Genomic imprinting of IGF2 is maintained in infantile hemangioma despite its high level of expression

Abstract

Hemangioma, the most common tumor of infancy, is characterized by rapid growth and slow regression. Increased mRNA expression of insulin-like growth factor 2 (IGF2) has been detected in the proliferating phase by cDNA microarray analysis, but the underlying mechanism causing the increase remains unknown. Here, using quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry, we show that IGF2 is highly expressed in both proliferating and involuting phase hemangioma, but is not detectable in other vascular lesions such as pyogenic granuloma, venous malformation, lymphatic malformation, or in normal infant skin. Loss of imprinting of the Igf2 gene has been associated with IGF2 overexpression in a variety of childhood tumors. To determine if loss of imprinting and consequent bi-allelic expression might contribute to the increased expression of IGF2, we examined the genomic imprinting status of Igf2 in 48 individual hemangiomas. We determined allele-specific Igf2 expression using reverse transcriptase-PCR combined with analysis of an Apa I-sensitive restriction fragment length polymorphism. Similar to heterozygous normal skin controls, all 15 informative hemangiomas showed uniform mono-allelic expression of Igf2. Therefore, loss of imprinting is not involved in the increased expression of IGF2 in infantile hemangioma.

Figure 1

mRNA expression of IGF2 and KDR in infantile hemangioma. (A) Using ³²P-labeled cDNA probes to IGF2 and KDR, Northern blotting was performed on tissue RNA of proliferating hemangioma 33 and 32 from patients of 4 and 7 mo of age, involuting hemangioma I-12 and I-14 from 2 patients of 3 y of age, and normal infant skin. Equivalent loading of RNA was verified by staining the gel with ethidium bromide to visualize 28s and 18s rRNA. (B) The intensity of IGF2 and KDR signals were quantified, normalized to that of I-14, and plotted as relative fold increase.

Figure 2

Quantitative real-time PCR analysis of IGF2 and KDR transcripts in hemangioma compared with levels in other vascular anomalies. In panel A, normalized RNA levels for IGF2 (black bars) and KDR (hatched bars) were determined from a standard curve generated from cycle threshold (Ct) values obtained from serial dilutions of cDNA from human dermal microvascular endothelial cells (panel B). Slopes were −5.8 for KDR (+) and −3.6 for IGF2 (−).

Figure 3

IGF2 protein expression in infantile hemangioma compared with other vascular anomalies. Cryosections of proliferating hemangioma (A and F), involuting hemangioma (B), normal infant skin (C), venous malformation (D), congenital hemangioma (G), and lymphatic malformation (H) were stained with a mouse anti-human IGF2 antibody (all panels except E). Note the presence of endothelial IGF2 expression associated with vascular channels and the lack of IGF2 associated with intralesional arterioles in hemangioma (A, B, and F). Panels E and F are serial sections of a proliferating hemangioma labeled with a rabbit anti-human GLUT1 antibody (E) for comparison to immunostaining observed with a mouse anti-human IGF2 antibody (F). Arrows in panels E and F highlight a GLUT1-positive vessel that appears to be IGF2-negative. (I) Normalized RNA levels for IGF2 (black bars) and KDR (hatched bars) in 5 different isolates of hemangioma-derived endothelial cells (hemEC) were determined by real-time PCR as described in Figure 2. Placental RNA served as a positive control.

Figure 4

Imprinting status of Igf2 in infantile hemangioma. (A) Agarose gel electrophoresis of ApaI-digested PCR products of Igf2 exon 9 amplified from genomic DNA. Proliferating hemangioma designated 39, 55, 57, involuting hemangioma I-18, I-21, I-24, and normal infant skin F3 were heterozygous (A and B alleles). I-13 (B allele) and I-19 (A allele) were homozygous. The results are representative of 27 proliferating hemangiomas, 21 involuting hemangiomas, and 11 normal infant skin specimens (see Table 1). (B) Agarose gel electrophoresis of ApaI-digested cDNA products of Igf2. Monoallelic Igf2 expression is shown in proliferating hemangiomas 35, 38, 51, 53, 57, 63, involuting hemangiomas I-16, I-18, I-20, I-21, I-24, I-25, I-30, and normal infant skin control F3 and F10. The results are representative of the analysis of 15 informative cases. Homozygous hemangioma I-13 and skin control F4 and F11 were included to verify that ApaI digestions had gone to completion.