Limits...
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 ~11 kb deletion silences oneAPCallele in the blood of affected individuals. Germline heterozygous SNPs were evaluated in the RNA isolated from peripheral blood, with sample traces shown here. dbSNP rs465899 was found to be heterozygous in the proband 0124 (III-5) gDNA but not in the cDNA, whereas this SNP was heterozygous in both gDNA and cDNA in the unaffected subject 0124 (III-4). A loss of cDNA heterozygosity was also observed in proband 0130 (IV-1) with SNP rs459552, and in proband 0163 (III-2) with SNP rs465899.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4418073&req=5

Fig6: The ~11 kb deletion silences oneAPCallele in the blood of affected individuals. Germline heterozygous SNPs were evaluated in the RNA isolated from peripheral blood, with sample traces shown here. dbSNP rs465899 was found to be heterozygous in the proband 0124 (III-5) gDNA but not in the cDNA, whereas this SNP was heterozygous in both gDNA and cDNA in the unaffected subject 0124 (III-4). A loss of cDNA heterozygosity was also observed in proband 0130 (IV-1) with SNP rs459552, and in proband 0163 (III-2) with SNP rs465899.

Mentions: APC is transcribed in a wide variety of tissues, including peripheral blood cells [6]. To assess the effect of the ~11 kb deletion on allelic transcription in these kindreds, we identified exonic SNPs in APC which were heterozygous in the germline of affected and unaffected individuals of these FAP kindreds. Silencing of an APC allele in individuals heterozygous for these SNPs would result in the detection of only one base at the corresponding complementary DNA (cDNA) site from expression of a single APC allele. Sanger sequencing of genomic DNA (gDNA) from proband III-5 of kindred 0124 showed heterozygousity at SNPs rs459552 and rs465899, but sequencing of cDNA derived from blood shows the presence of only one allele for both corresponding positions. This is in contrast to the unaffected individual III-4 of kindred 0124 whose gDNA is heterozygous for SNP rs465899, and in whom cDNA sequencing shows evidence of mRNA expression from two alleles (Table 3 and Figure 6). The silencing of one APC allele was also observed in affecteds from the other two kindreds with loss of cDNA heterozygosity at SNP rs459552 in proband IV-1 of kindred 0130, and SNPs rs459552 and rs465899 in proband III-2 of kindred 0163 (Table 3 and Figure 6).Table 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 ~11 kb deletion silences oneAPCallele in the blood of affected individuals. Germline heterozygous SNPs were evaluated in the RNA isolated from peripheral blood, with sample traces shown here. dbSNP rs465899 was found to be heterozygous in the proband 0124 (III-5) gDNA but not in the cDNA, whereas this SNP was heterozygous in both gDNA and cDNA in the unaffected subject 0124 (III-4). A loss of cDNA heterozygosity was also observed in proband 0130 (IV-1) with SNP rs459552, and in proband 0163 (III-2) with SNP rs465899.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4418073&req=5

Fig6: The ~11 kb deletion silences oneAPCallele in the blood of affected individuals. Germline heterozygous SNPs were evaluated in the RNA isolated from peripheral blood, with sample traces shown here. dbSNP rs465899 was found to be heterozygous in the proband 0124 (III-5) gDNA but not in the cDNA, whereas this SNP was heterozygous in both gDNA and cDNA in the unaffected subject 0124 (III-4). A loss of cDNA heterozygosity was also observed in proband 0130 (IV-1) with SNP rs459552, and in proband 0163 (III-2) with SNP rs465899.
Mentions: APC is transcribed in a wide variety of tissues, including peripheral blood cells [6]. To assess the effect of the ~11 kb deletion on allelic transcription in these kindreds, we identified exonic SNPs in APC which were heterozygous in the germline of affected and unaffected individuals of these FAP kindreds. Silencing of an APC allele in individuals heterozygous for these SNPs would result in the detection of only one base at the corresponding complementary DNA (cDNA) site from expression of a single APC allele. Sanger sequencing of genomic DNA (gDNA) from proband III-5 of kindred 0124 showed heterozygousity at SNPs rs459552 and rs465899, but sequencing of cDNA derived from blood shows the presence of only one allele for both corresponding positions. This is in contrast to the unaffected individual III-4 of kindred 0124 whose gDNA is heterozygous for SNP rs465899, and in whom cDNA sequencing shows evidence of mRNA expression from two alleles (Table 3 and Figure 6). The silencing of one APC allele was also observed in affecteds from the other two kindreds with loss of cDNA heterozygosity at SNP rs459552 in proband IV-1 of kindred 0130, and SNPs rs459552 and rs465899 in proband III-2 of kindred 0163 (Table 3 and Figure 6).Table 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