Characterization of a t(5;8)(q31;q21) translocation in a patient with mental retardation and congenital heart disease: implications for involvement of RUNX1T1 in human brain and heart development

Abstract

The chromosome break points of the t(8;21)(q21.3;q22.12) translocation associated with acute myeloid leukemia disrupt the RUNX1 gene (also known as AML1) and the RUNX1T1 gene (also known as CBFA2T3, MTG8 and ETO) and generate a RUNX1-RUNX1T1 fusion protein. Molecular characterization of the translocation break points in a t(5;8)(q32;q21.3) patient with mild-to-moderate mental retardation and congenital heart disease revealed that one of the break points was within the RUNX1T1 gene. Analysis of RUNX1T1 expression in human embryonic and fetal tissues suggests a role of RUNX1T1 in brain and heart development and support the notion that disruption of the RUNX1T1 gene is associated with the patient's phenotype.

Figure 1

Pictures of the patient at age of 1 year (a), 2 years (b), 4.5 years (c) and 27 years (d–f). He has minor craniofacial dysmorphism with dental anomalies and a coarse face with full eye-brows and full lips. The nose shows a broad tip, the forehead is high and the posterior hairline is low set.

Figure 2

Physical map of the break point region at 8q21.3. The chromosome 8 break point was within a 26.5 kb region (shaded gray) determined by the overlapping ends of the BAC clones RP11-280G8 and RP11-341C1. The break point thus mapped within intron 1b of RUNX1T1 near, and overlapping with some of the break points reported in AML (marked by vertical lines).

Figure 3

Expression of RUNX1T1 in human embryonic and fetal tissues. (a) Expression level in a panel of tissues. Expression level is shown as fold change compared to the expression in embryonic heart. The age of the embryo/fetus is indicated at each tissue. (b) Expression level in 25 human embryonic hearts. Expression level is shown as fold change compared to the expression in embryonic heart on day 40. The curve represents the average value of the data points in groups of three. Expression level was determined using QPCR. The C_T values used for calculation of the relative expression were in the range of 19–26 cycles. The data was normalized using the average value of two housekeeping genes (B2M and HPRT).

Figure 4

Expression of RUNX1T1 in the developing human brain. (a) shows a sagittal section through the midbrain of a 5-week-old embryo. At this early stage of development all cell nuclei in the CNS exhibit a positive-staining reaction, which is particularly strong in the perinuclear space. The reactivity is most pronounced in the ventricular zone (VZ). Outside the CNS blood vessels (BV) are strongly reacting whereas the mesenchyme shows a weak reactivity (arrow). (b) depicts a coronal section through the forebrain of an 8-week-old fetus. The layers of the frontal cortex show a differential reactivity depending of differentiation: The radial glial cells/neuroblasts of the ventricular zone (VZ) express an overall weak reactivity. The migrating young neurons in the subventricular and intermediate zone (IZ) express strong nuclear reactivity, which is also seen in the subplate zone (SP). Differentiating neurons in the cortical plate (CP) show a strong nuclear but also a cytoplasmic reactivity. Nuclei of Cajal-Retzius cells in the marginal zone (MZ) are also distinctly labeled. (c) is from a horizontal section of the lower brain stem of a 16-week-old fetus. The inset shows mature neurons in the ambiguous nucleus with strong cytoplasmic reactivity, distinct nucleoli but a lack of expression in the nuclei. Early differentiating glial cells (GC) show a marked staining of their nuclei.

Figure 5

Expression of RUNX1T1 in the developing human heart. This figure shows the overall reactivity for RUNXITI in the heart of a 6-week-old embryo (a). Note the positive-staining reaction in endocardial cells (EC) of the septum primum (b) and in the developing trabeculae (c). The nuclei of the cardiomyocytes (CM) are more weakly stained.

Figure 6

Expression of RUNX1T1 in the developing human heart. (a) In the heart of a 9-week-old fetus the endocardial cells (EC) express a very strong cytoplasmic reactivity (b) whereas the nuclei of the cardiomyocytes (CM) are distinctly stained.