Clinical and functional characterization of COL2A1 p.Gly444Ser variant: From a fetal phenotype to a previously undisclosed postnatal phenotype

doi:10.1016/j.bonr.2023.101728

Case Reports

. 2023 Nov 27:19:101728.

doi: 10.1016/j.bonr.2023.101728. eCollection 2023 Dec.

Clinical and functional characterization of COL2A1 p.Gly444Ser variant: From a fetal phenotype to a previously undisclosed postnatal phenotype

Enrica Marchionni¹, Maria Rosaria D'Apice¹, Viviana Lupo^{1

2}, Giovanna Lattanzi^{3

4}, Elisabetta Mattioli^{3

4}, Gina Lisignoli⁵, Elena Gabusi⁵, Gerardo Pepe⁶, Manuela Helmer Citterich⁶, Elena Campione⁷, Anna Maria Nardone¹, Paola Spitalieri², Noemi Pucci⁸, Dario Cocciadiferro⁹, Eliseo Picchi⁸, Francesco Garaci¹⁰, Antonio Novelli⁹, Giuseppe Novelli^{1

2}

Affiliations

¹ Medical Genetics Unit, Policlinico Tor Vergata, University of Rome Tor Vergata, Rome, Italy.
² Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.
³ CNR Institute of Molecular Genetics "Luigi Luca Cavalli-Sforza" Unit of Bologna, Bologna, Italy.
⁴ Istituto Ortopedico Rizzoli, Bologna, Italy.
⁵ IRCCS Istituto Ortopedico Rizzoli, Laboratorio di Immunoreumatologia e Rigenerazione Tissutale Bologna, Italy.
⁶ Department of Biology, University of Rome Tor Vergata, Rome, Italy.
⁷ Dermatologic Unit, Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy.
⁸ Diagnostic Imaging Unit, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.
⁹ Translational Cytogenomics Research Unit, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.
¹⁰ Neuroradiology Unit, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.

PMID: 38076483
PMCID: PMC10698255
DOI: 10.1016/j.bonr.2023.101728

Case Reports

Clinical and functional characterization of COL2A1 p.Gly444Ser variant: From a fetal phenotype to a previously undisclosed postnatal phenotype

Enrica Marchionni et al. Bone Rep. 2023.

. 2023 Nov 27:19:101728.

doi: 10.1016/j.bonr.2023.101728. eCollection 2023 Dec.

Authors

Affiliations

¹ Medical Genetics Unit, Policlinico Tor Vergata, University of Rome Tor Vergata, Rome, Italy.
² Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.
³ CNR Institute of Molecular Genetics "Luigi Luca Cavalli-Sforza" Unit of Bologna, Bologna, Italy.
⁴ Istituto Ortopedico Rizzoli, Bologna, Italy.
⁵ IRCCS Istituto Ortopedico Rizzoli, Laboratorio di Immunoreumatologia e Rigenerazione Tissutale Bologna, Italy.
⁶ Department of Biology, University of Rome Tor Vergata, Rome, Italy.
⁷ Dermatologic Unit, Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy.
⁸ Diagnostic Imaging Unit, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.
⁹ Translational Cytogenomics Research Unit, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.
¹⁰ Neuroradiology Unit, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.

