Morphologic and Immunohistopathologic Studies Maurice Godfrey,* Douglas R

Am. J. Hum. Genet. 43:894-903, 1988

Type I Achondrogenesis-Hypochondrogenesis: Morphologic and Immunohistopathologic Studies Maurice Godfrey,* Douglas R. Keene,* Eugene Blank,t Hisae Hori,* Lynn Y. Sakai,* Lydia A. Sherwin,$'1 and David W. Hollister* § *Shriners Hospital for Crippled Children; and Departments of TRadiology, tClinical Pathology, and §Biochemistry and Medicine, Oregon Health Sciences University, Portland

Summary A 32-wk-gestation female with type II achondrogenesis-hypochondrogenesis has been studied. The clinical features were typical, and radiographs revealed short ribs, hypoplastic ilia, absence of ossification of sacrum, pubis, ischia, tali, calcanei, and many vertebral bodies; the long bones were short with mild metaphyseal flaring. The femoral cylinder index was 6.3. Comparison with previous cases placed the patient toward the mild end of the achondrogenesis-hypochondrogenesis spectrum (Whitley-Gorlin prototype IV). Light microscopy revealed hypercellular cartilage with decreased matrix traversed by numerous fibrous vascular canals. The growth plate was markedly abnormal. Ultrastructural studies revealed prominently dilated rough endoplasmic reticulum containing a fine granular material with occasional fibrils in all chondrocytes. Immunohistologic studies indicated irregular large areas of cartilage matrix staining with monoclonal antibody to human type III collagen. The relative intensity of matrix staining for type II collagen appeared diminished. More striking, however, were intense focal accumulations of type II collagen within small rounded perinuclear structures of most chondrocytes but not other cell types. These results strongly suggest intraceliular retention of type II collagen within vacuolar structures, probably within the dilated rough endoplasmic reticulum observed in all chondrocytes by electron microscopy (EM), and imply the presence of an abnormal, poorly secreted type II collagen molecule. Biochemical studies (see companion paper) suggest that this patient had a new dominant lethal disorder caused by a structural abnormality of type II collagen.

