Chromosome 16q22-linked familial AML: Exclusion of candidate genes, and possible disease risk modification by NQO1 polymorphisms

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Pedigrees and samples

Pedigrees, numbering of individuals, DNA and sample preparation have been described in :
Family A: Horwitz M, Benson KF, Li FQ, Wolff J, Leppert MF, Hobson L, Mangelsdorf M, Yu S, Hewett D, Richards RI, Raskind WH. Genetic heterogeneity in familial acute myelogenous leukemia: evidence for a second locus at chromosome 16q21-23.2. Am J Hum Genet. 1997 Oct;61(4):873-81.
Family B: Gao Q, Horwitz M, Roulston D, Hagos F, Zhao N, Freireich EJ, Golomb HM, Olopade OI. Susceptibility gene for familial acute myeloid leukemia associated with loss of 5q and/or 7q is not localized on the commonly deleted portion of 5q. Genes Chromosomes Cancer. 2000 Jun;28(2):164-72.
Samples used in this study were affecteds IV-1, IV-3, unaffected carriers II-5 and III-5 for family A, and affecteds III-1, III-6, unaffecteds II-3, III-2, III-7, III-8 and III-9 for family B. All individuals had given informed consent. Lymphoblastic cell lines for RNA isolation were available from individuals in italics. Genomic Sequences, Gene Structures and Primer Design

Genomic DNA sequences and gene structures were obtained using public databases (http://www.ncbi.nlm.nih.gov/Genbank/index.html, http://genome.ucsc.edu and http://www.expasy.ch/sprot). Primers pairs were designed to amplify Single Nucleotide Polymorphisms (SNP) or exons and intronic flanking regions using Primer3 (http://www.broad.mit.edu/cgi-bin/primer/primer3_www.cgi) and are listed in supplementary information, Table 1: Genes and Primers.

PCR and Sequencing

Standard PCR conditions were used for most of the amplifications. Briefly, 50-100 ng DNA was amplified in a final volume of 50μl using 1.5mM MgCl₂, 2mM of each nucleotide, 3U Taq polymerase (all from Roche Diagnostics, Mannheim, Germany), 100ng/μl of each primer (Geneworks, Adelaide, Australia), and anti-Taq antibody (Clontech Laboratories, Palo Alto, CA, USA). PCR reactions were carried out on a Peltier Thermal Cycler PTC-200 (MJ Research, Waltham MA) and included initial denaturation at 95°C for 5min, then 40 cycles of denaturation at 95°C for 20sec, annealing for 30sec and elongation at 72°C for 30sec, and a final elongation step at 72°C for 10min. The amplicons were purified using QIAquick PCR purification kit (Qiagen, Clifton Hill, Australia), sequenced in both directions using BigDye terminator cycle sequencing (Perkin Elmer Corporation) and analyzed on an ABI377 sequencer.

Denaturing high pressure liquid chromatography (DHPLC)

Analysis of NFATC3 and NFAT5 genes was undertaken by a preliminary DHPLC screen followed by direct sequencing. BAC clones were used as genomic reference DNA. For the analysis of the NFAT5 gene, BAC clones RPCI11-140H17 covering exons 1 to 3 and RPCI11-311C24 covering exons 3 to 16 ( both from human BAC library RPCI-11) were used, and for the NFATC3 gene, BAC clones RPCI11-96D1 (RPCI human BAC library 11) covering exons 7 to 12 and CIT987SK-A-67A1 (CalTech human BAC library A) covering exons 1 to 9. DHPLC was used also for the analysis of NFAT5 exon 4 of DNA from all members of both families and from 30 unrelated non leukemic individuals. Briefly, DNA was amplified using 2.5 U Platinum Taq DNA polymerase (Invitrogen, Melbourne, Australia), 1.5mM MgCl₂, 2mM dNTP and 100ng/μl primers in a final volume of 50μl. Amplicons of patient DNA and reference BAC DNA were mixed in an equimolar ratio (for detection of homozygous mutations in patient samples). Mixed and non mixed samples, and negative control PCR samples without DNA were run in an MJ research PTC-200, Peltier Thermal Cycler (Geneworks, Adelaide, Australia) according to Varian’s PCR Guidelines for DHPLC (Varian Instruments, Melbourne, Australia) and analysed using the Varian ProStar DHPLC system. Temperatures for resolution of heteroduplex molecules were determined using a computer analysis package (http://insertion.stanford.edu/melt1.html). Any sample showing a chromatogram with variation in shape and retention time as compared to the control chromatograms was further analysed by direct sequencing.

Population studies of sequence variants

For NFAT5 exon 4 sequence variant IVS3-62A>G, DHPLC was performed as above using DNA from IV-1, IV-3, II-5 and III-5 of family A, II-1, III-6, II-3, III-2, III-7, III-8 and III-9 of family B, and from 30 unrelated non leukemic individuals.
For CDH1 exon 14 sequence variant 2292C>T, SinI restriction analysis was performed on amplified exon 14 DNA from individuals II-3, III-1, III-2, III-6, III-7, III-8 and III-9 of family B, and from 21 unrelated non leukemic individuals. Samples from family A were not tested, as prior sequencing did not show the sequence variation in affect individual IV-3. Twenty μl of the amplified DNA were digested with SinI (www.promega.com) for 2 hours at 37°C following the standard protocol of the manufacturer. The digests were analysed on a 1.5% agarose gel. Samples without the 2292C>T variation show bands of 295bp + 197bp, whereas samples with the sequence variation are not digested and show the 492bp band.

Semi-quantitative RT-PCR

Semi-quantitative RT-PCR was performed using RNA extracted from lymphoblastoid cell lines derived from peripheral blood from individuals IV-3 and III-5 of family A and from individuals II-3, III-1 and III-9 of family B. Total RNA was extracted from 3x107 cells using the RNeasy Midi Kit (Qiagen). cDNA synthesis was performed using 2μg of total RNA, T7-(dT)24 primer and SuperScript II (Invitrogen Life Technologies). The product was cleaned up using the GeneChip Sample Cleanup Module (Qiagen) and subsequently used for PCR with the following primers: for E2F-4 5’-TGCCACCACCTGAAGATTTG and 3’-GGACATCAACTCCTCCAGCA, for CTCF 5’-TCCACCCCAGGAAGATCCTA and 3’-GCTTCTCGTGGGTGTGTTTG, for NFATc3 5’-GGACAGCACTCAACTCAAGCA and 3’-GTTGGAAACCCAAGGTCCAA, for NFAT5 5’-AAACGGACCCTTTGGCTCTC and 3’-CTTGCATAGCCTTGCTGTCG, for NQO1 5’-ACCACGAGCCCAGCCAAT and 3’-ACTCCGGAAGGGTCCTTTGT, and for CDH1 5’-CTTACTGCCCCCAGAGGATG and 3’-AAAAACCACCAGCAACGTGA. PCR was carried out using the described standard conditions, with increasing cDNA dilutions up to 1:3000.

Northern Blots

Northern Blots were performed following standard procedures. RNA was extracted from 107 cells, and for each sample, 10μg of RNA was used for blotting. Probes were radiolabeled by PCR (for primers, see supplementary information, Table 2: Probes for Northern).