PMID: 38076483
PMCID: PMC10698255
DOI: 10.1016/j.bonr.2023.101728

Abstract

COL2A1 gene encodes the alpha-1 chain of type-II procollagen. Heterozygous pathogenic variants are associated with the broad clinical spectrum of genetic diseases known as type-II collagenopathies. We aimed to characterize the NM_001844.5:c.1330G>A;p.Gly444Ser variant detected in the COL2A1 gene through trio-based prenatal exome sequencing in a fetus presenting a severe skeletal phenotype at 31 Gestational Weeks and in his previously undisclosed mild-affected father. Functional studies on father's cutaneous fibroblasts, along with in silico protein modeling and in vitro chondrocytes differentiation, showed intracellular accumulation of collagen-II, its localization in external Golgi vesicles and nuclear morphological alterations. Extracellular matrix showed a disorganized fibronectin network. These results showed that p.Gly444Ser variant alters procollagen molecules processing and the assembly of mature type-II collagen fibrils, according to COL2A1-chain disorganization, displayed by protein modeling. Clinical assessment at 38 y.o., through a reverse-phenotyping approach, revealed limp gait, short and stocky appearance. X-Ray and MRI showed pelvis asymmetry with severe morpho-structural alterations of the femoral heads bilaterally, consistent with a mild form of type-II collagenopathy. This study shows how the fusion of genomics and clinical expertise can drive a diagnosis supported by cellular and bioinformatics studies to effectively establish variants pathogenicity.

Keywords: COL2A1; Exome sequencing; Functional characterization; Reverse-phenotyping; Type-II collagenopathies.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Fig. 1**
Thoracic (A) and lumbar (B) spine lateral x-ray views demonstrated a «pear-shaped» appearance of T10 (red open arrow) and T8 (red closed arrow) vertebrae. Antero-posterior (C and D) hip x-ray view; to note the severe remodeling of femoral heads with fragmentation and flattened profile (red dotted arrows). In (D) quantification of sopraelevation of the right iliac crest and ipsilateral upper profile of acetabulum. Coronal T1-TSE (E) and T2-STIR (F) MRI of hip: to note small amount of trabecular oedema of femoral heads.

**Fig. 2**
A) Representative images of Safranin O staining of chondrogenic differentiated cells at days 0, 21 and 28. Graph showing the quantification of Safranin O positive cells. Two-way ANOVA with Sidak's multiple comparison test, ** p < 0.002, **** p < 0.0001 B) Phase contrast microscope images (20× objective) of patient's cells at days 0, 21 and 28 of chondrogenic differentiation.

**Fig. 3**
Immunofluorescence analysis of human chondrocytes in control (CTR) and in a patient carrying COL2A1 (p.Gly444Ser) variant. (A) Immunostaining of collagen II (green) in cells at days 0, 21 and 28 of chondrogenic differentiation and high magnification on the right (high magnification). Statistical analysis is reported in the graphs on the right. (B) Double immunofluorescence analysis of collagen II (green), and golgin 97 (red) in human chondrocytes at day 28 of differentiation. High magnification in the panels below (high magnification). Statistical analysis is reported in the graph below. (C) Immunostaining of fibronectin (green) in human chondrocytes at day 28 of differentiation. 4,6-diamidino-2-phenylindole (DAPI, blue) was used to counterstained cell nuclei. Scale bars, 10 μm. Three biological replicates were used in each experiment and statistically significant differences (p < 0.001) between values are indicated.

See this image and copyright information in PMC

References

1. Acevedo-Jake A.M., Ngo D.H., Hartgerink J.D. Control of collagen triple Helix stability by phosphorylation. Biomacromolecules. 2017;18(4):1157–1161. doi: 10.1021/acs.biomac.6b01814. - DOI - PubMed
1. Barat-Houari M., Dumont B., Fabre A., et al. The expanding spectrum of COL2A1 gene variants IN 136 patients with a skeletal dysplasia phenotype. Eur. J. Hum. Genet. 2016;24(7):992–1000. doi: 10.1038/ejhg.2015.250. - DOI - PMC - PubMed
1. Chen J., Ma X., Zhou Y., et al. Recurrent c.G1636A (p.G546S) mutation of COL2A1 in a Chinese family with skeletal dysplasia and different metaphyseal changes: a case report. BMC Pediatr. 2017;17(1):175. doi: 10.1186/s12887-017-0930-9. (Published 2017 Jul 24) - DOI - PMC - PubMed
1. Chen M., Zhang W., Gou Y., et al. GPS 6.0: an updated server for prediction of kinase-specific phosphorylation sites in proteins. Nucleic Acids Res. 2023;51(W1):W243–W250. doi: 10.1093/nar/gkad383. - DOI - PMC - PubMed
1. Désir J., Cassart M., Donner C., et al. Spondyloperipheral dysplasia as the mosaic form of platyspondylic lethal skeletal dyplasia torrance type in mother and fetus with the same COL2A1 mutation. Am. J. Med. Genet. A. 2012;158A(8):1948–1952. doi: 10.1002/ajmg.a.35301. - DOI - PubMed