Introduction type II (Langer-Saldino), as first proposed by Spranger et al. (1974). Whitley and Gorlin (1983) have proposed Achondrogenesis is a rare skeletal dysplasia character- subdividing achondrogenesis into four prototypes by ized by severe short-limbed dwarfism leading to death using roentgenographic criteria and measurements. Pro- in utero or in the neonatal period. Considerable pheno- totype I appears to be identical to the Parenti-Fraccaro typic and radiographic heterogeneity are apparent in achondrogenesis. Prototypes II-IV constitute a spec- this lethal disorder. Typically, affected infants are prema- trum, decreasing in severity, of Langer-Saldino achon- ture and exhibit a relatively large head, short trunk, drogenesis. Recently, further heterogeneity has been and extreme micromelia. The traditional subgroups of delineated for type I achondrogenesis (Borochowitz et achondrogenesis consist oftype I (Parenti-Fraccaro) and al. 1988). Although achondrogenesis type II has been considered to represent a unique disease entity with variabil- Received April 6, 1988; revision received June 29, 1988. ity, recent data (Borochowitz et al. 1986, 1988) sug- 1. In memoriam. gests that it constitutes the severe end of a spectrum Address for correspondence and reprints: David W. Hollister, M.D., oflethal short-limbed dwarfism which includes the simi- Shriners Hospital for Crippled Children, 3101 S.W. Sam Jackson Park lar but less severe condition termed hypochondrogene- Road, Portland, OR 97201. Stanescu et al. Infants with type II © 1988 by The American Society of Human Genetics. All rights reserved. sis by (1977). 0002-9297/88/4306-0010$02.00 achondrogenesis-hypochondrogenesis share similar ro- 894 Achondrogenesis-Hypochondrogenesis 895 entgenographic and pathologic features (Hendrickx et are often thin and in an irregular orientation (Hendrickx al. 1983; Maroteaux et al. 1983; Borochowitz et al. et al. 1983; Borochowitz et al. 1986; Eyre et al. 1986). 1986). More generally, type II achondrogenesis has been In perichondral areas, collagen fibers appear thicker considered to represent the most severe member of a and more densely packed and more characteristic of bone dysplasia family consisting of type II achondro- type I collagen than of normal cartilage type II colla- genesis, hypochondrogenesis, and spondyloepiphyseal gen (Eyre et al. 1986). Using immunohistochemical dysplasia congenita (SEDc) (Spranger 1985). The so- techniques, Horton (1984) and Horton et al. (1987) called SEDc family exhibits a neonatal pattern of de- demonstrated the presence of type I collagen in the layed or absent ossification of vertebral bodies, pubis, fibrous material surrounding the vascular canals, in ad- and epiphyseal centers and variable shortening of long dition to an apparent decrease in type II collagen in bones. cartilage matrix. Achondrogenesis is generally considered an auto- In the present report we describe the clinical, roent- somal recessive disorder, on the basis of familial recur- genographic, histological, ultrastructural, and immuno- rence (Whitley and Gorlin 1983). However, the milder histologic features in a mild case of type II achon- varieties of this disorder (hypochondrogenesis) are vir- drogenesis-hypochondrogenesis (Whitley and Gorlin tually all sporadic, and the absence ofparental consan- prototype IV). The immunohistologic results demon- guinity is notable (Maroteaux et al. 1983; Borochowitz strate the apparent abnormal intracellular accumula- et al. 1986). tion of type II collagen within vacuolar structures of Histological examination of bone and cartilage from chondrocytes, suggesting the presence of abnormal, cases of type II achondrogenesis show distinct and char- poorly secreted type II collagen. In the following paper acteristic abnormalities. Endochondral ossification is (Godfrey and Hollister 1988) we present (a) biochemi- greatly disorganized, and normal columnization at the cal findings that indicate a molecular defect of type II growth plate is absent. Calcified chondrocytes are pres- collagen in the pathogenesis of this patient's disorder ent in the metaphyseal trabeculae. Many studies have and (b) evidence suggesting that a new dominant mu- shown hypercellularity ofcartilage with a concomitant tation may account for the observed phenotype. decrease in intervening matrix. Vascularization in the cartilage is increased, and each vascular canal is sur- Material and Methods rounded by broad bands of fibrous tissue. Finally, the Tissue Sources chondrocytes appear enlarged and ballooned, but a recent histopathologic study (Borochowitz et al. 1986) Skin, cartilage, bone, and other tissue were obtained of 20 cases concluded that the chondrocytes were of from the patient and a skeletally normal 36-wk fetus normal size but surrounded by enlarged lacunae (Sal- within 3 h of death. Other neonatal tissues were pro- dino 1971; Goard and Kozlowski 1973; Jimenez et al. cured at autopsy. 1973; Xanthakos and Rejent 1973; Rimoin et al. 1974; Wiedemann et al. 1974; Yang et al. 1974; Maroteaux Histological Preparation of Tissue et al. 1976, 1983; Stanescu et al. 1977; Horton et al. Patient and control tissues were fixed in 10% forma- 1979; Sillence et al. 1979; Bueno et al. 1980; Chen et lin, and decalcified, and 4-micron sections were cut and al. 1981; Hendrickx et al. 1983; Borochowitz et al. then stained by standard hematoxylin and eosin tech- 1986; Eyre et al. 1986). niques (Sheehan and Hrapchak 1980). Without exception, electron micrographs of achondrogenesis II-hypochondrogenesis cartilage depict Electron Microscopy chondrocytes exhibiting dilated rough endoplasmic Immediately following excision, diced cartilage sam- reticulum (RER) filled with granular material and oc- ples were fixed in 0.1 M cacodylate-buffered Karnovsky's casional fine fibrils; sporadic inclusion bodies have also (1965) fixative for 20 h at 4 C, rinsed in cold buffer been observed (Hendrickx et al. 1983; Maroteaux et three times over 48 h, and finally fixed in cacodylate- al. 1983; Borochowitz et al. 1986; Eyre et al. 1986). buffered 1% OS04 for 2 h at 4 C. Thereafter, the spec- Hendrickx et al. (1983) have identified these inclusion imens were embedded, cut, stained, and visualized as bodies as lipid droplets. The territorial matrix of the described elsewhere (Keene et al. 1987). chondrocytes usually reveals a paucity of collagen fibers and proteoglycan profiles (Borochowitz et al. 1986). lmmunofluorescent Staining of Cartilage Collagen fibers in the interterritorial cartilage matrix Unfixed patient and control cartilage were cryo- 896 Godfrey et al. sectioned at 10 microns (Leitz 1720 Kryostat), and air- terpreted as normal. At autopsy there was marked pul- dried sections were fixed for 5 min in freshly prepared monary hypoplasia (lung weight was 27% of normal) formaldehyde (Graham and Karnovsky 1966). Pro- and thymic hypoplasia (thymus weight was 50% of nor- teoglycans were removed by treating with Chon- mal). Other organs were grossly normal. The brain was droitinase ABC (Miles) (Poole et al. 1980) for 90 min. not examined. After being washed with PBS, the sections were incubated for 1 h with monoclonal antibody. The mu- Roentgenographic Findings rine monoclonal anti-type II collagen antibody has been A whole-body X-ray of the infant is shown in figure described elsewhere (Hollister et al. 1982). A mono- 1. The femoral cylinder index (Whitney and Gorlin clonal antibody to human type III collagen (unreactive 1983) is 4.8 on the right and 6.3 on the left, a dis- to human types I, II, and IV-VII) was produced in crepancy that results from the position of the legs dur- A.SW/SnJ mice (H. Hori, unpublished data). The tis- ing the examination. Normal term infants have femo- sue sections were washed in PBS, then incubated for ral cylinder indices of 12-14 (Borochowitz et al. 1986). 30 min with a goat anti-mouse IgG conjugated to phycoerythrin (Biomeda, Foster City, CA). The fluores- Histologic Features cent emission maximum of phycoerythrin is 575 nm Sections ofcartilage and bone demonstrated marked (yellow-gold). To visualize cells, the sections were abnormalities. The zones of resting cartilage contained washed with PBS and stained with a 1:5 dilution of chondrocytes that appeared larger and more numerous 0.0005% propidium iodide (Biomeda), which imparts a red fluorescence to nuclei under excitation conditions used for phycoerythrin. The sections were mounted in Gel/Mount (Biomeda) and viewed and photographed with a Zeiss Photoscope III fluorescence microscope. Fluorescence excitation was achieved with a fluorescein filter set and observed through a barrier filter passing wavelengths >520 nm.

