Mutations of protocadherin 19 in female epilepsy (PCDH19-FE) lead to allopregnanolone deficiency.

Protocadherin 19 (PCDH19) female limited epilepsy (PCDH19-FE; also known as epilepsy and mental retardation limited to females, EFMR; MIM300088) is an infantile onset epilepsy syndrome with or without intellectual disability (ID) and autism. We investigated transcriptomes of PCDH19-FE female and control primary skin fibroblasts, which are endowed to metabolize neurosteroid hormones. We identified a set of 94 significantly dysregulated genes in PCDH19-FE females. Intriguingly, 43 of the 94 genes (45.7%) showed gender-biased expression; enrichment of such genes was highly significant (P = 2.51E-47, two-tailed Fisher exact test). We further investigated the AKR1C1-3 genes, which encode crucial steroid hormone-metabolizing enzymes whose key products include allopregnanolone and estradiol. Both mRNA and protein levels of AKR1C3 were significantly decreased in PCDH19-FE patients. In agreement with this, the blood levels of allopregnanolone were also (P < 0.01) reduced. In conclusion, we show that the deficiency of neurosteroid allopregnanolone, one of the most potent GABA receptor modulators, may contribute to PCDH19-FE. Overall our findings provide evidence for a role of neurosteroids in epilepsy, ID and autism and create realistic opportunities for targeted therapeutic interventions.

Mutations in UPF3B, a member of the nonsense-mediated mRNA decay complex, cause syndromic and nonsyndromic mental retardation.

Nonsense-mediated mRNA decay (NMD) is of universal biological significance. It has emerged as an important global RNA, DNA and translation regulatory pathway. By systematically sequencing 737 genes (annotated in the Vertebrate Genome Annotation database) on the human X chromosome in 250 families with X-linked mental retardation, we identified mutations in the UPF3 regulator of nonsense transcripts homolog B (yeast) (UPF3B) leading to protein truncations in three families: two with the Lujan-Fryns phenotype and one with the FG phenotype. We also identified a missense mutation in another family with nonsyndromic mental retardation. Three mutations lead to the introduction of a premature termination codon and subsequent NMD of mutant UPF3B mRNA. Protein blot analysis using lymphoblastoid cell lines from affected individuals showed an absence of the UPF3B protein in two families. The UPF3B protein is an important component of the NMD surveillance machinery. Our results directly implicate abnormalities of NMD in human disease and suggest at least partial redundancy of NMD pathways.

Loss of FMR2 further emphasizes the link between deregulation of immediate early response genes FOS and JUN and intellectual disability.

Loss of FMR2 causes Fragile X E (FRAXE) site-associated intellectual disability (ID). FMR2 regulates transcription, promotes alternative splicing with preference for G-quartet structure harbouring exons and is localized to the nuclear speckles. In primary skin fibroblasts from FRAXE patients (n = 8), we found a significant reduction in the number, but a significant increase in the size, of nuclear speckles, when compared with the controls (n = 4). Since nuclear speckles are enriched with factors involved in pre-mRNA processing, we explored the consequence of these defects and the loss of FMR2 on the transcriptome. We performed whole genome expression profiling using total RNA extracted from these cell lines and found 27 genes significantly deregulated by at least 2-fold at P < 0.05 in the patients. Among these genes, FOS was significantly upregulated and was further investigated due to its established role in neuronal cell function. We showed that (i) 30% depletion of Fmr2 in mouse primary cortical neurons led to a 2-fold increase in Fos expression, (ii) overexpression of FMR2 significantly decreased FOS promoter activity in luciferase assays, and (iii) as FOS promoter contains a serum response element, we found that not FOS, but JUN, which encodes for a protein that forms a transcriptional activator complex with FOS, was significantly upregulated in the patients' cell lines upon mitogen stimulation. These results suggest that FMR2 is an upstream regulator of FOS and JUN, and further link deregulation of the immediate early response genes to the pathology of ID- and FRAXE-associated ID in particular.

Contribution of copy number variants involving nonsense-mediated mRNA decay pathway genes to neuro-developmental disorders.

The nonsense-mediated mRNA decay (NMD) pathway functions not only to degrade transcripts containing premature termination codons (PTC), but also to regulate the transcriptome. UPF3B and RBM8A, important components of NMD, have been implicated in various forms of intellectual disability (ID) and Thrombocytopenia with Absent Radius (TAR) syndrome, which is also associated with ID. To gauge the contribution of other NMD factors to ID, we performed a comprehensive search for copy number variants (CNVs) of 18 NMD genes among individuals with ID and/or congenital anomalies. We identified 11 cases with heterozygous deletions of the genomic region encompassing UPF2, which encodes for a direct interacting protein of UPF3B. Using RNA-Seq, we showed that the genome-wide consequence of reduced expression of UPF2 is similar to that seen in patients with UPF3B mutations. Out of the 1009 genes found deregulated in patients with UPF2 deletions by at least 2-fold, majority (95%) were deregulated similarly in patients with UPF3B mutations. This supports the major role of deletion of UPF2 in ID. Furthermore, we found that four other NMD genes, UPF3A, SMG6, EIF4A3 and RNPS1 are frequently deleted and/or duplicated in the patients. We postulate that dosage imbalances of these NMD genes are likely to be the causes or act as predisposing factors for neuro-developmental disorders. Our findings further emphasize the importance of NMD pathway(s) in learning and memory.