Platyspondylic Lethal Skeletal Dysplasia, San DiegoType, Is Caused by FGFR3 Mutations

Steven G. Brodie,1 Hiroshi Kitoh,1,2 Ralph S. Lachman,1,3,4 Loyda M. Nolasco,1Pertchoui B. Mekikian,1 and William R. Wilcox1,3*1Ahmanson Department of Pediatrics, Steven Spielberg Pediatric Research Center, Cedars-Sinai Burns and AllenResearch Institute, Los Angeles, California

2Department of Genetics, Institute for Developmental Research, Aichi Prefectural Colony, Aichi, Japan3Department of Pediatrics, UCLA School of Medicine, Los Angeles, California4Department of Radiology, UCLA School of Medicine, Los Angeles, California

The platyspondylic lethal skeletal dyspla-sias (PLSDs) are a heterogeneous group ofshort-limb dwarfing conditions. The mostcommon form of PLSD is thanatophoric dys-plasia (TD), which has been divided into twotypes (TD1 and TD2). Three other types ofPLSD, or TD variants (San Diego, Torrance,and Luton), have been distinguished fromTD. The most notable difference betweenTD and the variants is the presence of largerough endoplasmic reticulum (rER) inclu-sion bodies within chondrocytes of the vari-ants. We examined 22 cases of TD variantsfor the presence of missense mutations inthe fibroblast growth factor receptor 3(FGFR3) gene. All 17 cases of the San Diegotype (PLSD-SD) were heterozygous for thesame FGFR3 mutations found in TD1. Nomutations were identified in the Torranceand Luton types. Large inclusion bodieswere found in all 14 cases of PLSD-SD. Simi-lar inclusion bodies were present in two of72 TD1 cases, but not in 39 controls. The ma-terial retained within the rER stained onlywith antibody to the FGFR3 protein. The ra-diographic and morphologic differences be-tween TD and PLSD-SD may be a conse-quence of other genetic factors, perhaps inthe processing of mutant FGFR3 moleculeswithin the rER. The presence of rER inclu-sion bodies cannot reliably discriminate be-tween closely related skeletal dysplasias.Am. J. Med. Genet. 84:476–480, 1999.© 1999 Wiley-Liss, Inc.

KEY WORDS: fibroblast growth factor re-ceptor 3 (FGFR3); thanato-phoric dysplasia; platyspon-dylic lethal skeletal dysplasia;inclusion body; thanato-phoric variant

INTRODUCTION

The platyspondylic lethal skeletal dysplasias (PLSDs)are a heterogeneous group of disorders with short ribs,severe platyspondyly, and micromelia [Horton et al.,1979; Taybi and Lachman, 1996]. Thanatophoric dys-plasia (TD), the most common form of PLSD, is subdi-vided in its radiographic features into two types, TD1(MIM 187600) and TD2 (MIM 187610) [Langer et al.,1987; Tavormina et al., 1995; Wilcox et al., 1998]. Mis-sense mutations in the fibroblast growth factor recep-tor 3 (FGFR3) gene have been identified in both. TD2 isdue to a single missense mutation in the activation loopof the receptor (K650E), while 12 distinct missense mu-tations have been identified in TD1. These 12 muta-tions can create new cysteine residues in the extracel-lular domain (R248C, S249C, G370C, S371C, Y373C),eliminate the stop codon (J807G, J807R, J807C, J807L,J807W), creating a receptor predicted to have an addi-tional 141 amino acids, or result in a K650M substitu-tion [Tavormina et al., 1995; Rousseau et al., 1995,1996; Kitoh et al., 1998a; Wilcox et al., 1998;Tavormina et al., 1999]. The TD mutations cause li-gand-independent activation of the mutant receptor.Mutant FGFR3 with cysteine substitutions in the ex-tracellular domain can form abnormal disulfide-bonded dimers [Naski et al., 1996; Neilson and Friesel,1996; Webster and Donoghue, 1996; Webster et al.,1996; d’Avis et al., 1998].

Three other types of PLSD, or TD variants, are rec-ognized (San Diego [MIM 270230], Torrance, and Lu-ton [MIM 151210]) based on distinct radiologic andchondro-osseous morphologic characteristics [Hortonet al., 1979; Winter and Thompson, 1982; Spranger andMaroteaux, 1990; International Working Group on

S.G.B. and H.K. contributed equally to this work.Contract grant sponsor: NIH; Contract grant numbers: 5P01-

HD22657, M01-RR00425, and P30-HD34610; Contract grantsponsor: Human Growth Foundation.

