Novel deletion in 11p15.5 imprinting center region 1 in a patient with Beckwith-Wiedemann syndrome provides insight into distal enhancer regulation and tumorigenesis.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an early-onset overgrowth disorder with a high risk for embryonal tumors. It is mainly caused by dysregulation of imprinted genes on chromosome 11p15.5; however, the driving forces in the development of tumors are not fully understood. PROCEDURE: We report on a female patient presenting with macrosomia, macroglossia, organomegaly and extensive bilateral nephroblastomatosis. Adjuvant chemotherapy was initiated; however, the patient developed hepatoblastoma and Wilms tumor at 5 and 12 months of age, respectively. Subsequent radiofrequency ablation of the liver tumor and partial nephrectomy followed by consolidation therapy achieved complete remission. RESULTS: Molecular genetic analysis revealed a maternally derived large deletion of the complete H19-differentially methylated region (H19-DMR; imprinting control region-1 [ICR1]), the whole H19 gene itself as well as large parts of the distal enhancer region within the imprinting cluster-1 (IC1). Extended analysis showed highly elevated insulin-like growth factor 2 (IGF2) expression, possibly explaining at least in part the distinct BWS features and tumor manifestations. CONCLUSIONS: This study of a large maternal deletion encompassing the H19 gene and complete ICR1 is the first to demonstrate transcriptional consequences on IGF2 in addition to methylation effects resulting in severe overgrowth and occurrence of multiple tumors in a BWS patient. Studying this deletion helps to clarify the complex molecular processes involved in BWS and provides further insight into tumorigenesis.

Kaiso mediates human ICR1 methylation maintenance and H19 transcriptional fine regulation.

BACKGROUND: Genomic imprinting evolved in a common ancestor to marsupials and eutherian mammals and ensured the transcription of developmentally important genes from defined parental alleles. The regulation of imprinted genes is often mediated by differentially methylated imprinting control regions (ICRs) that are bound by different proteins in an allele-specific manner, thus forming unique chromatin loops regulating enhancer-promoter interactions. Factors that maintain the allele-specific methylation therefore are essential for the proper transcriptional regulation of imprinted genes. Binding of CCCTC-binding factor (CTCF) to the IGF2/H19-ICR1 is thought to be the key regulator of maternal ICR1 function. Disturbances of the allele-specific CTCF binding are causative for imprinting disorders like the Silver-Russell syndrome (SRS) or the Beckwith-Wiedemann syndrome (BWS), the latter one being associated with a dramatically increased risk to develop nephroblastomas. METHODS: Kaiso binding to the human ICR1 was detected and analyzed by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA). The role of Kaiso-ICR1 binding on DNA methylation was tested by lentiviral Kaiso knockdown and CRISPR/Cas9 mediated editing of a Kaiso binding site. RESULTS: We find that another protein, Kaiso (ZBTB33), characterized as binding to methylated CpG repeats as well as to unmethylated consensus sequences, specifically binds to the human ICR1 and its unmethylated Kaiso binding site (KBS) within the ICR1. Depletion of Kaiso transcription as well as deletion of the ICR1-KBS by CRISPR/Cas9 genome editing results in reduced methylation of the paternal ICR1. Additionally, Kaiso affects transcription of the lncRNA H19 and specifies a role for ICR1 in the transcriptional regulation of this imprinted gene. CONCLUSIONS: Kaiso binding to unmethylated KBS in the human ICR1 is necessary for ICR1 methylation maintenance and affects transcription rates of the lncRNA H19.