A homozygous pathogenic missense variant broadens the phenotypic and mutational spectrum of CREB3L1-related osteogenesis imperfecta

TL;DR: The first homozygous pathogenic missense variant is identified in a patient with lethal OI, which is located within the highly conserved basic leucine zipper domain, four amino acids upstream of the DNA binding domain, and affects a critical residue in this functional domain, thereby decreasing the type I collagen transcriptional binding ability.

Abstract: The cyclic adenosine monophosphate responsive element binding protein 3-like 1 (CREB3L1) gene codes for the endoplasmic reticulum stress transducer old astrocyte specifically induced substance (OASIS), which has an important role in osteoblast differentiation during bone development. Deficiency of OASIS is linked to a severe form of autosomal recessive osteogenesis imperfecta (OI), but only few patients have been reported. We identified the first homozygous pathogenic missense variant [p.(Ala304Val)] in a patient with lethal OI, which is located within the highly conserved basic leucine zipper domain, four amino acids upstream of the DNA binding domain. In vitro structural modeling and luciferase assays demonstrate that this missense variant affects a critical residue in this functional domain, thereby decreasing the type I collagen transcriptional binding ability. In addition, overexpression of the mutant OASIS protein leads to decreased transcription of the SEC23A and SEC24D genes, which code for components of the coat protein complex type II (COPII), and aberrant OASIS signaling also results in decreased protein levels of SEC24D. Our findings therefore provide additional proof of the potential involvement of the COPII secretory complex in the context of bone-associated disease.

Summary

Introduction

• Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous group of heritable bone dysplasias, with the severity of symptoms ranging from perinatal lethality to generalized osteopenia (1).
• This brittle bone disease affects one in 15,000-20,000 births and is characterized by typical clinical manifestations such as bone fragility, skeletal deformities, low bone mass and short stature.
• The CREB3L1 gene (cAMP Responsive Element Binding Protein 3 Like 1) encodes the endoplasmic reticulum (ER)-stress transducer ‘old astrocyte specifically induced substance’ , a basic leucine zipper (bZIP) transcription factor which belongs to the well-conserved family of the cyclic AMP responsive element binding protein/activating transcription factor (CREB/ATF) genes.

Clinical phenotype

• The authors report a consanguineous Turkish family of second cousins, who had a medically terminated pregnancy at 19 weeks of gestation due to skeletal changes highly suggestive for severe OI.
• Antenatal ultrasound findings of the female fetus (IV-3, Fig. 1) included short tubular bones, multiple rib fractures with beaded appearance, and a narrow thorax circumference of 81mm (2.5-5th percentile).
• The parents (III-7 and III-8, Fig. 1) did not show any overt clinical signs of OI and had no history of fractures.
• The affected alanine (Ala) residue is located within a highly conserved bipartite nuclear localization sequence (NLS) within the bZIP domain, and only 4 amino acids (AA) upstream of the DNA binding domain , in which the earlier reported in-frame deletion p.(Lys312del) is located , (Fig. 2, Fig. 3A) (22).
• Biochemical assays using a luciferase reporter were performed in order to validate the direct impact of the p.(Ala304Val) variant on the regulation of the expression of the downstream target genes of OASIS, using type I collagen expression as a representative example.

Discussion

• This is the first report linking a pathogenic missense variant to the CREB3L1-related AR form of OI, and the 4th case in total implicating this gene.
• In contrast to the two earlier reports, the child presented by Lindahl et al., survived infancy.
• Together, these findings suggest that both p.(Ala304Val) and p.(Lys312del) have similar working mechanisms; they both form stable mutant proteins, which subsequently might accumulate in the cytosol.
• Recent studies have shown that monoubiquitylation of SEC31A helps to regulate COPII size, that glycosylation of both SEC24 and SEC23 is important for organization and regulation of COPII vesicles, and that phosphorylation of SEC23 and SEC24 confers directionality on COPII vesicles from ER to Golgi (34, 38).
• Keller et al. first proposed that mutations in OASIS can lead to OI due to disruption of the important role this protein plays in the secretion of type I collagen and other bone matrix proteins from osteoblasts during osteogenesis (22, 23, 33).

Ethical considerations

• Written and signed informed consent was obtained from the parents of the patient participating in this study.
• Genomic DNA (gDNA) from the proband, siblings or parents was isolated from whole blood according to the standard procedures.

Molecular studies

• The authors used conventional Sanger sequencing and next generation panel sequencing (MiSeq platform – Illumina) for molecular screening of the COL1A1, COL1A2, CRTAP, LEPRE1, PPIB, CREB3L1, WNT1, PLS3, BMP1, FKBP10, IFITM5, PLOD2, SERPINF1, SERPINH1, SP7 and TMEM38B genes.
• For NGS, single bases (up to 20 bases intronic of all coding exons) were covered with a minimal of 30x.
• Confirmational Sanger sequencing and segregational analysis was performed using the BigDye Terminator Cycle Sequencing Kit (Life Technologies, Carlsbad, Ca, USA) and run on a ABI 3730XL DNA Analyzer (Life technologies).
• Nucleotide numbering reflects cDNA numbering, with +1 corresponding to the A nucleotide of the ATG translation initiation codon in the reference sequence of CREB3L1 (NM_052854.2).
• Variant nomenclature follows the Human Genome Variation Society (HGVS) guidelines (http://www.hgvs.org/mutnomen), and variant classification was done by using the Alamut Visual software (version 2.10) and according to the American College of Medical Genetics (ACMG) standards and guidelines (Genome Aggregation Database, http://gnomad.broadinstitute.org) (24, 39).

