Imprinting, expression, and localisation of DLK1 in Wilms tumours

Abstract

Background: Loss of imprinting (LOI) of the H19/IGF2 domain is a common feature of Wilms tumour. The GTL2/DLK1 domain is also imprinted and is structurally similar to H19/IGF2. The question arises as to whether DLK1 also undergoes LOI in Wilms tumour, or whether the LOI mechanism is restricted to the H19/IGF2 domain.

Aim: To investigate the imprinting status of DLK1 in Wilms tumours with IGF2 LOI. The cellular localisation of DLK1 in the tumours was also examined.

Methods: DLK1 expression was measured by quantitative real time polymerase chain reaction (Q-PCR) in 30 Wilms tumours that had previously been classified according to whether they had IGF2 LOI, WT1 mutations, or 11p15.5 loss of heterozygosity. Allele specific expression of DLK1 was examined by direct sequencing using a DLK1 exon 5 polymorphism (rs1802710). Immunohistochemical analysis of DLK1 was performed on 13 tumours and two intralobar nephrogenic rests, in addition to two fetal kidneys and one fetal skeletal muscle sample.

Results: Ten of 30 tumours were heterozygous for rs1802710 and all tumours showed retention of imprinting of DLK1. Moderate to high expression of DLK1 was detected by Q-PCR in nine of 13 tumours with myogenic differentiation. Immunohistochemical expression of DLK1 was detected in the myogenic elements.

Conclusion: LOI does not occur at the GTL2/DLK1 domain in Wilms tumour. This finding suggests that LOI at 11p15.5 does not reflect non-specific disruption of a shared imprinting mechanism. DLK1 expression in Wilms tumour might reflect the presence of myogenic differentiation, rather than an alteration of its imprinting status.

Figure 1

The structural, spatial, and regulatory features of IGF2/H19 and DLK1/GTL2 domains. Expressed and imprinted genes are shown as open boxes and black boxes, respectively. CTCF binding sites are indicated by vertical rectangles. Open circles indicate enhancer elements.

Figure 2

The tumour DNA is heterozygous for the C/T polymorphism. The father and mother are homozygous for the C and T allele, respectively. Therefore, exclusive expression of the C allele indicates paternal expression of DLK1.

Figure 3

Immunohistochemistry for DLK1 in (A) the developing kidney (21 weeks), (B) tumour bearing normal kidney, and (C) fetal skeletal muscle (8 weeks). Original magnification: A, ×200; B, C, ×400. (A) DLK1 immunostaining is faintly positive or negative in condensed mesenchyme (cm), renal vesicles (rv), an S shaped body (s), a presumptive podocyte (pp), and glomerular epithelium (gl). DLK1 was weakly positive in the cytoplasm of ureteric buds (ub) and ureteric bud ampullae (uba). DLK1 was moderately to highly expressed in the cytoplasm of developing tubules (dt, future proximal and distal convoluted tubules). (B) Expression of DLK1 was seen in the nucleus and cytoplasm of collecting ducts (cd), Henle’s loops (hl), proximal and distal convoluted tubules (pct and dct). (C) DLK1 is expressed in fetal skeletal muscle.

Figure 4

Immunohistochemistry for DLK1 in (A) Wilms tumour with myogenic differentiation, (B) Wilms tumour without myogenic differentiation, and (C) an intralobar nephrogenic rest (ILNR). Original magnification, ×200. (A) Tumour WT1-3. Immunostaining for DLK1 is seen in the cytoplasm of the myogenic elements (mu). Focal immunostaining is seen in the nucleus and cytoplasm of the blastemal (bl) and the epithelial cells (arrows). (B) Tumour LOH-3. A tumour showing blastemal predominant histology with focal epithelial differentiation (arrowheads). DLK1 is expressed in the nucleus and cytoplasm of the blastemal cells and epithelial elements. (C) An ILNR showing similar DLK1 staining patterns to that seen in Wilms tumours. Non-polarised epithelial structures (central and stromal tubules, indicated by arrowheads) and blastemal components (bl) show high expression of DLK1.