Objectives

• Definition of sex-specific methylation target genes by genome-wide DNA methylation analyses using DNA from genital skin fibroblasts in normal males and CAIS (task 1)
• Development of high-throughput platform for methylation analysis (task 2)
• Analysis of methylation signatures in patients comprising the whole spectrum of AIS (task 3)
• Analysis of methylation signatures in fibroblasts from 46,XY- DSD with defects of androgen biosynthesis (task 3)
• Correlation of methylation signatures with AIS-phenotypes, -genotypes, molecular AR function and puberty (task 3)

Workpackage Description

The androgen insensitivity syndrome (AIS) embraces a striking variability of phenotypic expressivity, which affects assessment of individual prognosis and clinical management. The AR-gene mutation and classical models of AR-function poorly reflect the functional capacity of the AR with respect to an individual.
We have previously discovered an AR-dependent “transcriptional memory” in cultured labioscrotal fibroblasts reflecting genital masculinization. It is known that epigenetic mechanisms are involved in establishing tissue-specific gene-expression patterns. We therefore hypothesized that the presence or absence of androgen action during sex-dimorphic development during embryogenesis is reflected by sex-specific methylation signatures. High-throughput methylation analysis in genital target tissues from an EU-wide patient cohort provides unique perspectives for understanding phenotypic expressivity in AIS.

Task 1: Definition of up to 100 sex-specific methylation target genes by genome-wide methylation analysis.

WP04 aims at definition of about 100 sex-specific methylation target genes by genome-wide DNA methylation analyses using DNA from normal male scrotal fibroblasts and labia majora fibroblasts from patients with CAIS. Analyses will be based on 10 CAIS strains and 10 strains of normal male scrotum. Analyses will be performed on the Affymetrix microarray facility in the Institute of Human Genetics in Kiel, Germany, located in the same building as the Division of Paediatric Endocrinology, Kiel, Germany. The methylation patterns of defined genes will be validated by methylation-specific PCR.

Task 2: Development of a custom platform for high-throughput methylation analysis of DSD-fibroblasts.

Subsequently, we will set up a custom platform for high-throughput methylation analyses. It will cover the PCR-validated gene promoters detected in task 1.

Task 3: Analysis and biological interpretation of methylation signatures in labioscrotal-derived fibroblasts of AIS-patients comprising the whole clinical and molecular spectrum using the new custom device.

The database in Glasgow will be screened for patients with AIS comprising the whole phenotypic and molecular spectrum and in whom labioscrotal fibroblasts are available. It is planned to analyze 80 - 100 samples. Based on our preliminary results, it is expected that methylation patterns will reflect individual phenotypic expressivity of androgen action based on the genital phenotype. Molecular determination of the methylation signatures in the DNA-samples will take from month 19 – month 36, data anaylsis and publication will start in month 25 and take until month 36.