J Med Genet 2000;37:263–271 263 Report of five novel and one recurrent COL2A1 mutations with analysis of genotype-phenotype J Med Genet: first published as 10.1136/jmg.37.4.263 on 1 April 2000. Downloaded from correlation in patients with a lethal type II collagen disorder Geert R Mortier, MaryAnn Weis, Lieve Nuytinck, Lily M King, Douglas J Wilkin, Anne De Paepe, Ralph S Lachman, David L Rimoin, David R Eyre, Daniel H Cohn Abstract osteoarthrosis and little or no skeletal growth Achondrogenesis II-hypochondrogenesis abnormality.2 Achondrogenesis II-hypochond- and severe spondyloepiphyseal dysplasia rogenesis and lethal spondyloepiphyseal dys- congenita (SEDC) are lethal forms of plasia congenita (SEDC) represent the more dwarfism caused by dominant mutations severe end of the spectrum.1 5–7 These entities in the type II collagen gene (COL2A1). To are characterised by severe disproportionate identify the underlying defect in seven short stature of prenatal onset. The distinction cases with this group of conditions, we between these phenotypes is mainly based used the combined strategy of cartilage upon clinical, radiographic, and morphological protein analysis and COL2A1 mutation features but considerable phenotypic overlap 1 6–8 analysis. Overmodified type II collagen often hampers proper classification. and the presence of type I collagen was All of the previously reported cases of found in the cartilage matrix of all seven achondrogenesis II-hypochondrogenesis in which the molecular defect was found were Department of cases. Five patients were heterozygous for Medical Genetics, a nucleotide change that predicted a heterozygous for a mutation in the COL2A1 University Hospital of glycine substitution in the triple helical gene resulting in a glycine substitution in the Gent, De Pintelaan triple-helical domain of the proá1(II) collagen domain (G313S, G517V, G571A, G910C, 9–16 185, B-9000 Gent, G943S). In all five cases, analysis of carti- chain. Biochemical analysis of cartilage usu- Belgium lage type II collagen suggested incorpora- ally showed the presence of type I collagen and G R Mortier post-translationally overmodified type II colla- L Nuytinck tion of the abnormal á1(II) chain in the A De Paepe extracellular collagen trimers. The G943S gen. Studies on cultured chondrocytes from mutation has been reported previously in one case of hypochondrogenesis with a G913C Department of mutation showed lack of secretion of type II another unrelated patient with a strikingly 16 Orthopedics, collagen in the medium. Mutations reported http://jmg.bmj.com/ similar phenotype, illustrating the possi- University of in cases of SEDC are more heterogeneous. ble specific eVect of the mutation. The Washington, Seattle, Single amino acid substitutions, deletions, or WA, USA radiographically less severely aVected pa- duplications in the triple helical domain of the MAWeis tient was heterozygous fora4bpdeletion D R Eyre proá1(II) collagen chain have been in the splice donor site of intron 35, likely 17–24 reported. Steven Spielberg to result in aberrant splicing. One case The aim of this study was to identify the Pediatric Research was shown to be heterozygous for a single mutation in the COL2A1 gene in seven cases of Center, Burns & Allen nucleotide change predicted to result in a achondrogenesis II-hypochondrogenesis or se- on October 1, 2021 by guest. Protected copyright. Cedars-Sinai Research T1191N substitution in the carboxy- vere SEDC based on the results of the Institute and propeptide of the proá1(II) collagen chain. Departments of biochemical analysis of cartilage tissue. Instead Pediatrics and Study of the clinical, radiographic, and of performing a “head to tail” mutation analy- Radiology, UCLA morphological features of the seven cases sis of the entire gene, we first started screening School of Medicine, supports evidence for a phenotypic con- those regions of the COL2A1 gene in which a Los Angeles, CA, USA tinuum between achondrogenesis II- mutation was most likely to reside, based on L M King hypochondrogenesis and lethal SEDC and the electrophoretic properties of the extracted R S Lachman suggests a relationship between the D L Rimoin cartilage collagen. The phenotype of each case D H Cohn amount of type I collagen in the cartilage was studied in relation to the molecular and and the severity of the phenotype. biochemical findings and compared with other Medical Genetics (J Med Genet 2000;37:263–271) reported cases of achondrogenesis II- Branch, National hypochondrogenesis and SEDC in which a Human Genome Keywords: type II collagen disorders; achondrogenesis Research Institute, II-hypochondrogenesis; spondyloepiphyseal dysplasia COL2A1 mutation was identified. National Institutes of congenita; COL2A1 Health, Bethesda, MD, Materials and methods USA PATIENTS D J Wilkin Several heritable osteochondrodysplasias have The seven patients in this study were referred now been recognised as members of the family Correspondence to: in the period 1982 to 1991 to the International Dr Mortier of type II collagen disorders, all of which result Skeletal