Genetic disruption of the oncogenic HMGA2-PLAG1-IGF2 pathway causes fetal growth restriction

Abstract

PurposeFetal growth is a complex process involving maternal, placental and fetal factors. The etiology of fetal growth retardation remains unknown in many cases. The aim of this study is to identify novel human mutations and genes related to Silver-Russell syndrome (SRS), a syndromic form of fetal growth retardation, usually caused by epigenetic downregulation of the potent fetal growth factor IGF2.MethodsWhole-exome sequencing was carried out on members of an SRS familial case. The candidate gene from the familial case and two other genes were screened by targeted high-throughput sequencing in a large cohort of suspected SRS patients. Functional experiments were then used to link these genes into a regulatory pathway.ResultsWe report the first mutations of the PLAG1 gene in humans, as well as new mutations in HMGA2 and IGF2 in six sporadic and/or familial cases of SRS. We demonstrate that HMGA2 regulates IGF2 expression through PLAG1 and in a PLAG1-independent manner.ConclusionGenetic defects of the HMGA2-PLAG1-IGF2 pathway can lead to fetal and postnatal growth restriction, highlighting the role of this oncogenic pathway in the fine regulation of physiological fetal/postnatal growth. This work defines new genetic causes of SRS, important for genetic counseling.

Figure 1

Pedigrees, growth charts, and photographs of patients carrying mutations affecting the HMGA2–PLAG1–IGF2 pathway. The patient with sporadic PLAG1 anomaly is next to her healthy twin brother. The patient with IGF2 anomaly shows that a prominent forehead evident in early childhood may become less obvious in later life. The patients’ growth charts between the ages of 2 and 20 years are included in the Supplementary Figure.

Figure 2

Functional characterization of the effects of HMGA2 and PLAG1 silencing/mutation on IGF2 expression. (a) HMGA2 is an upstream regulator of PLAG1, which in turn is an upstream regulator of IGF2. (b) The HMGA2–PLAG1 pathway regulates IGF2-P3 expression. (c) The IGF2 regulation by HMGA2 in a PLAG1-independent manner. (d) The effect of the PLAG1:c.439del on IGF2 expression in cultures of patient fibroblasts. A single asterisk indicates a P < 0.05, and four asterisks indicate a P < 0.0001. The histograms represent the fold change means of biological replicates. Three separate small interfering RNA (siRNA) and vector transfection assays were performed. Each transfection assay contained at least four transfected wells of control and siRNA or vector transfection (N ≥ 12 biological sample per group per assay). For fibroblast expression, five cultures of control fibroblasts derived from different donors were compared to five different passages of cultured fibroblasts from the patient carrying the PLAG1 single-nucleotide deletion. The error bars indicate the standard error of the mean.

Figure 3

Schematic representation of the HMGA2–PLAG1–IGF2 pathway in a normal individual and in Silver–Russell cases. Green arrows indicate normal expression, the sizes of red arrows are representative of IGF2 downregulation and red stars indicate functional impairment for HMGA2, PLAG1, or IGF2 expression. SRS, Silver–Russell syndrome.