*Correspondence to: William R. Wilcox, M.D., Ph.D., MedicalGenetics, Cedars-Sinai Medical Center, 8700 Beverly Blvd., SSB-3, Los Angeles, CA 90048. E-mail : wwilcox@cshs.org

Received 29 January 1999; Accepted 17 February 1999

American Journal of Medical Genetics 84:476–480 (1999)

© 1999 Wiley-Liss, Inc.

Constitutional Diseases of Bone, 1998]. At an early ges-tational age there is radiographic overlap betweenPLSD-SD and TD1 [Taybi and Lachman, 1996; Kitohet al., 1998b]. Both disorders are characterized by ashort, narrow skull base; short, bowed long bones;platyspondyly; hypoplastic ilia with a flat acetabularroof; and short ribs. PLSD-SD has metaphyseal flaring,which is not found in cases of TD1 (Fig. 1). In terms ofmorphologic features, both TD1 and PLSD-SD have

short, disorganized chondrocyte columns, but the ab-normalities tend to be less severe in the latter condition[Horton et al., 1979; Kitoh et al., 1998b]. The moststriking difference between TD and the variants is thepresence of large, dilated loops of rough endoplasmicreticulum (rER inclusion bodies) in the chondrocytes ofthe variants [Horton et al., 1979; Winter and Thomp-son, 1982; Kitoh et al., 1998b].

MATERIALS AND METHODSMolecular Analysis

Cases of TD (TD1: n 4 115; TD2: n 4 23), PLSD-SD(n 4 17), PLSD-Torrance (n 4 4), and PLSD-Luton (n4 1), collected from 1972 to 1997 by the InternationalSkeletal Dysplasia Registry (http://www.csmc.edu/genetics/skeldys), were analyzed for mutations in thecoding region of the FGFR3 gene by a combination ofpolymerase chain reaction amplification, restriction di-gestion, and sequencing. Screening methods for theR248C, S249C, G370C, S371C, Y373C, K650E, K650M,and stop codon mutations from either total RNA orgenomic DNA have been described elsewhere[Tavormina et al., 1995; Wilcox et al., 1998].

Clinical, Radiographic, and Histologic Analysis

All cases were submitted to the International Skel-etal Dysplasia Registry. The prenatal and postnatalclinical histories were reviewed for each case. Skeletalradiographs were evaluated for the degree of cranio-synostosis, platyspondyly, and curvature and short-ness of the femora. Histologic sections of long bones,when available, were examined for the extent of col-umn preservation, mesenchymal banding, and bonyabnormalities, as previously described [Wilcox et al.,1998]. Ultrastructural studies of resting cartilage wereperformed as previously described [Brodie et al., 1998].

Immunohistochemistry

Glycol-embedded sections were fixed with 4% para-formaldehyde, etched with 0.5 M NaOH in ethanol(50%) (37°C), digested with trypsin (0.05%), andwashed with phosphate-buffered saline (pH 7.4). Fro-zen sections (6 mm) of cartilage were fixed with 4%paraformaldehyde and washed with phosphate-buffered saline (pH 7.4). Endogenous enzyme activitywas blocked using Endo/Blocker (Biømeda, Foster City,CA), and the tissue was partially digested with Auto/Zyme solution (Biømeda) for 5 min. The sections wereblocked for 20 min with 0.1% bovine serum albuminand 1% nonfat dry milk and incubated with the pri-mary antibody overnight (4°C); this was followed by aperoxidase-tagged secondary antibody for 1 hr andABC solution (Vector, Burlingame, CA) for 30 min. Sec-tions were developed with a peroxidase substrate DABsolution (Biømeda) and mounted. Anti-type IX collagenmonoclonal antibody (Development Studies Hybrid-oma Bank, Iowa City, IA), anti-FGFR3 (Santa CruzBiotechnology, Santa Cruz, CA), anti-type II collagen(a gift from Dr. Robin Poole, Montreal, Canada), andanti-aggrecan (a gift from Dr. D. Heinegård, Lund,

Fig. 1. Anterior–posterior radiographs of the lower limbs in 21-week-old fetuses with TD1 (A) and PLSD-SD (B). Although there are similaritiesbetween the two cases, note the straighter femora and metaphyseal cup-ping in PLSD-SD.

