Functional Characterization of NIPBL Physiological Splice Variants and Eight Splicing Mutations in Patients with Cornelia de Lange Syndrome

María Teresa-Rodrigo,Gabriele Gillessen-Kaesbach,Pablo Lapunzina,María Gil-Rodríguez,Feliciano Ramos,Jolanta Wierzba,Juliane Eckhold,Milagros Ciero,Juan De Karam,María Hernández-Marcos,Diana Braunholz,César Casale,Beatriz Puisac,Fernando Santos-Simarro,Carolina Baquero,Andreas Dalski,Juan Pié,Frank Kaiser

doi:10.3390/ijms150610350

Abstract

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21) or functionally associated factors (NIPBL, HDAC8) of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ΔE10, ΔE12, ΔE33,34, and B’. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame.

Highlights

Cornelia de Lange syndrome (CdLS; OMIM 1227470, 300590, 610759, 614701, 300882) is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and abnormalities of other systems with variable expressivity [1]
By targeted Sanger sequencing of all 47 exons of the NIPBL gene we could identify nine allelic variants in our patients (Figure 1, Table 1) situated into the exon-intron junctions
We describe the first coordinated analysis of twelve CdLS–causing splice-site mutations in the NIPBL gene on DNA and RNA level as well as by in silico analyses

Summary

Introduction

Cornelia de Lange syndrome (CdLS; OMIM 1227470, 300590, 610759, 614701, 300882) is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and abnormalities of other systems with variable expressivity [1]. Mutations in five genes, encoding structural components of the cohesin complex (SMC1A, SMC3, and RAD21) and its regulators (NIPBL and HDAC8) have been found in patients with CdLS [2,3,4,5,6,7,8,9]. 60% of patients with CdLS carry an identifiable mutation in NIPBL [2,4,5]. This gene is located on chromosome 5p13.2 and contains 47 exons. Only two splicing isoforms have been detected in embryonic tissues, there may be more variants due to the large size of NIPBL [10,11]. In the main isoform (A), exons 2–47 codify for 2804 amino acids

Results

Discussion

Conclusion