Severe Progressive Autism Associated with Two de novo Changes: A 2.6-Mb 2q31.1 Deletion and a Balanced t(14;21)(q21.1;p11.2) Translocation with Long-Range Epigenetic Silencing of LRFN5 Expression.

In a 19-year-old severely autistic and mentally retarded girl, a balanced de novo t(14;21)(q21.1;p11.2) translocation was found in addition to a de novo 2.6-Mb 2q31.1 deletion containing 15 protein-encoding genes. To investigate if the translocation might contribute to developmental stagnation at the age of 2 years with later regression of skills, i.e. a more severe phenotype than expected from the 2q31.1 deletion, the epigenetic status and expression of genes proximal and distal to the 14q21.1 breakpoint were investigated in Ebstein Barr Virus-transformed lymphoblast and primary skin fibroblast cells. The 14q21.1 breakpoint was found to be located between a cluster of 7 genes 0.1 Mb upstream, starting with FBXO33, and the single and isolated LRFN5 gene 2.1 Mb downstream. Only expression of LRFN5 appeared to be affected by its novel genomic context. In patient fibroblasts, LRFN5 expression was 10-fold reduced compared to LRFN5 expressed in control fibroblasts. In addition, a relative increase in trimethylated histone H3 lysine 9 (H3K9M3)-associated DNA starting exactly at the translocation breakpoint and going 2.5 Mb beyond the LRFN5 gene was found. At the LRFN5 promoter, there was a distinct peak of trimethylated histone H3 lysine 27 (H3K27M3)-associated DNA in addition to a diminished trimethylated histone H3 lysine 4 (H3K4M3) level. We speculate that dysregulation of LRFN5, a postsynaptic density-associated gene, may contribute to the patient's autism, even though 2 other patients with 14q13.2q21.3 deletions that included LRFN5 were not autistic. More significantly, we have shown that translocations may influence gene expression more than 2 Mb away from the translocation breakpoint.

A 2.1 Mb deletion adjacent but distal to a 14q21q23 paracentric inversion in a family with spherocytosis and severe learning difficulties.

A familial q21.1q23.2-inversion on chromosome 14 that co-segregated with spherocytosis and learning difficulties or mild mental retardation was extensively investigated by bacterial artificial chromosome fluorescence in situ hybridization and array-comparative genomic hybridization. As expected, a deletion of the beta-spectrin gene SPTB, a known cause of spherocytosis, was found. More unexpectedly, this deletion was approximately 1.6 Mb distal to the 14q23.2-inversion breakpoint. The deletion spanned approximately 2.1 Mb and contained 15 annotated genes in addition to SPTB, among them PLEKHG3, a guanide nucleotide exchange factor for Rho GTPases. This gene is highly expressed in the brain and our best candidate for causing the mild mental retardation. The case illustrates that inversions can be associated with microdeletions close to but not including one of the inversion breakpoints.

Rasp21 sequences opposite the nucleotide binding pocket are required for GRF-mediated nucleotide release.

The substrate requirements for the catalytic activity of the mouse Cdc25 homolog Guanine nucleotide Release Factor, GRF, were determined using the catalytic domain of GRF expressed in insect cells and E. coli expressed H-Ras mutants. We found a requirement for the loop 7 residues in Ras (amino acids 102 to 105) for stimulation of guanine nucleotide release. The dependence on the switch I and II regions of Rasp21 (encompassing the residues that shift position in the GTP- versus GDP-bound protein), which had been seen with Sdc25-mediated exchange, was also found for GRF. In addition, the sensitivity of H-Ras to GRF was abolished when residues 130-139 were replaced by proline-aspartic acid-glutamine, whereas substitution of the entire loop 8 (residues 123-130 replaced by leucine-isoleucine-arginine) had no effect on the stimulation of guanine nucleotide release by GRF. Substrate activity of Ras proteins were independent of their post-translational processing, GDP release was stimulated threefold more effectively by GRF than was GTP release, and no major differences were found between the mammalian N-, H- and K-Ras proteins. Examining the responsiveness of the Ras protein from S. pombe and the human Ras like proteins RhoA, Rap1A, Rac1 and G25K revealed a strict Ras specificity; of these only S. pombe Ras was GRF sensitive.