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Screening in silico predicted remotely acting NF1 gene regulatory elements for mutations in patients with neurofibromatosis type 1.

Hamby SE, Reviriego P, Cooper DN, Upadhyaya M, Chuzhanova N - Hum. Genomics (2013)

Bottom Line: These regions were then sequenced in 47 NF1 patients in whom no mutations had been found in either the NF1 or SPRED1 gene regions.Five patients were found to harbour DNA sequence variants in the distal H3K27ac-enriched region.Although these variants are of uncertain pathological significance and still remain to be functionally characterized, this approach promises to be of general utility for the detection of mutations underlying other inherited disorders that may be caused by mutations in remotely acting regulatory elements.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK.

ABSTRACT
Neurofibromatosis type 1 (NF1), a neuroectodermal disorder, is caused by germline mutations in the NF1 gene. NF1 affects approximately 1/3,000 individuals worldwide, with about 50% of cases representing de novo mutations. Although the NF1 gene was identified in 1990, the underlying gene mutations still remain undetected in a small but obdurate minority of NF1 patients. We postulated that in these patients, hitherto undetected pathogenic mutations might occur in regulatory elements far upstream of the NF1 gene. In an attempt to identify such remotely acting regulatory elements, we reasoned that some of them might reside within DNA sequences that (1) have the potential to interact at distance with the NF1 gene and (2) lie within a histone H3K27ac-enriched region, a characteristic of active enhancers. Combining Hi-C data, obtained by means of the chromosome conformation capture technique, with data on the location and level of histone H3K27ac enrichment upstream of the NF1 gene, we predicted in silico the presence of two remotely acting regulatory regions, located, respectively, approximately 600 kb and approximately 42 kb upstream of the NF1 gene. These regions were then sequenced in 47 NF1 patients in whom no mutations had been found in either the NF1 or SPRED1 gene regions. Five patients were found to harbour DNA sequence variants in the distal H3K27ac-enriched region. Although these variants are of uncertain pathological significance and still remain to be functionally characterized, this approach promises to be of general utility for the detection of mutations underlying other inherited disorders that may be caused by mutations in remotely acting regulatory elements.

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Schematic of the occurrence and level of H3K27ac enrichment within interacting and NF1-containing DNA fragments. The level of enrichment is shown by the height of the curves (not to scale).
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Figure 1: Schematic of the occurrence and level of H3K27ac enrichment within interacting and NF1-containing DNA fragments. The level of enrichment is shown by the height of the curves (not to scale).

Mentions: The Hi-C data indicated that a 1-Mb fragment of chromosome 17, starting at position 29,000,000 and ending at position 29,999,999, termed the NF1-containing fragment since it contained the entire NF1 gene (positions 29,421,945–29,709,134), was found to interact most strongly with the adjacent 1-Mb fragment on chromosome 17 (703 and 255 intra-chromosomal interactions with the NF1-containing fragment recorded in HindIII- and NcoI-maps, respectively) which occupies positions 28,000,000–28,999,999 upstream of the NF1-containing fragment (Figure 1). The ENCODE data indicated the presence of an H3K27ac-enriched region somewhere between positions 28,846,790 and 28,847,790 in the interacting fragment (region A in Figure 1). This region is distal (approximately 600 kb upstream) to the NF1 gene. Another peak in H3K27ac enrichment was observed within the NF1-containing fragment (region B between positions 29,378,421 and 29,379,750) which is proximal (approximately 42 kb) to the NF1 gene. The level of enrichment was higher in the distal region A than in the proximal region B. ENCODE data also revealed the presence in region A of three overlapping binding sites for the transcription factors, p300 (positions 28,846,840–28,847,158), c-Fos (28,846,815–28,847,158) and c-Jun (28,846,819–28,847,094), all of which were identified by in vitro ChIP-seq assay (http://genome.ucsc.edu/index.html).