Structural modeling of the variant

• By means of the I-TASSER server, which is an integrated platform for automated protein structure and function prediction based on the sequence-to-structure-to-function paradigm, 5 different three dimensional structural protein models were generated of the full length WT, p.(Ala304Val), and p.(Lys312del) protein sequences (26-28).
• The homology model of the CREB bZIP-CRE complex (PDB: 1DH3 – Mus musculus – generated in the expression system of Escherichia coli) was used as a template (30).
• The UCSF Chimera software package (version 1.13, build 41780) was used to visualize, study the localization, and model the effect of the specific protein variant (Dunbrack rotamers and FindHBond function), respectively (32, 40) .

Expression vectors

• The primers for sitedirected mutagenesis were designed using the QuikChange Primer Design tool and were purchased as HPLC-purified primers (primer sequences are listed in the Supplementary Table S1) (Integrated DNA Technologies).
• A pCMV-3Tag-2 empty vector (cat240196, Agilent) was purchased to use as a transfection control in their experiments (‘Empty’).
• Final constructs were sequenced, and a control- digestion was performed to confirm correct vector structure (data not shown).

Luciferase reporter assay

• For the luciferase experiments, 20,000 HEK293 cells were seeded in clear bottom 96 well plates (CLS3603-48EA, Sigma-Aldrich) in triplicate at day 1 and transiently co-transfected at day 3 using FuGene HD transfection reagent (E2311, Promega).
• Twenty-four hours post transfection, cells were lysed according to the manufacturers guidelines (Dual-Glo Luciferase Assay System, Promega) and luciferase activity was measured using a GloMax-Multi Detection System (E7031, Promega).
• Graphs display data-points normalized to WT values.
• In brief, 200,000 HEK293 cells were seeded in 6-well plates in triplicate at day 1 and transiently transfected at day 2 using FuGene HD transfection reagent (E2311, Promega) at a 3:1 ratio (3l reagent: 1g plasmid) per well and incubated for 48 hours before harvesting.
• These cells were subsequently processed for quantitative reverse-transcription PCR (RT-qPCR) or immunoblotting.

Quantitative reverse transcription PCR

• Total RNA was extracted from transfected HEK293 cells using the RNeasy Kit .
• Starting from 2g of RNA, cDNA was subsequently synthesized with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories).
• RT-qPCR reactions were prepared with the addition of RealTime ready DNA Probes Master mix and ResoLight Dye and were run in duplicate on a LightCycler 480 System.
• Data were analyzed with qbase+ software (version 3.0, Biogazelle) (42), and expression was normalized to the housekeeping genes HPRT1, RPL13A and YWHAZ.
• Graphs display data-points normalized to WT values.

Legends to Figures

• Pedigree of the Turkish CREB3L1 OI family, also known as (A).
• The proband is indicated with an arrow, asterisks denote family members available for molecular testing.
• (B): Postmortem examination of fetus IV:3 showed bowed extremities with bilateral angulation of the forearms due to fractures, bilateral femoral and tibial bowing.
• Figure 2 Protein structure and function of OASIS.

Full text

biblio.ugent.be
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Except for items where current copyright restrictions apply, these papers are available in Open
Access.
This item is the archived peer-reviewed author-version of:
A homozygous pathogenic missense variant broadens the phenotypic and mutational
spectrum of CREB3L1-related osteogenesis imperfecta
Guillemyn B, Kayserili H, Demuynck L, Sips P, De Paepe A, Syx D, Coucke PJ, Malfait F,
Symoens S
Human Molecular Genetics, 28 (11), 1801-1809, 2019.
This is a pre-copyedited, author-produced version of an article accepted for publication in
Human Molecular Genetics following peer review. The version of record is available online
at: https://doi.org/10.1093/hmg/ddz017.
To refer to or to cite this work, please use the citation to the published version:
Guillemyn B, Kayserili H, Demuynck L, Sips P, De Paepe A, Syx D, Coucke PJ, Malfait F, and
Symoens S (2019). A homozygous pathogenic missense variant broadens the phenotypic
and mutational spectrum of CREB3L1-related osteogenesis imperfecta. Hum Mol Genet
28(11) 1801-1809. doi: 10.1093/hmg/ddz017

