The fetal FMR1 premutation phenotype: clues from the amniotic fluid transcriptome. L. M. Zwemer1, S. L. Nolin2, P. Okamoto3, M. Eisenberg4, D. W. Bianchi1 1) Mother Infant Research Institute, Tufts Medical Center, Boston, MA, USA; 2) New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA; 3) Integrated Genetics/Laboratory Corporation of America Holdings, Westborough, MA, USA; 4) Laboratory Corporation of America Holdings, Research Triangle Park, NC, USA.

   Second trimester amniotic fluid supernatant (AFS) cell-free fetal (cff) RNA is a pure source of functional and developmental information about many organs, including the fetal brain. Prior studies have demonstrated transcriptome-wide differences in gene expression between unaffected and affected fetuses for both genetic and non-genetic diseases. Trinucleotide repeat expansion in the FMR1 (fragile X mental retardation 1) gene results in a diagnosis of either a premutation (55-200 CGG repeats) or full mutation (200 repeats). Little is known about the human fetal phenotype for either full mutation or premutation alleles; here we focused on fetuses diagnosed with the premutation allele, which is frequently associated with adult-onset diseases. Residual AFS from six pairs (four male, two female) of age- and gender-matched premutation and unaffected fetuses was collected after diagnostic testing for fragile X syndrome. cff RNA was extracted and prepared for hybridization to the Affymetrix HG-U133 Plus 2.0 GeneChip expression microarray. Seven hundred fifty-seven (757) probe sets representing 587 genes were significantly differentially expressed consistently in the six pairs (two-sided paired t-test with Benjamini-Hochberg (BH) adjusted p0.05). This includes several genes specifically expressed in the testis, the immune, and central nervous systems, as defined by the BioGPS GeneAtlas database. Manual curation of the top differentially expressed genes using Online Mendelian Inheritance in Man identified several candidate genes of interest, including: CDKL5, CDNF, and CPLX2, which have all previously been implicated in neurological dysregulation. Among them, CDKL5 is involved in multiple disorders that map to the X-chromosome or are involved in intellectual disability. Functional analysis using Ingenuity Pathway Analysis showed that several pathways of interest were enriched at a false discovery rate of 0.25 (right-tailed Fishers exact test with BH correction) including: cell-mediated immune response, nervous system and endocrine system development. This pilot study provides novel transcriptomic evidence to suggest that a molecular phenotype already exists in fetuses that are carriers of the FMR1 premutation allele, including dysregulation of several candidate genes. The differential regulation of tissue-specific genes is consistent with the later involvement of these organ systems in adult-onset diseases associated with the FMR1 premutation.

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