Novel Mutation of the RUNX2 Gene in Patients with Cleidocranial Dysplasia

Hordyjewska, Ewa; Jaruga, Anna; Kandzierski, Grzegorz; Tylzanowski, Przemko

doi:10.1159/000477307

Cited by 6 publications

(12 citation statements)

References 36 publications

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“…Recent studies have shown that mutations in the Runt domain of RUNX2 severely affect its DNA‐binding ability . Therefore, we tested the transactivation potential of five identified variants carrying out Luciferase reporter assay with a reporter plasmid containing six osteoblast specific elements from the osteocalcin promoter (OSE2) placed upstream of a luciferase reporter gene .…”

Section: Resultsmentioning

confidence: 99%

“…Missense mutants c.391C>T p.(Arg131Cys) (p.R131C), c.407T>A p.(Leu136Gln) (p.L136Q), c.652A>G p.(Lys218Glu) (p.K218E), c.659C>G p.(Thr220Arg) (p.T220R) were generated by site‐directed mutagenesis (QuickChange Lightning Site‐Directed Mutagenesis Kit, STRATAGENE) using WT (wild type) clone tagged with hemagglutinin (HA) (WT‐HA‐RUNX2‐pcDNA3.1[+]) as a template . The results of mutagenesis were confirmed by Sanger sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…The TurboFect transfection reagent (Thermo Scientific) was used for transfections of plasmids (WT‐HA‐RUNX2‐pcDNA3.1(+), R131C, L136Q, K218E, T220R, and Δe4) together with the pOSE2‐6Luc reporter plasmid and CMV‐LacZ, to HEK293 cells as described in Ref. . Transfections were performed in triplicates.…”

Section: Methodsmentioning

confidence: 99%

“…All the procedures were performed as in Ref. using antibodies: 1:1000 dilution of HA‐Tag (C29F4) Rabbit mAb (Cell Signaling; #3724) and bovine anti‐rabbit IgG‐HRP (Santa Cruz Biotechnology; sc‐2370) diluted 1 in 5000.…”

Section: Methodsmentioning

confidence: 99%

“…The 350 ng of WT‐, R131C‐, L136Q‐, K218E‐, T220R‐ or Δe4‐RUNX2‐pcDNA3.1(+) expression clones were transfected into HEK293 cells. Following 48 hours of incubation, cells were washed with PBS and subjected to fixation, quenching, permeabilization and blocking . Next, the cells were incubated with antibodies: primary HA‐Tag (C29F4) Rabbit mAb (Cell Signaling; #3724) in 1:1000 dilution, and with secondary AlexaFluor 555 Donkey Anti‐Rabbit IgG (H+L) (Thermo Scientific; Cat# A‐31572, ) in 1:200 dilution.…”

Section: Methodsmentioning

confidence: 99%

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Functional analysis of novel RUNX2 mutations identified in patients with cleidocranial dysplasia

Hordyjewska-Kowalczyk

Sowińska‐Seidler

Olech

et al. 2019

Clinical Genetics

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RUNX2 (Runt‐related transcription factor 2) is a master regulator of osteoblast differentiation, cartilage and bone development. Pathogenic variants in RUNX2 have been linked to the Cleidocranial dysplasia (CCD), which is characterized by hypoplasia or aplasia of clavicles, delayed fontanelle closure, and dental anomalies. Here, we report 11 unrelated Polish patients with CCD caused by pathogenic alterations located in the Runt domain of RUNX2. In total, we identified eight different intragenic variants, including seven missense and one splicing mutation. Three of them are novel: c.407T>A p.(Leu136Gln), c.480C>G p.(Asn160Lys), c.659C>G p.(Thr220Arg), additional three were not functionally tested: c.391C>T p.(Arg131Cys), c.580+1G>T p.(Lys195_Arg229del), c.652A>G p.(Lys218Glu), and the remaining two: c.568C>T p.(Arg190Trp), c.673C>T p.(Arg225Trp) were previously reported and characterized. The performed transactivation and localization studies provide evidence of decreased transcriptional activity of RUNX2 due to mutations targeting the Runt domain and prove that impairment of nuclear localization signal (NLS) affects the subcellular localization of the protein. Presented data show that pathogenic variants discovered in our patients have a detrimental effect on RUNX2, triggering the CCD phenotype.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Functional analysis of novel RUNX2 mutations identified in patients with cleidocranial dysplasia

Hordyjewska-Kowalczyk

Sowińska‐Seidler

Olech

et al. 2019

Clinical Genetics

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Whole-exome sequencing identification of a novel splicing mutation of RUNX2 in a Chinese family with cleidocranial dysplasia

Zhang

Zhao

et al. 2019

Archives of Oral Biology

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Missense substitutions at a conserved 14-3-3 binding site in HDAC4 cause a novel intellectual disability syndrome

Wakeling

McEntagart

Bruccoleri

et al. 2021

Human Genetics and Genomics Advances

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Summary Histone deacetylases play crucial roles in the regulation of chromatin structure and gene expression in the eukaryotic cell, and disruption of their activity causes a wide range of developmental disorders in humans. Loss-of-function alleles of HDAC4 , a founding member of the class IIa deacetylases, have been reported in brachydactyly-mental retardation syndrome (BDMR). However, while disruption of HDAC4 activity and deregulation of its downstream targets may contribute to the BDMR phenotype, loss of HDAC4 function usually occurs as part of larger deletions of chromosome 2q37; BDMR is also known as chromosome 2q37 deletion syndrome, and the precise role of HDAC4 within the phenotype remains uncertain. Thus, identification of missense variants should shed new light on the role of HDAC4 in normal development. Here, we report seven unrelated individuals with a phenotype distinct from that of BDMR, all of whom have heterozygous de novo missense variants that affect a major regulatory site of HDAC4, required for signal-dependent 14-3-3 binding and nucleocytoplasmic shuttling. Two individuals possess variants altering Thr244 or Glu247, whereas the remaining five all carry variants altering Pro248, a key residue for 14-3-3 binding. We propose that the variants in all seven individuals impair 14-3-3 binding (as confirmed for the first two variants by immunoprecipitation assays), thereby identifying deregulation of HDAC4 as a pathological mechanism in a previously uncharacterized developmental disorder.

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Novel Mutation of the RUNX2 Gene in Patients with Cleidocranial Dysplasia

Cited by 6 publications

References 36 publications

Functional analysis of novel RUNX2 mutations identified in patients with cleidocranial dysplasia

Functional analysis of novel RUNX2 mutations identified in patients with cleidocranial dysplasia

Whole-exome sequencing identification of a novel splicing mutation of RUNX2 in a Chinese family with cleidocranial dysplasia

Missense substitutions at a conserved 14-3-3 binding site in HDAC4 cause a novel intellectual disability syndrome

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