X-inactivation modifies disease severity in female carriers of murine X-linked Alport syndrome

Abstract

Background: Female carriers of X-linked Alport syndrome (XLAS) demonstrate variability in clinical phenotype that, unlike males, cannot be correlated with genotype. X-inactivation, the method by which females (XX) silence transcription from one X chromosome in order to achieve gene dosage parity with males (XY), likely modifies the carrier phenotype, but this hypothesis has not been tested directly.

Methods: Using a genetically defined mouse model of XLAS, we generated two groups of Alport female (Col4a5(+/-)) carriers that differed only in the X-controlling element (Xce) allele regulating X-inactivation. We followed the groups as far as 6 months of age comparing survival and surrogate outcome measures of urine protein and plasma urea nitrogen.

Results: Preferential inactivation of the mutant Col4a5 gene improved survival and surrogate outcome measures of urine protein and plasma urea nitrogen. In studies of surviving mice, we found that X-inactivation in kidney, measured by allele-specific mRNA expression assays, correlated with surrogate outcomes.

Conclusions: Our findings establish X-inactivation as a major modifier of the carrier phenotype in X-linked Alport syndrome. Thus, X-inactivation patterns may offer prognostic information and point to possible treatment strategies for symptomatic carriers.

Fig. 1

Groups 1 and 2. Representation of paired 129 (white) and B6 (black) X chromosomes illustrating map positions and genotypes. Strain-specific polymorphisms in the Srpx, Rpgr and Aff2 genes were used in X-inactivation assays.

Fig. 2

(a) Representative Srpx-based assays for Xi. Mouse kidneys from the indicated strains were analysed by multiplex RT-PCR and subsequent digestion as described in Animals and Methods and Table 1. Sau96I digests the B6, but not the 129 Srpx RT-PCR product, generating fragments of 154 and 221 bp from the original 375-bp product. (b) Distribution by group of Xi, measured as allele-specific Srpx expression. (c) Correlation between Xi and 6-month urine protein-to-creatinine ratio. Group 1 mice are indicated by filled grey circles and Group 2 by open circles. (d) Correlation between Xi and 6-month plasma urea nitrogen. Group 1 mice are indicated by filled grey circles and Group 2 by open circles.

Fig. 3

Predicted mean level and slope change in urine protein-to-creatinine ratio based on longitudinal analysis including Xi as a moderator of slope (i.e. including an Xi-by-age interaction). Specific examples of trajectories are given for Xi = 0.6 and Xi = 0.4. Vertical bars represent 95% CI of predicted mean urine protein-to-creatinine ratio at each time point. At 6 months, the 95% CIs do not overlap with the other mean values and this difference is statistically significant (P = 0.039).

Fig. 4

Correlation between Xi and Col4a5 mRNA levels. Group 1 mice are indicated by filled grey circles and Group 2 by open circles.