Dysplasia Registry at Cedars-Sinai from dominant mutations in the COL2A1 Medical Center, Los Angeles for diagnosis. Revised version received gene.1–4 Phenotypes within this group range The clinical features and radiographs were sent 12 July 1999 Accepted for publication from severe lethal dwarfism at birth to to the Registry for evaluation and tissue speci- 5 August 1999 relatively mild conditions with precocious mens, taken during necropsy, were collected Table 1 Clinical, radiographic, and morphological features of seven patients with a lethal type II collagen disorder 264 Patient R82-121 R83-32 R85-109 R86-6 R86-153 R87-75 R91-68 COL2A1 mutation T1191N? Gly571Ala Gly910Cys Gly313Ser Gly943Ser Gly517Val IVS35+4delAGTG Clinical Sex Male Female Male Female Female Female Female Birth length (gestational age) 39 cm (36 wk) 39 cm (40 wk) ? (37 wk) ? (34 wk) 29 cm (35 wk) 38 cm (42 wk) 34 cm (38 wk) Short limbs Yes Yes Yes Yes Yes Yes Yes Small thorax Yes Yes Yes Yes Yes Yes Yes Cleft palate No No No Yes Yes No No Respiratory insuYciency at birth No Yes Yes Yes Yes Yes Yes Ageatdeath 1y1mth 63d 1d 10h 6h 2d 4d Radiographic Long bonesa ++* hands and feet +++* ++* +++* ++++* ++++* +† Pelvisb ? Yes Yes Yes Yes Yes No Spinec Cervical Cervical Cervical, sacral Cervical Entire spine Cervical, sacral Only platyspondyly Thoraxd Ye s Ye s Ye s Ye s Ye s Ye s Ye s Pubic bonese ?NoNoNoNoNoNo Talus and calcaneusf Yes No Only talus No No ? Only talus Morphological Resting cartilage Mild hypervascular ? Very hypervascular Mild hypervascular Very hypervascular Very hypervascular Slightly increased vascular canals Chondrocytes LM Inclusion bodies Inclusion bodies Inclusion bodies, Inclusion bodies, Inclusion bodies Inclusion bodies, Inclusion bodies, EM dilated RER‡ dilated RER‡ dilated RER‡ dilated RER‡ Growth plate Abnormal Abnormal ? Abnormal Abnormal Abnormal Abnormal aDegree of shortening of the long tubular bones with* or without† metaphyseal cupping. bPresence of flared iliac wings. cPlatyspondyly with level of incomplete ossification of vertebral bodies. dSmall thorax with short ribs. eOssification of pubic bones. fOssification of talus and calcaneus. All radiographs were taken in the neonatal period except for patient R82-121 where the radiographs were taken at 1 month of age. ‡Electron micrographs show dilated rough endoplasmic reticulum containing granular material. blue as described previously. by SDS-PAGE andresulting stained collagen CB withdigested peptides with Coomassie were cyanogen bromide analysed a (CB) and control. the Forwithout peptide signs of analysis, a skeletal cartilage dysplasia was was used as were dried and(AT Biochem) exposed for to 11 hoursrated at by 3.5 W. Thereduced electrophoresis gels to through 2.5and MDE µmol/l. Fragments gels the were sepa- amount of unlabelled dCTP was For DNA sequencewere visualised analysis by PCR ethidium0.6 bromide generated staining. MDE gel supplemented withsamples 10% were glycerol separated in xylene on cyanol MDE(50% sucrose, in gels 0.1% bromophenol (1 for water) blue, 60 0.1% was minutesdenatured at at added. 98 68 heteroduplex The analysis,hours at PCR −70 products were cartilage collagen fro phoresis (SDS-PAGE).dodecyl Human sulphate-polyacrylamidetype gel epiphyseal II electro- molecules were analysed by sodium 200 µmol/l of each dNTP,DNA 2 mmol/l polymerase, MgCl 2.5containing pmol 100 ng of ofplifications each genomic were DNA, primer, performed 2fibroblasts in U by 25 µl standardGenomic reactions procedures. DNA PCR was isolated am- fromNUCLEIC cultured skin ACID ANALYSIS described previously. extracted, washed, and digested withsis pepsin of as pepsin solubilisedCartilage collagen, stored cartilage at was −70 PROTEIN ANALYSIS DNA samples were not available. studies. All patientsfor morphological, were biochemical, and molecular sporadic. Parental initial two minutesThe amplification at conditions 95 mmol/l consisted Tris-HCl, pH 8.3, of and an 50 mmol/l KCl. á polymorphism (SSCP)TBE analysis, bu mide 5 gels µCi (29:1examined acrylamide:bis on of ratio) non-denaturingrequest. in 6% The polyacryla- 1 PCRsequences generated for the fragments otherACTTTAGGACCTG-3' were exons for are exon available 51.GCTTGCTCCACTC-3' on Primer and 5'-TCCTGTC CCC-3' forGG-3' exon and 46;exon 5'-AGGAGGCCTCGGGAAGT and 5'-TCCTCTGA 35;GGCCTCGGGCAGAGCCAGGC-3' 5'-CCCTGACCTGACTCAATC ACCTTCTTACCCCAGCTCTTC-3' and 5'- for TGCCCAGCAGTCCAG-3' for exonGGCTGAGGCGGCTG-3' 33; and 5'- 5'-TCCTAA CAGGCTAC-3' forCTTGGTG-3' exon and 31; 5'-CTCAGTGGGACTC TCAG-3' 5'-GTGCCC for exon 22; 5'-GGTTGATCACTT GCCTC-3' andsequences 5'-GGATACCATGTGACC werea 5'-CTGTTCTCACTCACT nineminute, minute and 72 incubationcycles at of 95 72 -[ 32 × P] dCTP was added to the PCR reaction TBE) overnight at 250 V. The fragments V er. For single strand conformation ° ° C for one minute, 60 C with an intensifying screen. For ° ° C for one minute, followed by C for five minutes, renatured 11 ma4dayold ° ° C, and loading bu The resulting collagen Mortier, Weis, Nuytinck, et al C was used. For analy- ° C followed by 36 11 x ray film for 48 ° ° C.