FGFR3 Mutations in Thanatophoric Variants 477

Sweden) polyclonal antibodies were used at dilutions of1:1,000, 1:5,000, 1:1,500 and 1:1,000, respectively.

Human Subjects

This study was approved by the Human Subjects In-stitutional Review Board at Cedars-Sinai.

RESULTS

Seventeen cases of San Diego, four cases of Torrance,and one case of Luton were screened for the 12 knownTD1 mutations in the FGFR3 gene. All cases of PLSD-SD had mutations in FGFR3 (seven R248C, two S249C,six Y373C, and two stop codon), but no mutations wereidentified in cases of the Torrance or Luton types.Large chondrocyte inclusion bodies were present in 14cases of PLSD-SD (Fig. 2b). Three cases of PLSD-SDhad inadequate tissue preservation, and for this reasonthe presence or absence of chondrocyte inclusions couldnot be determined.

The identification of FGFR3 mutations in PLSD-SDprompted us to examine the chondrocyte ultrastruc-ture in 72 cases of TD for the presence of inclusions (46R248C, four S249C, 16 Y373C, two K650M, and four

stop codon). Large chondrocyte inclusions were foundin two cases (3%) (R248C). Small to medium-size inclu-sions were observed in 23 cases (32%) (16 R248C, oneS249C, four Y373C, and two stop codon), and no inclu-sions were observed in 11 cases of TD2 (Fig. 2a).. Wethen studied cases of osteogenesis imperfecta (n 4 21),hypophosphatasia (n 4 7), and normal controls (n 411). Small to medium-size inclusion bodies were pre-sent in 52% of cases of osteogenesis imperfecta, 14% ofhypophosphatasia cases, and 27% of normal controls,suggesting that small to medium-size chondrocyte in-clusions are a normal variant of cellular trafficking.Small to medium-size inclusions were more common atearlier gestational ages.

Immunohistochemical studies using antibodies spe-cific for collagens (types II, IX, and X), aggrecan, ver-sican, cartilage oligomeric matrix protein (COMP), andFGFR3 were performed on frozen or glycol-embeddedtissues. There were no detectable differences in matrixstaining compared with controls, and there was nostaining of inclusion bodies except with antibody toFGFR3 in PLSD-SD (Fig. 3).

The gestational ages of the cases ranged from 18 to37 (24.8 ± 5.7) weeks and 18 to 40 (33.1 ± 6.4) weeks forTD with and without inclusion bodies, respectively,and 17 to 21 (19.0 ± 1.2) weeks for PLSD-SD. Therewere no significant differences in radiographic abnor-malities between TD1 and PLSD-SD (at identical ges-tational ages), other than the metaphyseal flare of thelong bones in PLSD-SD. The chondro-osseous morpho-logic abnormalities were less severe in PLSD-SD, irre-spective of the specific FGFR3 mutation. Cases of TD1with inclusions had radiographic findings identical tothose of cases without, but the chondro-osseous mor-phologic features were less abnormal.

DISCUSSION

The TD variants were originally distinguished fromTD based on radiographic and histologic differences.While PLSD-Torrance and -Luton are quite different,PLSD-SD shares many characteristics with TD. PLSD-SD has been defined as a separate disorder largely be-cause of the metaphyseal spikes, better preservation ofthe growth plate, and large rER inclusions. Therefore,we were surprised to identify FGFR3 mutations iden-tical to those found in TD1 in 17 cases of PLSD-SD.

Nerlich et al. [1996] identified Y373C and R248Csubstitutions in two fetuses (21 weeks of gestation)with “variant” TD. Their second case is similar in ra-diographic features to PLSD-SD, while the first wastypical of TD at that gestational age. No ultrastruc-tural data were presented. The pelvic features of thesecond case, said to be different from TD, are in factcommonly seen in TD and PLSD-SD cases under 24weeks of gestation [Kitoh et al., 1998b]. The chondro-cyte columns were relatively preserved compared withtypical TD, also a common finding at an early gesta-tional age [Wilcox et al., 1998]. Nerlich et al. [1996]further differentiated their cases from TD because of alack of mesenchymal ingrowth across the physis (so-called fibrous band) in the chondro-osseous materialthey studied (anatomical site not specified). We have

Fig. 2. Ultrastructural features in a representative case of TD1 (A)with small/medium-size inclusion bodies and in PLSD-SD (B). Note themultiple loops of dilated rough endoplasmic reticulum observed in cases ofTD1 with inclusions (also found in normal controls) and the larger, abnor-mal inclusions in the San Diego variant. (A: ×12,960; B:×19,200.)

