyzed using three cutoffs:70:30,75:25 and 80:20.
RESULTS:We identified 307 individuals distributed in 43 families;among them,156 were females(48 affected).We found skewing in excess of 80:20 is significantly more prevalent in affected than unaffected females(p=0.004,odds-ratio 3.3[1.4-7.4]).In 19 females(11.3%),skewing seems to be protective based on possible segregation of an X-linked disease allele.When analyzing allele size, tendency is seen towards a protective or deleterious effect of certain allele-sizes action(allele-sizes 247bp and 236bp,n=18 and n=10,respectively;X2 247=4.3,X2 236=3.6;p=0.08).
CONCLUSIONS: Skewed X inactivation significantly contributes to LVOTO in affected females.In families compatible with X-linked inheritance,inactivation of affected allele can be protective against disease in women.These findings should facilitate search for LVOTO loci on X-chromosome and prospective use of XCI as clinical marker for disease progression.