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[1] Acevedo-Jake A.M., Ngo D.H., Hartgerink J.D. Control of collagen triple Helix stability by phosphorylation. Biomacromolecules. 2017;18(4):1157–1161. doi: 10.1021/acs.biomac.6b01814. - DOI - PubMed

[2] Acevedo-Jake A.M., Ngo D.H., Hartgerink J.D. Control of collagen triple Helix stability by phosphorylation. Biomacromolecules. 2017;18(4):1157–1161. doi: 10.1021/acs.biomac.6b01814. - DOI - PubMed

[3] Barat-Houari M., Dumont B., Fabre A., et al. The expanding spectrum of COL2A1 gene variants IN 136 patients with a skeletal dysplasia phenotype. Eur. J. Hum. Genet. 2016;24(7):992–1000. doi: 10.1038/ejhg.2015.250. - DOI - PMC - PubMed

[4] Barat-Houari M., Dumont B., Fabre A., et al. The expanding spectrum of COL2A1 gene variants IN 136 patients with a skeletal dysplasia phenotype. Eur. J. Hum. Genet. 2016;24(7):992–1000. doi: 10.1038/ejhg.2015.250. - DOI - PMC - PubMed

[5] Chen J., Ma X., Zhou Y., et al. Recurrent c.G1636A (p.G546S) mutation of COL2A1 in a Chinese family with skeletal dysplasia and different metaphyseal changes: a case report. BMC Pediatr. 2017;17(1):175. doi: 10.1186/s12887-017-0930-9. (Published 2017 Jul 24) - DOI - PMC - PubMed

[6] Chen J., Ma X., Zhou Y., et al. Recurrent c.G1636A (p.G546S) mutation of COL2A1 in a Chinese family with skeletal dysplasia and different metaphyseal changes: a case report. BMC Pediatr. 2017;17(1):175. doi: 10.1186/s12887-017-0930-9. (Published 2017 Jul 24) - DOI - PMC - PubMed

[7] Chen M., Zhang W., Gou Y., et al. GPS 6.0: an updated server for prediction of kinase-specific phosphorylation sites in proteins. Nucleic Acids Res. 2023;51(W1):W243–W250. doi: 10.1093/nar/gkad383. - DOI - PMC - PubMed

[8] Chen M., Zhang W., Gou Y., et al. GPS 6.0: an updated server for prediction of kinase-specific phosphorylation sites in proteins. Nucleic Acids Res. 2023;51(W1):W243–W250. doi: 10.1093/nar/gkad383. - DOI - PMC - PubMed

[9] Désir J., Cassart M., Donner C., et al. Spondyloperipheral dysplasia as the mosaic form of platyspondylic lethal skeletal dyplasia torrance type in mother and fetus with the same COL2A1 mutation. Am. J. Med. Genet. A. 2012;158A(8):1948–1952. doi: 10.1002/ajmg.a.35301. - DOI - PubMed

[10] Désir J., Cassart M., Donner C., et al. Spondyloperipheral dysplasia as the mosaic form of platyspondylic lethal skeletal dyplasia torrance type in mother and fetus with the same COL2A1 mutation. Am. J. Med. Genet. A. 2012;158A(8):1948–1952. doi: 10.1002/ajmg.a.35301. - DOI - PubMed

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Clinical and functional characterization of COL2A1 p.Gly444Ser variant: From a fetal phenotype to a previously undisclosed postnatal phenotype

Affiliations

Clinical and functional characterization of COL2A1 p.Gly444Ser variant: From a fetal phenotype to a previously undisclosed postnatal phenotype

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

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Abstract

Conflict of interest statement

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