478 Brodie et al.

found it necessary, however, to make multiple sectionsto identify this abnormality in the young fetus, and it ismost easily identified in the long bones of the limbs.

The rER is the initial site for synthesis and matura-tion of membrane and soluble proteins destined for se-cretion or export to other organelles and the cell sur-face. Proteins shuttled through the rER that are mis-folded or improperly oligomerized are prevented fromexport and are subsequently degraded. Inclusion bodydisorders demonstrate selective retention of mutantproteins within the rER. Immunohistochemical, bio-chemical, and molecular analysis has shown that sub-sets of mutations in alpha1-antitrypsin, alpha1-antichymotrypsin, alpha2-antiplasmin, fibrinogen,complement factors (C3 and C4), CFTR (cystic fibrosistransmembrane regulator), and the LDL (low-densitylipoprotein) receptor are abnormally retained [Lehr-man et al., 1987; Pathak et al., 1988; Miura et al., 1989;Ng et al., 1989; Lindmark et al., 1990; Teckman et al.,

1996; Ashkenas and Byers, 1997]. The presence of in-clusion bodies within chondrocytes is a consistent find-ing in a number of chondrodysplasias, including thetype II collagenopathies and COMP disorders [Godfreyet al., 1988; Maddox et al., 1997]. Osteogenesis imper-fecta has retention of mutant type I collagen withinfibroblasts and osteoblasts [Bonadio and Byers, 1985].PLSD-SD is the first example of an inclusion bodychondrodysplasia where the underlying defect is notassociated with an extracellular matrix protein butrather a plasma membrane protein.

Misfolding and retention of mutant FGFR3 could bedue to abnormal intra- or intermolecular interactions.Most cases of TD1, however, did not demonstrate thepresence of inclusions. Other genetic factors associatedwith protein trafficking through the ER, such as vari-able activity of molecular chaperones or disulfide isom-erase, may explain the presence of rER inclusion bodiesin some cases. Identical mutations resulting in dilatedrER in some cases and none in others have also beennoted in a1-antitrypsin deficiency. Individuals with theZZ genotype and hepatocyte inclusions are susceptibleto liver disease, while those without inclusions are not.The inclusions appear to be due to defective clearanceof mutant molecules from the rER in susceptible indi-viduals [Teckman et al., 1996].

Radiographic and morphologic characteristics havebeen useful in delineating specific skeletal dysplasias[Horton et al., 1979; Spranger and Maroteaux, 1990;Taybi and Lachman, 1996]. Since (a) all cases of PLSD-SD were in fetuses of less than 22 weeks’ gestation, (b)large inclusions are a rare finding in TD1, and (c)PLSD-SD is much less common than TD1, we hypoth-esize that the radiographic appearance of PLSD-SDevolves into TD1 at a later gestational age. The pres-ence of inclusions may explain the radiographic differ-ences and decreased severity of morphologic changes inPLSD-SD because less mutant FGFR3 may reach thecell surface and participate in signaling. The pheno-type of TD1 should now include PLSD-SD. The pres-ence or absence of inclusion bodies can no longer beused to discriminate closely related skeletal dysplasias.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the donation ofspecimens to the International Skeletal Dysplasia Reg-istry by referring physicians and families. We thankMaryAnn Priore, Sheilah Levin, and Roberta Bonac-quisti for administering the Registry; Christine Kimfor technical support; and Dr. David Rimoin for usefuldiscussions.

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Fig. 3. Immunohistochemical staining of chondrocytes using FGFR3antibody. A: control; B: PLSD-SD. In control tissues there is perinuclearand membrane staining, while in PLSD-SD there is also staining of theendoplasmic reticulum (×40). Growth plate cartilage from four normal con-trols and four cases of PLSD-SD were studied with antibodies specific forcollagens (type II, IX, and X), aggrecan, versican, COMP, and FGFR3.Another 27 cases of TD1 were studied only with FGFR3 antibody. Positivecytoplasmic staining was seen with FGFR3 antibody in all cases of PLSD-SD and in one case of TD1 (with large inclusions by electron microscopy).

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