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Novel APC promoter and exon 1B deletion and allelic silencing in three mutation-negative classic familial adenomatous polyposis families.

Lin Y, Lin S, Baxter MD, Lin L, Kennedy SM, Zhang Z, Goodfellow PJ, Chapman WC, Davidson NO - Genome Med (2015)

Bottom Line: Furthermore, this same deletion with identical breakpoints was found in the probands of two additional APC mutation-negative classical FAP kindreds.Phasing analysis of single nucleotide polymorphisms (SNPs) around the deletion site in the three probands showed evidence of a shared haplotype, suggesting a common founder deletion in the three kindreds.These results support the causal role of a novel promoter deletion in FAP and suggest that non-coding deletions, identifiable using second-generation sequencing methods, may account for a significant fraction of APC mutation-negative classical FAP families.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Washington University School of Medicine, St. Louis, Missouri USA.

ABSTRACT

Background: The overwhelming majority (approximately 80%) of individuals with classic familial adenomatous polyposis (FAP) exhibit mutations in the coding sequence of the adenomatous polyposis coli (APC) tumor suppressor gene. Families without detectable APC mutations are unable to benefit from the use of genetic testing for clinical management of this autosomal dominant syndrome.

Methods: We used exome sequencing and linkage analysis, coupled with second-generation sequencing of the APC locus including non-coding regions to investigate three APC mutation-negative classical FAP families.

Results: We identified a novel ~11 kb deletion localized 44 kb upstream of the transcription start site of APC that encompasses the APC 1B promoter and exon. This deletion was present only in affected family members of one kindred with classical FAP. Furthermore, this same deletion with identical breakpoints was found in the probands of two additional APC mutation-negative classical FAP kindreds. Phasing analysis of single nucleotide polymorphisms (SNPs) around the deletion site in the three probands showed evidence of a shared haplotype, suggesting a common founder deletion in the three kindreds. SNP analysis within the coding sequence of APC, revealed that this ~11 kb deletion was accompanied by silencing of one of the APC alleles in blood-derived RNA of affected individuals.

Conclusions: These results support the causal role of a novel promoter deletion in FAP and suggest that non-coding deletions, identifiable using second-generation sequencing methods, may account for a significant fraction of APC mutation-negative classical FAP families.

No MeSH data available.


Related in: MedlinePlus

The heterozygous ~11 kbAPCdeletion is present in all affected members of the 0124 kindred. (A) Primers were designed to bind upstream of the ~11 kb deletion (primer 1), within the deletion (primer 2), and downstream of the deletion (primer 3). In the presence of a normal allele, primers 1 and 2 result in a 373 bp PCR product. Primers 1 and 3 do not form a product in the absence of the ~11 kb deletion but form a 298 bp PCR product when the deletion is present. (B) In the five affected members of kindred 0124 (III-5, IV-4, V-6, III-3, and IV-2), PCR using these primers resulted in two fragments of the expected sizes. In the three unaffected members (III-4, V-1, and IV-3), PCR produced only one fragment. (C) Sanger sequencing of the PCR product from affected member III-5 using primer 3 confirmed the presence of the ~11 kb deletion. The sequencing trace transitions from coordinate chr5:112,034,825 to chr5:112,045,846 (GRCh37/hg19), a span of 11,020 bp. (D) Sequencing the same PCR product using primer 2 showed the presence of a normal allele and the absence of the gap with a transition from coordinate chr5:112,034,825 to chr5:112,034,826.
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Fig3: The heterozygous ~11 kbAPCdeletion is present in all affected members of the 0124 kindred. (A) Primers were designed to bind upstream of the ~11 kb deletion (primer 1), within the deletion (primer 2), and downstream of the deletion (primer 3). In the presence of a normal allele, primers 1 and 2 result in a 373 bp PCR product. Primers 1 and 3 do not form a product in the absence of the ~11 kb deletion but form a 298 bp PCR product when the deletion is present. (B) In the five affected members of kindred 0124 (III-5, IV-4, V-6, III-3, and IV-2), PCR using these primers resulted in two fragments of the expected sizes. In the three unaffected members (III-4, V-1, and IV-3), PCR produced only one fragment. (C) Sanger sequencing of the PCR product from affected member III-5 using primer 3 confirmed the presence of the ~11 kb deletion. The sequencing trace transitions from coordinate chr5:112,034,825 to chr5:112,045,846 (GRCh37/hg19), a span of 11,020 bp. (D) Sequencing the same PCR product using primer 2 showed the presence of a normal allele and the absence of the gap with a transition from coordinate chr5:112,034,825 to chr5:112,034,826.

Mentions: We verified the presence of the deletion using primers designed to create a PCR product of 373 bp when a genomic template with normal APC promoter is present (primers 1 and 2, Figure 3A). A second primer pair creates a 298 bp PCR product only when a genomic template with the ~11 kb deletion is present (primers 1 and 3, Figure 3A). We used these primers to assess the eight members of kindred 0124 (Figure 3B). PCR in all five affected individuals generated both 298 bp and 373 bp products, indicating heterozygosity for the promoter deletion allele in APC. In the three unaffected individuals, only the 373 bp fragment was generated indicating the absence of the promoter deletion.Figure 3


Novel APC promoter and exon 1B deletion and allelic silencing in three mutation-negative classic familial adenomatous polyposis families.

