© American College of Medical Genetics and Genomics ORIGINAL RESEARCH ARTICLE Monoallelic and biallelic CREB3L1 variant causes mild and severe osteogenesis imperfecta, respectively Rachel B. Keller, BS1, Thao T. Tran, BS1, Shawna M. Pyott, PhD1, Melanie G. Pepin, MS, CGC1, Ravi Savarirayan, MD, FRACP2,3, George McGillivray, MBChB2, UW Center for Mendelian Genomics, Deborah A. Nickerson, PhD4, Michael J. Bamshad, MD4 and Peter H. Byers, MD1,5 Purpose: Osteogenesis imperfecta (OI) is a heritable skeletal heterozygous family members. CREB3L1 encodes old astrocyte dysplasia. Dominant pathogenic variants in COL1A1 and COL1A2 specifically induced substance (OASIS), an endoplasmic reticulum explain the majority of OI cases. At least 15 additional genes have stress transducer. The variant disrupts a DNA-binding site and been identified, but those still do not account for all OI phenotypes prevents OASIS from acting on its transcriptional targets including that present. We sought the genetic cause of mild and lethal OI SEC24D, which encodes a component of the coat protein II complex. phenotypes in an unsolved family. Conclusion: This report confirms that CREB3L1 is an OI-related Methods: We performed exome sequencing on seven members of gene and suggests the pathogenic mechanism of CREB3L1-associated the family, both affected and unaffected. OI involves the altered regulation of proteins involved in cellular Results: We identified a variant in cyclic AMP responsive element secretion. CREB3L1 binding protein 3-like 1 ( ) in a consanguineous family. The Genet Med advance online publication 17 August 2017 variant caused a prenatal/perinatal lethal OI in homozygotes, similar to that seen in OI type II as a result of mutations in type I collagen Key Words: bone; COPII; CREB3L1; OASIS; osteogenesis genes, and a mild phenotype (fractures, blue sclerae) in multiple imperfecta INTRODUCTION fully understood, cause autosomal dominant forms of OI with Osteogenesis imperfecta (OI; MIM 166200, 166210, 259420, unique features, e.g., hyperplastic callus formation (MIM and 166220), is a genetically and phenotypically heteroge- 610967).4,5 Recently, an X-linked form of OI caused by neous disorder characterized by brittle bones and variable mutations in MBTPS2 (MIM 300294) was reported.6 bone deformity.1 Severity ranges from intrauterine fractures Even with this expanded catalog of OI disease genes, the and significantly shortened long bones with perinatal lethality underlying cause of OI in a subset of families has still not been to few fractures with normal stature and life expectancy.1 identified. From exome sequence analysis of members of a Extraskeletal features may include blue sclerae, dentinogenesis consanguineous family of Lebanese descent (Figure 1a), we imperfecta, adult-onset hearing loss, joint hypermobility, and identified a trinucleotide deletion in cyclic AMP responsive hyperlaxity of skin.1 In the majority (>90%) of affected element binding protein 3–like 1 (CREB3L1; MIM 616215) that individuals, mutations in COL1A1 (MIM 120150) and corresponds to an in-frame single amino acid deletion in old COL1A2 (MIM 120160), which encode the proα1(I) and astrocyte specifically induced substance (OASIS), and results in proα2(I) chains of mature type I procollagen, respectively, a mild OI phenotype in heterozygotes and a lethal phenotype in account for the phenotype.2 Genes involved in collagen homozygotes. Previously, homozygosity for whole-gene deletion processing (CRTAP (MIM 605497), P3H1 (MIM 610339), of CREB3L1 was shown to result in lethal OI (MIM 616229) in PPIB (MIM 123841), FKBP10 (MIM 607063), SERPINH1 a Turkish family.7 Our study confirms that mutations in (MIM 600943), BMP1 (MIM 112264), and PLOD2 (MIM CREB3L1 result in an OI phenotype wherein disease severity is 601865)), bone cell differentiation and signaling (SP7 (MIM determined by whether mutations are monoallelic or biallelic, 606633) and WNT1 (MIM 164820)), bone cell association and identifies a potential mechanism of action. with extracellular matrix (SPARC (MIM 182120)), and matrix mineralization (SERPINF1 (MIM 172860) and TMEM38B MATERIALS AND METHODS (MIM 611236)) have been implicated in rarer forms of Exome and confirmatory sequencing recessively inherited disease.2,3 Additionally, mutations in Written informed consent was obtained for all individuals IFITM5 (MIM 614757), through mechanisms that are not in the study. Exome sequencing was performed by the 1Department of Pathology, University of Washington, Seattle, Washington, USA; 2Victorian Clinical Genetics Services, Murdoch Children’s Research Institute, Melbourne, Victoria, Australia; 3Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia; 4Center for Mendelian Genomics, University of Washington, Seattle, Washington, USA; 5Department of Medicine (Medical Genetics), University of Washington, Seattle, Washington, USA. Correspondence: Rachel B. Keller ([email protected]) Submitted 9 March 2017; accepted 5 June 2017; advance online publication 17 August 2017. doi:10.1038/gim.2017.115 GENETICS in