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

Schematic of primer design for haplotype phasing of SNP genotypes in relation to the ~11 kbAPCdeletion. The primer pairs P14/P15 and P8/P13 flanked the ~11 kb deletion and relevant SNPs. Only amplification of the APC allele with the ~11 kb deletion would result in an amplicon A size of 5.5 kb and an amplicon B size of 6.9 kb. Amplification of the allele without the ~11 kb deletion would not be efficient, as the resultant amplicons would be greater than 16 kb. Amplicons A and B were used as templates for Sanger sequencing reactions to reveal polymorphic variations in the diagrammed SNPs flanking the ~11 kb deletion.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Schematic of primer design for haplotype phasing of SNP genotypes in relation to the ~11 kbAPCdeletion. The primer pairs P14/P15 and P8/P13 flanked the ~11 kb deletion and relevant SNPs. Only amplification of the APC allele with the ~11 kb deletion would result in an amplicon A size of 5.5 kb and an amplicon B size of 6.9 kb. Amplification of the allele without the ~11 kb deletion would not be efficient, as the resultant amplicons would be greater than 16 kb. Amplicons A and B were used as templates for Sanger sequencing reactions to reveal polymorphic variations in the diagrammed SNPs flanking the ~11 kb deletion.

Mentions: As alluded to above, these three kindreds reside in geographically distinct regions and no known relatedness was discerned by questionnaire. Nevertheless, we examined genetic evidence of a relationship between these kindreds by determining the genetic haplotype associated with the ~11 kb deletion in each of the three probands. We first queried 11 SNPs flanking the deletion coordinates (Table 2), seven of which ranged from 229 to 2,408 bp 5′ of the deletion, and four of which ranged from 4,276 to 6,151 bp 3′ of the deletion. SNPs rs10463643, rs10051624, rs2900066, rs6594643, rs6594644, rs4705559, rs7704618, and rs1661035 were heterozygous in the probands of the 0124 and 0163 kindreds. These findings suggested that these SNPs could distinguish the haplotype associated with the ~11 kb deletion from the haplotype from the chromosomal strand without the deletion. Accordingly we phased the SNP genotypes by using a PCR strategy to selectively amplify the DNA from the chromosomal strand containing the ~11 kb deletion (Figure 5). Flanking primers were designed such that amplicons of 5.5 kb and 6.9 kb would reflect the deletion, whereas amplification from the wild type chromosomal strand would yield an amplicon 11 kb larger. Accordingly, we then isolated the 5.5 and 6.9 kb amplicons for Sanger sequencing as a result of which we were able to phase the SNP genotypes associated with the deletion. The sequencing results showed that the haplotype associated with the ~11 kb deletion was identical across the queried SNPs in each of the three probands (Table 2), providing evidence to suggest that these three kindreds share a common ancestor.Table 2


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)

Schematic of primer design for haplotype phasing of SNP genotypes in relation to the ~11 kbAPCdeletion. The primer pairs P14/P15 and P8/P13 flanked the ~11 kb deletion and relevant SNPs. Only amplification of the APC allele with the ~11 kb deletion would result in an amplicon A size of 5.5 kb and an amplicon B size of 6.9 kb. Amplification of the allele without the ~11 kb deletion would not be efficient, as the resultant amplicons would be greater than 16 kb. Amplicons A and B were used as templates for Sanger sequencing reactions to reveal polymorphic variations in the diagrammed SNPs flanking the ~11 kb deletion.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Schematic of primer design for haplotype phasing of SNP genotypes in relation to the ~11 kbAPCdeletion. The primer pairs P14/P15 and P8/P13 flanked the ~11 kb deletion and relevant SNPs. Only amplification of the APC allele with the ~11 kb deletion would result in an amplicon A size of 5.5 kb and an amplicon B size of 6.9 kb. Amplification of the allele without the ~11 kb deletion would not be efficient, as the resultant amplicons would be greater than 16 kb. Amplicons A and B were used as templates for Sanger sequencing reactions to reveal polymorphic variations in the diagrammed SNPs flanking the ~11 kb deletion.
Mentions: As alluded to above, these three kindreds reside in geographically distinct regions and no known relatedness was discerned by questionnaire. Nevertheless, we examined genetic evidence of a relationship between these kindreds by determining the genetic haplotype associated with the ~11 kb deletion in each of the three probands. We first queried 11 SNPs flanking the deletion coordinates (Table 2), seven of which ranged from 229 to 2,408 bp 5′ of the deletion, and four of which ranged from 4,276 to 6,151 bp 3′ of the deletion. SNPs rs10463643, rs10051624, rs2900066, rs6594643, rs6594644, rs4705559, rs7704618, and rs1661035 were heterozygous in the probands of the 0124 and 0163 kindreds. These findings suggested that these SNPs could distinguish the haplotype associated with the ~11 kb deletion from the haplotype from the chromosomal strand without the deletion. Accordingly we phased the SNP genotypes by using a PCR strategy to selectively amplify the DNA from the chromosomal strand containing the ~11 kb deletion (Figure 5). Flanking primers were designed such that amplicons of 5.5 kb and 6.9 kb would reflect the deletion, whereas amplification from the wild type chromosomal strand would yield an amplicon 11 kb larger. Accordingly, we then isolated the 5.5 and 6.9 kb amplicons for Sanger sequencing as a result of which we were able to phase the SNP genotypes associated with the deletion. The sequencing results showed that the haplotype associated with the ~11 kb deletion was identical across the queried SNPs in each of the three probands (Table 2), providing evidence to suggest that these three kindreds share a common ancestor.Table 2

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