Results Clinical Summary The patient was a 1,190-g female, the product of a 32-33-wk gestation to a gravida 4, para 3 35-year-old white mother and nonconsanguineous father. The mother's three previous children by a different father were normal. Prenatal care was good; ultrasound at 16 wk was interpreted as normal. The infant required immediate resuscitation and in- tubation. On examination, she was a short-limbed dwarfwith a relatively large head, flat nasal bridge, cleft palate, micrognathia, low-set ears, short neck, small thorax with protuberant abdomen, and marked micromelia. The short limbs were not bent, and polydactyly was not present. Measurements were as follows: length 28 cm (72% ofage-matched normal); occipital-frontal Figure I Portable X-ray obtained antemortem. The calvaria circumference 29.5 cm (upper range ofnormal); crown- is normal. All the long bones, including the ribs, are proportionately to-rump length 20.5 cm (79% of normal); chest cir- shortened with mild metaphyseal flaring but are longer and better cumference 22.5 cm; abdominal circumference 24 cm; modeled than more severe varieties of achondrogenesis-hypochon- leg length (symphysis to heel) right = 8.0 cm, left = drogenesis. The ilia are small and irregular, and the bases are flat. The ossification centers of the thoracic vertebral bodies are small 7.5 cm; arm span 29 cm. and widely separated. Ossification centers are present in the pedicles The infant died within 12 h of birth. Chromosome of the lumbar vertebrae but not in the bodies. The sacrum, ischia, analysis was subsequently reported as 46,XX and in- pubic bones, calcanei, and tali are not ossified. Achondrogenesis-Hypochondrogenesis 897

