Lymphatic and angiogenic candidate genes predict the development of secondary lymphedema following breast cancer surgery

Abstract

The purposes of this study were to evaluate for differences in phenotypic and genotypic characteristics in women who did and did not develop lymphedema (LE) following breast cancer treatment. Breast cancer patients completed a number of self-report questionnaires. LE was evaluated using bioimpedance spectroscopy. Genotyping was done using a custom genotyping array. No differences were found between patients with (n = 155) and without LE (n = 387) for the majority of the demographic and clinical characteristics. Patients with LE had a significantly higher body mass index, more advanced disease and a higher number of lymph nodes removed. Genetic associations were identified for four genes (i.e., lymphocyte cytosolic protein 2 (rs315721), neuropilin-2 (rs849530), protein tyrosine kinase (rs158689), vascular cell adhesion molecule 1 (rs3176861)) and three haplotypes (i.e., Forkhead box protein C2 (haplotype A03), neuropilin-2 (haplotype F03), vascular endothelial growth factor-C (haplotype B03)) involved in lymphangiogensis and angiogenesis. These genetic associations suggest a role for a number of lymphatic and angiogenic genes in the development of LE following breast cancer treatment.

Figure 1. FOXC2 Gene Structure and Linkage Disequilibrium.

An ideogram of forkhead box C2 (FOXC2) is presented above the white bar that represents the physical distance along human chromosome 16 (chr16: 85,158,358–85,160,040; genome assembly 36.3, NM_005251.2). Exons are represented as thick bars. Reference sequence identifiers (rsID) for each single nucleotide polymorphism (SNP) are plotted both in terms of their physical distance (i.e., the white bar at the top of the figure) and equidistantly to render the pairwise linkage disequilibrium (LD) estimates that were calculated and visualized with Haploview 4.2. The gene structure for FOXC2 was rendered with FancyGene 1.4. The correlation statistic (r² and D') is provided in the heatmap. LD-based haplotype block definition was based on the D' confidence interval method. The haploblock is indicated in a bolded triangle and its component SNPs are rendered in bold font. Pairwise D' value (range: 0–1, inclusive) was rendered in color, with darker red diamond representing D' value approaching 1.0. When the r² value (range of 0–100, inclusive) is not equal to 0 or 100, it is provided in a given diamond. The 2-SNP haplotype associated with LE is composed of one rare and one common allele of two SNPs located in the immediate early promoter (rs34221221; rare “C” allele) and immediately downstream of the FOXC2 coding region (rs1035550; common “C” allele).

Figure 2. Differences between the lymphedema and no lymphedema groups.

A – Differences between the lymphedema and no lymphedema groups in the percentages of patients who were homozygous for the common allele (AA) or heterozygous or homozygous for the rare allele (AG+GG) for rs315721 in lymphocyte cytosolic protein 2 (LCP2). B – Differences between the lymphedema and no lymphedema groups in the percentages of patients who were homozygous or heterozygous for the common allele (TT+TG) or homozygous for the rare allele (GG) for rs849530 in neuropilin-2 (NRP2). C – Differences between the lymphedema and no lymphedema groups in the percentages of patients who were homozygous or heterozygous for the common allele (AA+AT) or homozygous for the rare allele (TT) for rs158689 in protein tyrosine kinase (SYK). D – Differences between the lymphedema and no lymphedema groups in the percentages of patients who were homozygous for the common allele (CC) or heterozygous or homozygous for the rare allele (CT+TT) for rs3176861 in vascular cell adhesion molecule 1 (VCAM1).

Figure 3. NRP2 Gene Structure and Linkage Disequilibrium.

An ideogram of neuropilin 2 (NRP2) is presented above the white bar that represents the physical distance along human chromosome 2 (chr2: 206,255,469–206,371,102; genome assembly 36.3, NM_201266.1). Exons are represented as thick bars. Gray lines connecting the exons represent introns. Reference sequence identifiers (rsID) for each single nucleotide polymorphism (SNP) are plotted both in terms of their physical distance (i.e., the white bar at the top of the figure) and equidistantly to render the pairwise linkage disequilibrium (LD) estimates that were calculated and visualized with Haploview 4.2. The gene structure for NRP2 was rendered with FancyGene 1.4. The correlation statistics (r² and D') are provided in the heatmap. LD-based haplotype block definition was based on the D' confidence interval method. The haploblock is indicated in a bolded triangle and its component SNPs are rendered in bold font. Pairwise D' values (range: 0–1, inclusive) were rendered in color, with darker red diamonds representing D' values approaching 1.0. When the r² values (range of 0–100, inclusive) are not equal to 0 or 100, they are provided in a given diamond. The 3-SNP haplotype associated with LE consists of one rare and two common alleles of three SNPs (rs849530 “G” rare allele, rs950219 “G” common allele, rs3771052 “G” common allele) located in intron 1 of the gene.

Figure 4. VEGFC Gene Structure and Linkage Disequilibrium.

An ideogram of vascular endothelial growth factor C (VEGFC) is presented above the white bar that represents the physical distance along human chromosome 4 (chr4: 177,841,685–177,950,889; genome assembly 36.3, NM_005429.2). Exons are represented as thick bars. Gray lines connecting the exons represent introns. Reference sequence identifiers (rsID) for each single nucleotide polymorphism (SNP) are plotted both in terms of their physical distance (i.e., the white bar at the top of the figure) and equidistantly to render the pairwise linkage disequilibrium (LD) estimates that were calculated and visualized with Haploview 4.2. The gene structure for VEGFC was rendered with FancyGene 1.4. The correlation statistics (r² and D') are provided in the heatmap. LD-based haplotype block definition was based on the D' confidence interval method. The haploblock is indicated in a bolded triangle and its component SNPs are rendered in bold font. Pairwise D' values (range: 0–1, inclusive) were rendered in color, with darker red diamonds representing D' values approaching 1.0. When the r² values (range of 0–100, inclusive) are not equal to 0 or 100, they are provided in a given diamond. The 2-SNP haplotype associated with LE consists of one rare and one common allele of two SNPs (rs3775202 “G” rare allele, rs3775195 “C” common allele) located in intron 4 of the gene. Of note, the strong linkage disequilibrium estimates observed in public databases (i.e., HapMap) resulted in the selection of 8 SNPs that tagged the entire coding region of the VEGFC gene.