healthmedicinet

Study subjects

Within our FAP registry, we identified three APC mutation-negative FAP kindreds with classical phenotypes. Participants from these
kindreds gave written informed consent for participation in a study approved by the
Institutional Review Board of Washington University, St. Louis (IRB# 201105464) and
which conforms to the Helsinki Declaration. These kindreds were concentrated in three
different states: Missouri, Illinois, and Idaho, and were not known to be related.
The probands from each kindred underwent clinical genetic testing with Colaris AP®
(Myriad Genetic Laboratories, Salt Lake City, UT, USA), which sequences 15 exons of
APC and exons 7 and 13 of MUTYH to detect the mutations Y165C and G382D. Southern blot analysis on all exons of APC is also performed to detect large rearrangements. No known pathologic mutations in
APC and MUTYH were detected by this method in the probands of the three kindreds.

Blood from participants was collected in tubes without additives for DNA extraction
and in PAXgene Blood RNA Tubes (PreAnalytiX; Qiagen, Valencia, CA, USA) for RNA preservation
and extraction. DNA blood extraction was performed using the Gentra Puregene Blood
Kit (Qiagen), and RNA blood extraction was performed using the PAXgene Blood RNA Kit
(Qiagen).

Exome sequencing and linkage analysis

Exome capture was performed with the SureSelect 38 Mb All Exon kit (Agilent, Salta
Clara, CA, USA) for members of kindred 0124 III-5, III-3, III-4, and the SureSelect
72 Mb (V4?+?UTR) kit was used for individuals 0124 IV-4, V-6, IV-2, V-1, and IV-3.
Paired-end sequencing (2?× 101 bp) was performed on the HiSeq 2000 platform (Illumina,
San Diego, CA, USA). The sequences were aligned against the GRCh37/hg19 human reference
genome using Novoalign (Novocraft, Selangor, Malaysia), optical duplicate sequence
removal was performed using samtools 13], and variant calls were identified using samtools and GATK 14]. Local realignment and base quality recalibration using GATK were performed prior
to variant calling, and a genotype quality GQ threshold of 20 was used to retain single
nucleotide variants (SNVs) for further analysis. All reported genomic coordinates
are in relation to the GRCh37/hg19 reference human genome. The sequencing data is
available at dbGaP with accession number phs000904.v1.p1.

To search for candidate exon mutations which segregated with the FAP phenotype in
kindred 0124, we applied a series of filters to the identified SNVs. We required that
a candidate SNV follow an autosomal pattern of inheritance and retained only those
which were heterozygous in affected individuals and homozygous in unaffected ones.
The SNVs were annotated for function using ANNOVAR 15], and only non-synonymous mutations were retained. Finally, single nucleotide polymorphisms
(SNPs) in dbSNP132 16] and identified in the 1000 Genomes project 17] were removed. To perform linkage analysis, the unfiltered, aggregated SNVs were analyzed
with MERLIN using parametric linkage analysis with a rare dominant disease model 18].

Coloseqâ„¢ testing and deletion validation

Subjects IV-4 of kindred 0124, IV-1 of kindred 0130, and III-2 of kindred 0163 underwent
sequencing of the APC locus with the ColoSeqâ„¢ Polyposis Panel, which performs second-generation sequencing
of APC and MUTYH to cover all exons, introns and flanking sequences 12]. An ~11 kb deletion encompassing exon and promoter 1B was detected in all three individuals
between coordinates chr5:112,034,824-112,045,845. To verify the presence of this deletion,
PCR using standard conditions and primers P1, P2, and P3 (Table 1) were used. In the presence of template genomic DNA with the ~11 kb deletion, primers
P1 and P3 form a 298 bp PCR product, whereas primers P1 and P2 do not result in a
product because the binding site for primer P2 is absent. With a normal genomic DNA
template, primers P1 and P2 form a 373 bp product, whereas primers P1 and P3 do not
form a product because the distance of their binding sites exceeds 11 kb. The PCR
amplicons were sequenced with Sanger chemistry using the ABI BigDye Terminator Mix
(version 3, Applied Biosystems, Foster City, CA, USA).

Table 1. Primer sequences and coordinates

SNP genotyping and haplotype phasing

To query SNPs flanking the promoter deletion, the following primer pairs were used
(Table 1): primers P4/P5 for rs76768628, rs60905866, and rs10463643; primers P6/P7 for rs10051624;
primers P8/P9 for rs2900066, rs6594643, and rs6594644; primers P10/P11 for rs4705559
and rs7704618; and primers P12/P13 for rs1661035 and rs76028897. To amplify DNA specifically
from the chromosomal strand harboring the ~11 kb deletion, two primer pairs were used:
P8/P13 and P14/P15 (Table 1). On chromosomes with the ~11 kb deletion, these primer pairs resulted in amplicons
of 5,541 bp and 6,956 bp in size, respectively. Because these amplicons represent
DNA amplified only from the chromosomal strand with the ~11 kb deletion, all of the
SNP genotypes determined from these amplicons were used as templates for Sanger sequencing
(and haplotype phasing) using primers P4, P6, P8, P10, and P12 (Table 1).

Allelic expression assessment

dbSNP was searched for SNPs residing in the coding region of APC, and rs459552 and rs465899 were found to be heterozygous in the exome sequence data
of one or more individuals of kindred 0124. After extraction of RNA from blood collected
in PAXgene Blood RNA Tubes, reverse transcription was performed using SuperScript
III Reverse Transcriptase (Invitrogen, Grand Island, NY, USA). The following primers
were used to perform PCR and subsequent sequencing for SNP assays (Table 1): primers P16/P17 for rs459552 (producing amplicon chr5:112,176,294-112,176,979)
and primers P18/P19 for rs465899 (producing amplicon chr5:112,176,956-112,177,213).