Lin Y, Lin S, Baxter MD, Lin L, Kennedy SM, Zhang Z, Goodfellow PJ, Chapman WC, Davidson NO - Genome Med (2015)

The heterozygous ~11 kbAPCdeletion is present in all affected members of the 0124 kindred. (A) Primers were designed to bind upstream of the ~11 kb deletion (primer 1), within the deletion (primer 2), and downstream of the deletion (primer 3). In the presence of a normal allele, primers 1 and 2 result in a 373 bp PCR product. Primers 1 and 3 do not form a product in the absence of the ~11 kb deletion but form a 298 bp PCR product when the deletion is present. (B) In the five affected members of kindred 0124 (III-5, IV-4, V-6, III-3, and IV-2), PCR using these primers resulted in two fragments of the expected sizes. In the three unaffected members (III-4, V-1, and IV-3), PCR produced only one fragment. (C) Sanger sequencing of the PCR product from affected member III-5 using primer 3 confirmed the presence of the ~11 kb deletion. The sequencing trace transitions from coordinate chr5:112,034,825 to chr5:112,045,846 (GRCh37/hg19), a span of 11,020 bp. (D) Sequencing the same PCR product using primer 2 showed the presence of a normal allele and the absence of the gap with a transition from coordinate chr5:112,034,825 to chr5:112,034,826.
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Related In: Results  -  Collection

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Fig3: The heterozygous ~11 kbAPCdeletion is present in all affected members of the 0124 kindred. (A) Primers were designed to bind upstream of the ~11 kb deletion (primer 1), within the deletion (primer 2), and downstream of the deletion (primer 3). In the presence of a normal allele, primers 1 and 2 result in a 373 bp PCR product. Primers 1 and 3 do not form a product in the absence of the ~11 kb deletion but form a 298 bp PCR product when the deletion is present. (B) In the five affected members of kindred 0124 (III-5, IV-4, V-6, III-3, and IV-2), PCR using these primers resulted in two fragments of the expected sizes. In the three unaffected members (III-4, V-1, and IV-3), PCR produced only one fragment. (C) Sanger sequencing of the PCR product from affected member III-5 using primer 3 confirmed the presence of the ~11 kb deletion. The sequencing trace transitions from coordinate chr5:112,034,825 to chr5:112,045,846 (GRCh37/hg19), a span of 11,020 bp. (D) Sequencing the same PCR product using primer 2 showed the presence of a normal allele and the absence of the gap with a transition from coordinate chr5:112,034,825 to chr5:112,034,826.
Mentions: We verified the presence of the deletion using primers designed to create a PCR product of 373 bp when a genomic template with normal APC promoter is present (primers 1 and 2, Figure 3A). A second primer pair creates a 298 bp PCR product only when a genomic template with the ~11 kb deletion is present (primers 1 and 3, Figure 3A). We used these primers to assess the eight members of kindred 0124 (Figure 3B). PCR in all five affected individuals generated both 298 bp and 373 bp products, indicating heterozygosity for the promoter deletion allele in APC. In the three unaffected individuals, only the 373 bp fragment was generated indicating the absence of the promoter deletion.Figure 3

Bottom Line: Furthermore, this same deletion with identical breakpoints was found in the probands of two additional APC mutation-negative classical FAP kindreds.Phasing analysis of single nucleotide polymorphisms (SNPs) around the deletion site in the three probands showed evidence of a shared haplotype, suggesting a common founder deletion in the three kindreds.These results support the causal role of a novel promoter deletion in FAP and suggest that non-coding deletions, identifiable using second-generation sequencing methods, may account for a significant fraction of APC mutation-negative classical FAP families.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Washington University School of Medicine, St. Louis, Missouri USA.

ABSTRACT

Background: The overwhelming majority (approximately 80%) of individuals with classic familial adenomatous polyposis (FAP) exhibit mutations in the coding sequence of the adenomatous polyposis coli (APC) tumor suppressor gene. Families without detectable APC mutations are unable to benefit from the use of genetic testing for clinical management of this autosomal dominant syndrome.

Methods: We used exome sequencing and linkage analysis, coupled with second-generation sequencing of the APC locus including non-coding regions to investigate three APC mutation-negative classical FAP families.

Results: We identified a novel ~11 kb deletion localized 44 kb upstream of the transcription start site of APC that encompasses the APC 1B promoter and exon. This deletion was present only in affected family members of one kindred with classical FAP. Furthermore, this same deletion with identical breakpoints was found in the probands of two additional APC mutation-negative classical FAP kindreds. Phasing analysis of single nucleotide polymorphisms (SNPs) around the deletion site in the three probands showed evidence of a shared haplotype, suggesting a common founder deletion in the three kindreds. SNP analysis within the coding sequence of APC, revealed that this ~11 kb deletion was accompanied by silencing of one of the APC alleles in blood-derived RNA of affected individuals.

Conclusions: These results support the causal role of a novel promoter deletion in FAP and suggest that non-coding deletions, identifiable using second-generation sequencing methods, may account for a significant fraction of APC mutation-negative classical FAP families.

No MeSH data available.


Related in: MedlinePlus