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

Abstract

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.

Keywords: Deletion 2q31.1; Epigenetics; Gene silencing; LRFN5; Translocation.

Fig. 1

Translocation breakpoint mapping. A Idiograms (courtesy of David Adler, University of Washington) of the normal and derivative chromosomes 14 and 21 with horizontal black lines indicating the translocation breakpoints. FISH results are shown below to verify the identity of centromeres and telomeres. B Results of BAC-FISH using probes flanking the 14q21.1 breakpoint. C Detailed breakpoint map including the adjacent genes and the position of the BAC-FISH probes. D BAC probes flanking centromere 21 are present on both the p-arm and the q-arm of the derivative chromosome 21, indicating that the centromere 21 is intact and that the breakpoint is in 21p11.

Fig. 2

Affymetrix 250K GeneChip data showing the 2.6-Mb 2q31.1 microdeletion (A), and a detailed map of the deletion breakpoints (B). A Visualization of the microdeletion by raw log2 intensity ratios (blue dots) with a running average (black line) and the CNAG-derived copy numbers (red dots). The microdeletion is further highlighted by a long stretch of loss of heterozygosity (blue square; LOH) in the SNP genotypes (green dots). The proximal and distal breakpoints are indicated as dotted lines. B Detailed map of the microdeletion breakpoint regions and the breakpoint-flanking SNPs. CNAG-derived copy numbers (red dots) and SNP genotypes (green dots) show that the proximal breakpoint is located within a 13.0-kb region encompassing the 3′-end of the uncharacterized DCAF17 gene. The distal breakpoint is located within a 21.5-kb region encompassing the 3′-end of the OLA1 gene. Exons of both genes are shown as numbered boxes, and the 3′UTRs are shown as dotted boxes.

Fig. 3

Expression analysis of the β-actin control (ACTB) and three 14q21.1 breakpoint-flanking genes on der(14) (PNN and FBXO33) and der(21) (LRFN5) in Epstein-Barr virus transformed lymphoblasts (EBV) and skin fibroblasts (Fib) of control (C) and patient (P). PNN and FBXO33 expression was detected in all samples and did not differ significantly between patient and control cells. LRFN5 expression was readily found in control fibroblasts, but was almost undetectable in patient fibroblasts. Bar graphs show the expression of PNN, FBXO33 and LRFN5 determined by real-time RT-PCR on control and patient fibroblasts. Expression levels are means of 4 replicate PCRs, error bars indicate the standard deviations, the scale is logarithmic.

Fig. 4

Results of patient fibroblast ChIP-on-CHIP experiment: Chromatin from patient fibroblast cell lysates was immunoprecipitated by antibodies against K3K4M3, H3K9M2, H3K9M3 and H3K27M3. DNA from these ChIP samples was amplified and hybridized to tile-path chromosome 14 oligonucleotide arrays, using input DNA as the comparative control. The curves show the average ratio of the hybridization signals of 2 independent immunoprecipitates, compared to the input DNA (for details, see the Methods section). The 14q21.1 translocation breakpoint region, the same as in figure 1, is marked with a vertical double line. There is a marked increase in H3K9M3-associated and a smaller increase in H3K4M3-associated DNA starting at the breakpoint and including the LRFN5 gene, and peaking at the LRFN5 promoter for H3K4M3- and H3K27M3-associated DNA.

Fig. 5

Results of real-time PCRs of the PNN, FBXO33 and LRFN5 promoters on H3K4M3 and H3K9M3 ChIP material from control and patient fibroblasts. The FBXO33 promoter-associated levels of H3K4M3 and H3K9M3 were not significantly different between control and patient (A) in contrast to the LRFN5 promoter-associated H3K4M3 and H3K9M3 levels (B). The decreased level of H3K4M3 and increased level of H3K9M3 that was found at the LRFN5 promoter in patient fibroblasts corresponded well with reduced LRFN5 expression (fig. 3). C A more elaborate analysis of H3K4M3 levels at the PNN, FBXO33 and LRFN5 promoters, and the LRFN5-associated CpG island, of control (light grey) and patient (dark grey) fibroblasts. These data demonstrated that the patient-specific decrease of H3K4M3 levels was more widespread within the LRFN5 promoter region. Levels of histone modifications are expressed as arbitrary (raw) values in A and B, or as percentage of the input DNA in C. Bar graphs show average values of independent replicates, with standard deviations as error bars.