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 (3 min read)
- 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.
- 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.
- 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).
- 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.
- 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) .
- 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.
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Cites background from "A homozygous pathogenic missense va..."
...CREB3L1 critical contribution in bone formation was also confirmed by its role as a genetic cause of autosomal recessive osteogenesis imperfecta in humans (Symoens et al., 2013; Keller et al., 2018; Guillemyn et al., 2019)....
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