Integrated genomic analysis implicates haploinsufficiency of multiple chromosome 5q31.2 genes in de novo myelodysplastic syndromes pathogenesis

Abstract

Deletions spanning chromosome 5q31.2 are among the most common recurring cytogenetic abnormalities detectable in myelodysplastic syndromes (MDS). Prior genomic studies have suggested that haploinsufficiency of multiple 5q31.2 genes may contribute to MDS pathogenesis. However, this hypothesis has never been formally tested. Therefore, we designed this study to systematically and comprehensively evaluate all 28 chromosome 5q31.2 genes and directly test whether haploinsufficiency of a single 5q31.2 gene may result from a heterozygous nucleotide mutation or microdeletion. We selected paired tumor (bone marrow) and germline (skin) DNA samples from 46 de novo MDS patients (37 without a cytogenetic 5q31.2 deletion) and performed total exonic gene resequencing (479 amplicons) and array comparative genomic hybridization (CGH). We found no somatic nucleotide changes in the 46 MDS samples, and no cytogenetically silent 5q31.2 deletions in 20/20 samples analyzed by array CGH. Twelve novel single nucleotide polymorphisms were discovered. The mRNA levels of 7 genes in the commonly deleted interval were reduced by 50% in CD34+ cells from del(5q) MDS samples, and no gene showed complete loss of expression. Taken together, these data show that small deletions and/or point mutations in individual 5q31.2 genes are not common events in MDS, and implicate haploinsufficiency of multiple genes as the relevant genetic consequence of this common deletion.

Figure 1. Chromosome 5q31.2 commonly deleted segment and array CGH plots.

A. Diagram of the chromosome 5q31.2 commonly deleted segment (CDS) (136.3–138.6 megabases) and the 5q33.1 CDS associated with the 5q minus syndrome (148.6–151.1 megabases). The centromere is located at the left and the telomere to the right. B. Whole chromosome 5 log₂ ratio (test/reference) plot for sample #315529 which contains del(5q) in 1/20 bone marrow cell metaphases, and has a blast count of 12%. No deletion was identified in this sample. To allow visualization of the data, each data point on the plot represents the average log₂ ratio for consecutive probes in a 20,000 base pair region (median number of probes per bin = 46). The dashed lines indicate the 5q31.2 and 5q33.1 CDS locations depicted in panel A. C. (Upper panel) Whole chromosome 5 log₂ ratio (test/reference) plot for sample #176267. An interstitial deletion on chromosome 5 produces a negative log₂ ratio for probes located within the deletion. The deletion boundaries are indicated by the arrowheads. The deletion spans from base 86,152,517–155,672,191 (determined using segMNT). The log₂ ratio deviation for the segment is consistent with the deletion of one allele. Sample #176267 contains del(5q) in 20/20 bone marrow cell metaphases (100%), and has a blast count of 7%. (Lower panel) Chromosome 5 log₂ ratio (test/reference) plot for sample #176267 from 136.3–138.6 Mb, corresponding to the 5q31.2 CDS. No biallelic deletion was identified in this region.

Figure 2. 5q31.2 SNP loss of heterozygosity in del(5q) samples.

A. Sequence traces for SNP rs1059110 in tumor samples from an individual homozygous for the common allele (left), a heterozygote (middle) with a normal allele trace peak height ratio = 0.44 (i.e., no allelic imbalance), and a heterozygote (right) with an abnormal allele trace peak height ratio = 0.20 (i.e., allelic imbalance). B. Heat map of allelic imbalance for 23 SNPs from 8 del(5q) and 9 non-del(5q) MDS samples. Each column represents a sample, and each row represents a SNP. red, allelic imbalance; yellow, no evidence of allelic imbalance; grey, homozygous SNP; white, no data. Base pair location is listed in the left column. C. (left panel) Proportion of abnormal metaphases is highly correlated with allelic skewing in heterozygous SNPs. (right panel) Blast count is a poor predictor of clonality. Two samples with bone marrow myeloblast counts less than 5% are clonal, indicated by marked SNP allelic skewing. In contrast, four samples with myeloblast counts greater than or equal to 5% were non-clonal as measured by SNP allelic ratio.

Figure 3. Gene expression levels of del(5q) genes in CD34+ cells.

mRNA expression levels were determined for 24 del(5q) genes that had probesets on the Affymetrix U133plus2 array using total RNA extracted from CD34+ purified cells from 6 MDS patients with del(5q), 20 MDS patients without del(5q), and 6 normal control individuals. Genes were rank ordered based on the median signal intensity in control samples. The average signal intensity of MDS samples (± standard deviation) with or without del(5q) is plotted relative to control samples. Each tick mark on the y-axis represents 1 fold change relative to control samples. The color bar indicates the Affymetrix signal intensity values for the control samples for each gene. Genes with 70–100% of samples called absent are plotted with red lines. When multiple probesets were called present for a gene, probesets were averaged (probesets were included in the average signal if they were present in at least 4 of 6 control samples and had a median expression in controls greater than 1,500, the scaled target intensity of arrays). If all probesets were called absent for a gene, the highest median expressing probeset was used for plotting. * p<0.05 compared to control.