MEDICINE | Volume 20 | Number 4 | April 2018 411 ORIGINAL RESEARCH ARTICLE KELLER et al | CREB3L1 recessive OI a b ** V V-3 3 4 V-4 365 370 375 380 365 370 375 380 5,100 5,150 5,200 5,250 5,100 5,150 5,200 5,250 proα1(I) * * * * * proα2(I) VI 12345678 VI-1 VI-7 365 370 375 380 365 370 375 380 VI-5 365 370 375 380 5,100 5,150 5,200 5,250 5,100 5,150 5,200 5,250 VI-4 VI-8 365370375380 365 370 375 380 5,100 5,150 5,200 5,250 5,100 5,150 5,200 5,250 5,100 5,150 5,200 5,250 Mild Ol- Lethal Ol- Unaffected like like cd α1(I) α2(I) R C C C C OI II VI-7 OI II VI-7 MEDIA CELLS Figure 1 Homozygosity and heterozygosity for a 3-bp deletion in CREB3L1 that results in a single amino acid deletion in OASIS (c.934_936delAAG [p.Lys312del]) are consistent with the variable severity of osteogenesis imperfecta (OI) phenotypes. (a) Pedigree of the CREB3L1 OI family. Arrow indicates the proband. Asterisks denote the family members whose exomes were sequenced. Chromatograms from Sanger sequence analysis confirming the exome findings are shown. (b) Effect of the CREB3L1 variant on type I collagen biochemistry. Collagen alpha chains synthesized by skin fibroblasts from the proband (VI-7) are structurally equivalent to those from control fibroblasts (c), in contrast to those from a typical OI type II patient (OI II) with a COL1A1 glycine substitution (p.Gly818Cys), in which overmodification results in both delayed procollagen mobility (upper panel; arrows) and delayed α1(I) and α2(I) chain mobility when procollagens are digested with pepsin (lower panel; arrows). We do not see abnormal retention of procollagens nor of alpha chains in the proband fibroblasts; as skin fibroblasts do not express CREB3L1, we would not expect OASIS to have a role in regulating secretion in these particular cells. (c) Anterior–posterior (left) and lateral (right) radiographs from VI-3 (homozygous for the 3- bp deletion in CREB3L1) following 18 weeks gestation termination. There is virtually no calvarial mineralization. Bones in all visible extremities are telescoped and very irregular. The ribs are thin and wavy and there is mild platyspondyly. (d) Radiographs of VI-4 (heterozygous for the 3-bp deletion in CREB3L1) at birth. The calvarium is thin and the anterior and posterior fontanelles are large, but there are no Wormian bones. The ribs are thin and there is mild platyspondyly. There is marked bowing of the right femur. University of Washington Center for Mendelian Genomics protocol. Sequencing reactions were set up using Big Dye v3.1 as previously described.8 Variants were sorted as described (Applied Biosystems) with the following program: 96 °C in the text. The variant in CREB3L1 was confirmed with for 10 seconds, 50 °C for 5 seconds, 60 °C for 4 minutes for Sanger sequencing. Primers for amplification of exon 7 are 40 cycles. Sequencing was run on an ABI 3730. Sequences listed in Supplementary Table S1 online (Integrated DNA were analyzed using Mutation Surveyor v4.0.9 software Technologies, Coralville, IA). Exon 7 was amplified using (Softgenetics, State College, PA). AmpliTaq Gold Polymerase (Applied Biosystems, Foster City, CA). The program used for cycling was: 95 °C for 12 minutes, 95 °C for 10 seconds, 61 °C for 40 seconds, Collagen analysis 72 °C for 50 seconds for 35 cycles, then, 72 °C for 7 minutes. Culture of skin fibroblasts and analysis of procollagens was Amplicons were treated with ExoSAP according to a standard performed as previously described.9 412 Volume 20 | Number 4 | April 2018 | GENETICS in MEDICINE CREB3L1 recessive OI | KELLER et al ORIGINAL RESEARCH ARTICLE In vitro transcription/translation and western blot are listed in Supplementary Table S1 (Integrated DNA In vitro transcription/translation (IVT) was performed using Technologies, Coralville, IA). Constructs were transiently the 1-Step Human Coupled IVT Kit–DNA (ThermoFisher transfected into HEK293 using XtremeGene 9 Transfection Scientific, Waltham, MA). Templates for IVT were generated Reagent (Roche, Indianapolis, IN) at a 3:1 (μl reagent:μg using a polymerase chain reaction (PCR)-based method.10 plasmid) ratio and incubated for 48 hours before harvest. Wild-type (WT) and variant (VT312) full-length CREB3L1 For the dimerization experiment, “WT312” and “MU312” complementary DNA sequences were amplified from plas- were transfected at the indicated ratios equaling 1 μg total of mids “WT312” and “MU312,” generated for overexpression plasmid (3:1 = 0.75 μg WT312 + 0.25 μg MU312; 1:1 = 0.5 μg (OE) experiments in mammalian cell lines (see “Over- WT312 + 0.5 μg MU312, 1:3 = 0.25 μg WT312 + 0.75 μg expression” below). Primers for creation of IVT templates MU312). are listed in Supplementary Table S1 (Integrated DNA Technologies, Coralville, IA). IVT reactions were assembled Quantitative reverse transcription PCR according to For quantitative reverse transcription PCR (RT-qPCR), total kit instructions and incubated for 6 hours at 30 °C. 1 μlof RNA was isolated from cells using the RNeasy Mini Kit each IVT reaction was run on 10% sodium dodecyl sulfate– (Qiagen, Valencia, CA).