A amount of calcified cartilage at the growth plate, but the trabeculae immediately adjacent to this zone were completely ossified (fig. 3B). This was markedly different from the situation in normal controls, in whom the adjacent trabeculae were cartilage undergoing calcification (fig. 3A). Sections of humerus, costochondral junc- tion, long bone from the foot, and spine were examined, and all showed similar abnormalities. The lung showed very poorly aerated air spaces lined by smudgy eosinophilic material, probably early hyaline membranes. The liver showed more residual ex- tramedullary hematopoiesis than did age-matched controls. Sections of thymus, thyroid, adrenal gland, and pancreas were unremarkable. Okim'm'eW;.s''''"':. Cartilage Ultrastructure Electron microscopy ofcartilage revealed the uniform presence of various-sized vacuoles of dilated RER in all chondrocytes (fig. 4A). In contrast, other cells (fibroblasts and endothelial cells) did not exhibit RER dilation (not shown). Figure 4B demonstrates a typical profile of RER observed, and figure 4C shows a higher- power view of the dilated RER with its characteristic bilaminar membranes studded with ribosomes. The material contained within the RER appears granular, with some delicate, short fibrils. Many chondrocytes contained osmophilic circular profiles identified as possible lipid droplets (fig. 4A) and non-membrane-bound granular material which was thought to be glycogen. Lysosomes and other intracellular organelles were not increased. Chondrocytes can be distinguished from F g r 2Phtomicrographoft scula canalsge ndogeneisc ar-e other cell types by their delicate foot processes, and

BPaoaiviwopainhylncatilage(88xx).Multiple vasclrsaeofvyigdmtraecn-retention of this feature suggests adequate tissue fixa- tuaine eachu debrou fband. ss eaeboh m r n m r u tion and absence of autolytic changes. The perichondrocytic matrix contained both collagen fibers and proteoglycan profiles similar to those ob- enum eration)ula than those in age controlCHge-o e inw served in the interterritorial matrix. There was no evi- o vaddition theres aonto icareia se inx).D aige - onw th e- dence of lacunar enlargement beyond the extent of the cato ilaoger a c vascularn l ina h dr g espacsc r chondrocyte, nor was there transmission EM (TEM) atilage(fig)2Aul2tiptevascularspcsovayn dim tr re o- evidence for absence of collagen fibers or proteogly- cain winthen pathi entu bappaenode mr.n cans in the pericellular spaces. The appearance of matrix collagen fibers changed in different regions. In areas (andrexhibitedy an etraorodinarcereae ofr sunirroundin removed from the fibrovascular canals, the collagen fibrosisa(ig.)2Bt2)aMnyothesei loosatcedfibtrous.band fibers were thin and sparse, as opposed to those collagen fibers close to the vascular canals, which appear containaed multrip.Ulietethin-walledbod vesseulsao vpary- thicker and were more numerous (data not shown). Cartilage Immunofluorescence ing diameters. Hypertrophic chondrocytes were pres- Figure 5 depicts the immunofluorescent staining pat- ent at the growth plate; however, column formation was terns observed in control and patient cartilage when absent or markedly defective (fig. 3 ). There was a scant the type III antibody is used and is followed by incuba- 898 Godfrey et al.

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Figure 3 Photomicrographs of growth-plate cartilage from the control (A) and the patient (B). Note the disorganization in columnization in the patient and the irregular zones of calcification and ossification (149x).