For Peer Review
1
A homozygous pathogenic missense variant broadens the
phenotypic and mutational spectrum of CREB3L1-related
osteogenesis imperfecta
Brecht Guillemyn
1
, Hülya Kayserili
2
, Lynn Demuynck
1
, Patrick Sips
1
, Anne De Paepe
1
,
Delfien Syx
1
, Paul J. Coucke
1
, Fransiska Malfait
1
, Sofie Symoens
1,*
1
Center for Medical Genetics Ghent, Ghent University Hospital, Department of Biomolecular
Medicine, Ghent 9000, Belgium
2
KOÇ University School of Medicine (KUSoM) Medical Genetics Department, Topkapi
Zeytinburnu, 34010 Istanbul, Turkey
*To whom correspondence should be addressed: Department of Biomolecular Medicine,
Center for Medical Genetics Ghent, Ghent University Hospital, Corneel Heymanslaan 10,
Medical Research Building 1, 9000 Ghent, Belgium. Tel: 0032/9 332 02 33; Email:
Sofie.Symoens@UGent.be
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Abstract
The cyclic AMP responsive element binding protein 3-like 1 (CREB3L1) gene codes for the
endoplasmic reticulum stress transducer old astrocyte specifically induced substance (OASIS),
which has an important role in osteoblast differentiation during bone development. Deficiency
of OASIS is linked to a severe form of autosomal recessive osteogenesis imperfecta (OI), but
only few patients have been reported. We identified the first homozygous pathogenic missense
variant (p.(Ala304Val)) in a patient with lethal OI, which is located within the highly conserved
basic leucine zipper domain, four amino acids upstream of the DNA binding domain. In vitro
structural modeling and luciferase assays demonstrate that this missense variant affects a
critical residue in this functional domain, thereby decreasing the type I collagen transcriptional
binding ability. In addition, overexpression of the mutant OASIS protein leads to decreased
transcription of the SEC23A and SEC24D genes, which code for components of the coat protein
complex type II (COPII), and aberrant OASIS signaling also results in decreased protein levels
of SEC24D. Our findings therefore provide additional proof of the potential involvement of the
COPII secretory complex in the context of bone-associated disease.
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Introduction
Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous group of heritable
bone dysplasias, with the severity of symptoms ranging from perinatal lethality to generalized
osteopenia (1). This brittle bone disease affects one in 15,000-20,000 births and is characterized
by typical clinical manifestations such as bone fragility, skeletal deformities, low bone mass
and short stature. Extraskeletal features, including blue sclerae, dentinogenesis imperfecta,
adult-onset hearing loss, joint hypermobility, restrictive pulmonary disease, cardiovascular
abnormalities and easy bruising, contribute to the multisystemic disorder (1-3). The
predominant autosomal dominant (AD) forms are caused by mutations in either COL1A1 (MIM
120150) or COL1A2 (MIM 120160), encoding the α1- and α2-chains of type I procollagen
respectively. Another rare AD OI subtype is associated with mutations in interferon–induced
transmembrane protein 5 (IFITM5, MIM 614757), which is involved in bone mineralization. In
approximately 10% of OI cases, the disease has an autosomal recessive (AR) inheritance.
Several genes have been associated with these AR forms of OI, and they are classified according
to their mechanism and pathophysiology: collagen post-translational modification (CRTAP,
MIM 605497; P3H1, MIM 610339; PPIB, MIM 123841), collagen processing and crosslinking
(SERPINH1, MIM 600943; FKBP10, MIM 607063; PLOD2, MIM 601865; BMP1, MIM
112264), bone mineralization (SERPINF1, MIM 172860) and osteoblast
differentiation/function (SP7, MIM 606633; TMEM38B, MIM 611236; WNT1, MIM 164820;
CREB3L1, MIM 616215; SPARC, MIM 182120; MBTPS2, MIM 300294; TAPT1, MIM
616897) (1, 2, 4-18).
The CREB3L1 gene (cAMP Responsive Element Binding Protein 3 Like 1) encodes the
endoplasmic reticulum (ER)-stress transducer ‘old astrocyte specifically induced substance’
(OASIS), a basic leucine zipper (bZIP) transcription factor which belongs to the well-conserved
family of the cyclic AMP responsive element binding protein/activating transcription factor
(CREB/ATF) genes. OASIS is processed by regulated intramembrane proteolysis (RIP) in
response to ER stress, and is highly expressed in osteoblasts (19, 20). OASIS
-/-
mice exhibit
severe osteopenia and spontaneous fractures, resulting from a decrease in type I collagen in the
bone matrix and a decline in the activity of osteoblasts. More recently, Col1a1 was identified
as a target of OASIS, and Murakami et al. demonstrated with murine studies that OASIS
activates the transcription of Col1a1 through an unfolded protein response element (UPRE)-
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4
like sequence in the Col1a1 promoter region, thereby revealing its critical role in bone
formation (19-21).
Hitherto, only 3 reports have associated homozygous CREB3L1 defects to an AR form of OI (a
whole gene deletion, the in-frame deletion (c.934_936delAAG, p.(Lys312del)) and the
nonsense variant (c.1284C>A, p.(Tyr428*))), which is currently classified as OI type XVI (2,
15, 22, 23).
Here, we present a Turkish family, in which molecular analysis of the proband revealed a
previously unreported homozygous missense variant (c.911C>T, p.(Ala304Val)).
We applied structural modeling to study the effects of this missense variant on the OASIS
protein. We then performed further in vitro studies to investigate the functional consequences
regarding regulation of type I collagen and COPII component gene expression.
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