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Mutations in ZBTB20 cause Primrose syndrome

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Primrose syndrome and 3q13.31 microdeletion syndrome are clinically related disorders characterized by tall stature, macrocephaly, intellectual disability, disturbed behavior and unusual facial features, with diabetes, deafness, progressive muscle wasting and ectopic calcifications specifically occurring in the former. We report that missense mutations in ZBTB20, residing within the 3q13.31 microdeletion syndrome critical region, underlie Primrose syndrome. This finding establishes a genetic link between these disorders and delineates the impact of ZBTB20 dysregulation on development, growth and metabolism.

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Figure 1: ZBTB20 is mutated in Primrose syndrome.

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NCBI Reference Sequence

Protein Data Bank

Change history

  • 20 July 2014

    In the version of this article initially published online, the name of author Marcel M. Mannens was misspelled. The error has been corrected for the print, PDF and HTML versions of this article.


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We are grateful to the patients and their families. We thank M.L. Motta and S. Venanzi (Istituto Superiore di Sanità) for technical assistance, the CINECA Consortium for computational resources, and K. Riabowol (University of Calgary), H. Nakabayashi (Hokkaido Information University) and Y. Miura (Graduate School of Medical Science, Nagoya, Japan) for providing the AFP promoter luciferase construct and the HNF1 and ATBF1 expression vectors, respectively. This work was supported by funding from the Istituto Superiore di Sanità (RC2013) to M.T.

Author information

Authors and Affiliations



V.C. and B.R. performed exome sequencing data validation and mutation analysis and wrote the manuscript. A.J., T.E.J.B., A. Ciolfi, A.v.K., F.B. and M.M.M. carried out the exome sequencing data processing and analyses. E.S., A.F., V.M. and L.B. performed the functional studies and genotyping. M. Anselmi, G.B. and L.S. designed and performed the molecular dynamics simulations and structural analyses. S.C. performed the confocal laser scanning microscopy analysis. M. Azage, D.R.C., A.J.E., A.M., A.-M.M., R.P., C.B., A. Casertano, D.M. and R.C.H. recruited and clinically characterized the study subjects and collected blood samples. M.T. and R.C.H. conceived the project, designed and supervised the experiments, analyzed and interpreted the data, and wrote the manuscript. All authors contributed to the final manuscript.

Corresponding authors

Correspondence to Marco Tartaglia or Raoul C Hennekam.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Germline ZBTB20 mutations causing Primrose syndrome.

Sequence electropherograms showing the de novo origin of the identified ZBTB20 missense change in sporadic case KND_01 and documenting the heterozygous state of the mutation in peripheral leukocytes, hair bulb cells and skin fibroblasts. A panel of representative disease-causing mutations is also shown.

Supplementary Figure 2 Partial amino acid sequence alignment of human ZBTB20 with representative orthologs showing conservation of the residues altered in Primrose syndrome.

Black circles on top of the alignment mark amino acids affected by Primrose syndrome–causative ZBTB20 mutations. The amino acid stretches constituting the four, tandemly arranged ZnFs (ZnF I to ZnF IV) are indicated.

Supplementary Figure 3 Molecular dynamics simulations (MDS) performed on the second ZnF (residues 606–633) predict an indirect effect of the L621Y change on DNA binding affinity.

MDS were performed on the wild-type (left) and mutant (right) ZnFs, free in solution. Although no differences in the general folding of the domain were observed between simulations, a substantial change in the orientation of His606 was documented in the domain with the disease-causing L621Y substitution. In contrast to what is observed in the wild-type domain, the introduction of Tyr621 promotes a conformational rearrangement of His606 due to a stacking interaction with the tyrosine residue at codon 621. This is exemplified by the χ1 torsional angle of the His606 side chain, which in the wild-type domain populates almost exclusively a value of about –60° (93%), whereas this population drops to 40% in the mutant. Conserved aromatic residues in the position corresponding to His606 are known to have a role in the stabilization of the orientation of the linker in the DNA-bound protein, through interactions with other hydrophobic residues12. These MDS suggest that the L621Y amino acid substitution could destabilize the arrangement between the first and the second ZnF domains corresponding to the DNA-bound conformation. This, in turn, might have an indirect effect on the DNA binding affinity of the protein.

Supplementary Figure 4 ZBTB20 mutants display nuclear localization but do not stably bind to chromatin.