tion with propidium iodide to visualize nuclei. In con- Similar immunofluorescent studies using the type II trast to the uniform absence of type III staining in all monoclonal antibody yielded striking results, as shown areas of control cartilage (fig. 5B), irregular, large areas in figure 6. The matrix ofcontrol cartilage, as expected, of patient cartilage matrix exhibited positive staining stained with this antibody (figs. 6A, 6B). Some accen- (fig. SA); other large areas exhibited no staining and tuation of fluorescent intensity was observed around resembled normal cartilage or control tissue in which chondrocytes at the lacunar rims; an occasional cell culture media was substituted for the type III antibody. contained a bright focal accumulation of type II stain- In general, patient matrix close to perichondrial areas ing within the cytoplasm, and a much larger number was variably stained, and some areas adjacent to the exhibited a fine, diffuse, dustlike fluorescence in the fibrovascular canals exhibited weak staining (fig. SC), cytoplasm. Rare perinuclear bright focal accumulations but this was not uniformly observed (fig. SD). The of fluorescence were observed in control cartilage. As fibrous materials of the fibrovascular canals stained in- expected, the perichondrium did not stain for type II tensely with the type III antibody (fig. SC, SD). Similar collagen (fig. 6B). Similar to control cartilage matrix, studies of the delicate vascular canals of normal carti- the patient's matrix exhibited variable type II staining, lage revealed only a faint rim of type III staining. The but the relative intensity of staining appeared to be less perichondrium of both the control and the patient tis- than that in control tissue in most areas. The most strik- sue was strongly positive for type III staining (data not ing findings were bright, rounded to oval, focal accumu- shown). No evidence of intracellular staining was ob- lations of yellow phycoerythrin fluorescence in a served in either patient or control tissue. perinuclear pattern beside, above or below chondro- Achondrogenesis-Hypochondrogenesis 899

Discussion The phenotypic features ofthe patient described here are characteristic oftype II achondrogenesis, specifically its milder variants (Wiedemann et al. 1974). Borocho- witz et al. (1986) observed a distinctly higher incidence of cleft palate in the milder variants of achondrogenesis, and the presence of such clefting in our patient is consistent with the relatively less severe phenotype. The clinical course was typical, although yet milder cases have survived for days or months. The cardinal roentgenographic features were apparent in the present case. The femoral cylinder index was 6.3, and this value, together with more sculptured ilia, places the patient in the category ofWhitley and Gorlin prototype IV achondrogenesis-hypochondrogenesis. Light-microscopic studies of cartilage and bone gave results similar to those previously observed in other such patients and confirm the diagnosis. Ultrastructural studies of cartilage revealed dilated RER in all chondrocytes, a feature that has been seen in all reported TEM studies of cartilage in achondrogenesis (Hendrickx et al. 1983; Maroteaux et al. 1983; Borochowitz et al. 1986; Eyre et al. 1986). Some varia- tions in the size of the dilated RER were seen, in contrast to the more uniform "marbles" described by Borochowitz et al. (1986). The perilacunar matrix was specifically examined be- cause of previous reports of marked abnormalities, including a paucity of collagen fibers and proteoglycan profiles around the chondrocytes (Borochowitz et al. Figure 4 Electron micrographs ofchondrocytes from femoral 1986). Our patient did not demonstrate these changes, hyaline cartilage of the patient. A, View of chondrocytes containing and there was no significant difference between the ter- vacuoles within the cytoplasm (final magnification 829 x; bar = 10 ritorial matrix and interterritorial matrix in terms of gm). B, Chondrocyte in panel A (arrow) at higher magnification (final collagen and/or proteoglycan profiles. We did not ob- magnification 4,781x; bar = 2 gm). C, Dilated RER (final magnificat- serve in the TEM that would suggest a dila- ion 41,293x; bar = 0.25 gm). Note granular contents with occa- changes sional fibrillar profiles contained in the dilated RER. tion of the chondrocytic lacunae, and it appeared that each chondrocyte filled its lacuna entirely. The expla- nation for these discrepancies is not known but may relate to time-dependent postmortem autolytic changes cyte nuclei which could be easily distinguished from prior to sample acquisition. the pink-red propidium iodide fluorescence ofthe nuclei In contrast to normal cartilage, the apparent content (figs. 5C-SE). The majority of chondrocytes contained ofcollagen-fiber profiles varied in different areas ofhya- such perinuclear staining, which appeared to localize line cartilage. Regions remote from the vascular canals to intracellular vacuolar structures. Typically, these appeared hypofibrillar, whereas cartilage adjacent to fluorescent vacuoles were brighter than adjacent ma- these structures was densely fibrillar. The latter areas trix staining. Frequently, multiple stained vacuoles could resembled the cartilage-perichondrium transition zone, be visualized within a single chondrocyte by focusing in which the larger collagen bundles and more densely through the specimen. In contrast to these findings in packed fibers presumably represent type I collagen. chondrocytes, no staining was observed for cells con- Our immunohistochemical results extend previous tained within either fibrovascular canals (fig. 6E) or observations (Horton 1984; Horton et al. 1987) with perichondrium (fig. 6F). respect to the distribution of type II collagen and pro- ~~~~~~o*-i*W=C - -o O4u 4.