Screening in silico predicted remotely acting NF1 gene regulatory elements for mutations in patients with neurofibromatosis type 1.

Hamby SE, Reviriego P, Cooper DN, Upadhyaya M, Chuzhanova N - Hum. Genomics (2013)

Schematic of the occurrence and level of H3K27ac enrichment within interacting and NF1-containing DNA fragments. The level of enrichment is shown by the height of the curves (not to scale).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750751&req=5

Figure 1: Schematic of the occurrence and level of H3K27ac enrichment within interacting and NF1-containing DNA fragments. The level of enrichment is shown by the height of the curves (not to scale).
Mentions: The Hi-C data indicated that a 1-Mb fragment of chromosome 17, starting at position 29,000,000 and ending at position 29,999,999, termed the NF1-containing fragment since it contained the entire NF1 gene (positions 29,421,945–29,709,134), was found to interact most strongly with the adjacent 1-Mb fragment on chromosome 17 (703 and 255 intra-chromosomal interactions with the NF1-containing fragment recorded in HindIII- and NcoI-maps, respectively) which occupies positions 28,000,000–28,999,999 upstream of the NF1-containing fragment (Figure 1). The ENCODE data indicated the presence of an H3K27ac-enriched region somewhere between positions 28,846,790 and 28,847,790 in the interacting fragment (region A in Figure 1). This region is distal (approximately 600 kb upstream) to the NF1 gene. Another peak in H3K27ac enrichment was observed within the NF1-containing fragment (region B between positions 29,378,421 and 29,379,750) which is proximal (approximately 42 kb) to the NF1 gene. The level of enrichment was higher in the distal region A than in the proximal region B. ENCODE data also revealed the presence in region A of three overlapping binding sites for the transcription factors, p300 (positions 28,846,840–28,847,158), c-Fos (28,846,815–28,847,158) and c-Jun (28,846,819–28,847,094), all of which were identified by in vitro ChIP-seq assay (http://genome.ucsc.edu/index.html).

Bottom Line: These regions were then sequenced in 47 NF1 patients in whom no mutations had been found in either the NF1 or SPRED1 gene regions.Five patients were found to harbour DNA sequence variants in the distal H3K27ac-enriched region.Although these variants are of uncertain pathological significance and still remain to be functionally characterized, this approach promises to be of general utility for the detection of mutations underlying other inherited disorders that may be caused by mutations in remotely acting regulatory elements.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK.

ABSTRACT
Neurofibromatosis type 1 (NF1), a neuroectodermal disorder, is caused by germline mutations in the NF1 gene. NF1 affects approximately 1/3,000 individuals worldwide, with about 50% of cases representing de novo mutations. Although the NF1 gene was identified in 1990, the underlying gene mutations still remain undetected in a small but obdurate minority of NF1 patients. We postulated that in these patients, hitherto undetected pathogenic mutations might occur in regulatory elements far upstream of the NF1 gene. In an attempt to identify such remotely acting regulatory elements, we reasoned that some of them might reside within DNA sequences that (1) have the potential to interact at distance with the NF1 gene and (2) lie within a histone H3K27ac-enriched region, a characteristic of active enhancers. Combining Hi-C data, obtained by means of the chromosome conformation capture technique, with data on the location and level of histone H3K27ac enrichment upstream of the NF1 gene, we predicted in silico the presence of two remotely acting regulatory regions, located, respectively, approximately 600 kb and approximately 42 kb upstream of the NF1 gene. These regions were then sequenced in 47 NF1 patients in whom no mutations had been found in either the NF1 or SPRED1 gene regions. Five patients were found to harbour DNA sequence variants in the distal H3K27ac-enriched region. Although these variants are of uncertain pathological significance and still remain to be functionally characterized, this approach promises to be of general utility for the detection of mutations underlying other inherited disorders that may be caused by mutations in remotely acting regulatory elements.

Show MeSH
Related in: MedlinePlus