Confocal microscopy analysis was performed in cells transiently expressing wild-type ZBTB20 or three disease-causing mutants (green), without (left) or with (right) treatment with CSK buffer before fixation. Nuclei are stained with DAPI (blue). Bars correspond to 10 μm. Images are representative of 300 (left) and 500 (right) analyzed cells (Supplementary Table 2).

Supplementary Figure 5 Impact of Primrose syndrome–causing mutations on ZBTB20 function.

(a) ZBTB20 mutants exhibit reduced binding to DNA. Pull-down assays were performed on lysates from transfected cells, using biotinylated oligonucleotides encompassing the minimal responsive sequence of the AFP promoter. The amount of DNA-bound ZBTB20 protein normalized to its expression level is reported as the fold decrease (mean ± s.d.) relative to the wild-type protein. Protein blots of a representative experiment of three performed are shown. Reduced DNA binding is also documented in extracts from cells coexpressing each mutant with the wild-type protein (right). In all comparisons, P values were calculated using the Student’s t test (one-sided distribution). *P < 0.05, **P < 0.01, ***P < 0.005. (b) The H596R ZBTB20 mutant exhibits impaired function in repressing luciferase reporter expression under the control of the phAFP[–178/+45] promoter and has a dominant-negative impact on wild-type ZBTB20. Transactivation assays were performed in the absence of exogenous ZBTB20 (white bar) and in the presence of exogenous wild-type ZBTB20 (black bar) or increasing amounts of the ZBTB20 mutant, alone (light gray bars) or in the presence of a fixed amount of wild-type ZBTB20 (dark gray bars). The normalized luciferase activity (mean ± s.d.) of the three experiments performed is reported as the fold decrease relative to cells not expressing exogenous ZBTB20. For all comparisons, P values were calculated using the two-sided Student’s t test. n.s. indicates statistically non-significant differences, and asterisks are used as above. (c) Disease-causing ZBTB20 mutants have impaired repressive function and dominant-negative impact on wild-type ZBTB20. Transactivation assays were performed in the absence of exogenous ZBTB20 (white bar) or in the presence of exogenous wild-type ZBTB20 (black bar) or individual disease-causing mutants, alone (light gray bars) or together with wild-type ZBTB20 (dark gray bars). The normalized luciferase activity (mean ± s.d.) of the three experiments performed is reported as above. All comparisons (one-sided Student’s t test) are in reference to wild-type ZBTB20.

Supplementary Figure 6 Facial features of subjects with 3q13.31 microdeletion syndrome.

The two individuals are representative of those exhibiting the shortest deleted genomic region encompassing the minimal region of overlapping deletion and were heterozygous for 2.79-Mb (chr. 3: 113,680,819–116,466,363, hg18) (left) and 2.67-Mb (chr. 3: 113,764,648–116,429,950, hg18) (right) deletions2. Both deletions were de novo. Note the resemblance to the facial characteristics in Primrose syndrome: macrocephaly, broad face, ptosis, full nasal tip, full lower lip and large jaw. Permission was obtained to publish pictures of both subjects (courtesy of M. Rio, Département de Génétique et INSERM U781, Université Paris Descartes, Hôpital Necker–Enfants Malades, Paris, France, and C. LeCaignec and A. David, CHU Nantes, Service de Genetique Medicale, Nantes, France).

Supplementary Figure 7 Dose-dependent inhibitory activity of wild-type ZBTB20 on the AFP promoter minimal responsive sequence.

Transactivation assays were performed using transiently transfected HEK 293T cells expressing wild-type ZBTB20 in the presence of the phAFP[–178/+45] promoter minimal region of the human AFP gene. Cells were transfected with 500 ng of phAFP[–178/+45]-Luc and 50 ng of pRL-Act-Renilla, together with the reported amount of construct encoding Xpress-tagged ZBTB20. Maximum inhibition was attained with a 1:1 ratio. Higher ZBTB20 levels did not influence maximal repressive activity. In all comparisons, P values were calculated using the Student’s t test (one-sided distribution). n.s. indicates statistically non-significant differences; *P < 0.05, **P < 0.005, ***P < 0.001.

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Cordeddu, V., Redeker, B., Stellacci, E. et al. Mutations in ZBTB20 cause Primrose syndrome. Nat Genet 46, 815–817 (2014).

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