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Figure 6 Fluorescence photomicrographs of type II monoclonal antibody staining of control and patient cartilage processed in parallel. A, Control cartilage matrix. The matrix exhibits type 11 staining with accentuated staining of the lacunar rims. The perinuclear areas are devoid of yellow phycoerythrin staining, and only rare bright inclusion vacuoles are observed. B, Control cartilage-perichondrial (PC) junc- tion. No perichondrial staining or intracellular inclusions of phycoerythrin staining are observed. C and D, Patient cartilage matrix. The matrix exhibits variable and decreased staining for type 11 collagen. Nearly every chondrocyte demonstrates phycoerythrin perinuclear staining of vacuolar structures (bright yellow focal staining contrasting with the pink-red nuclear staining). E, Edge of a fibrovascular (VC) canal in patient cartilage. Very reduced type II staining of adjacent cartilage matrix is shown together with multiple perinuclear inclusion bodies within chondrocytes; no inclusions are observed within cells of the fibrous canal. F, Patient perichondrium. No matrix staining or intracellular inclusions are present. Original magnification 500 x.

901 902 Godfrey et al. vide new information regarding the distribution oftype reflects decreased accumulation of this collagen type III collagen. Monoclonal antibodies specific for human within the observed hypofibrillar matrix or, possibly, type III collagen bound specifically to the loose fibrous dilution ofthis collagen type by codistribution of other tissue of the vascular canals. This finding is expected, collagens, such as types I and III. since in previous studies type I collagen has been local- On the basis of characteristic and typical clinical, ized to this perivascular material by immunohistochem- X-ray, and morphologic findings, this patient had type ical studies (Horton 1984; Horton et al. 1987) and since II achondrogenesis-hypochondrogenesis, Whitley-Gor- types I and III collagen are usually codistributed in var- lin prototype IV, and fits into the milder end of this ious tissues (Burgeson and Morris 1987). An unexpected disease spectrum. We interpret the immunohistologic finding was large, irregular areas of type III staining findings to indicate inappropriate deposition of type within otherwise typical cartilage matrix. Normally, III collagen within cartilage matrix, decreased deposi- type III collagen is not found in hyaline cartilage, and tion of type II collagen in matrix, and intracellular ac- it was not identified by immunohistology in normal con- cumulation oftype II collagen within chondrocytes, very trol cartilage. Since type III is usually codistributed with probably within the dilated RER observed in all cells type I, this finding may suggest deposition of both of by TEM studies. These interpretations suggests a num- these collagens within matrix. Notably, Eyre et al. ber of possible pathogenetic mechanisms for this dis- (1986) and Murray et al. (1987) have demonstrated the ease, including the presence of abnormal and poorly presence of type I collagen in achondrogenesis carti- secreted type II collagen. In the following paper (God- lage in the absence of type II collagen; these data sug- frey and Hollister 1988), we present evidence that an gest that type I can be an authentic component of ab- abnormal type II collagen is present. normal cartilage matrix. The finding of type III in matrix might further suggest that the contained cells are either incompletely differentiated chondrocytes or, Acknowledgments alternatively, chondrocytes undergoing dedifferentiation whose collagen biosynthetic repertoire yields this un- We wish to thank Dr. Everett Lovrien, who referred the patient. The technical assistance of Don Andersen, N. Donna usual cartilage matrix. Gaudette, Linda Germain, Elaine Roux, Brian Demings, and The most notable immunohistologic findings were Marie Spurgin is greatly appreciated. We thank Jose Perdomo, intense fluorescent staining, with anti-type II antibody, Biomeda Corp., for immunofluorescent reagents and guid- of small round or oval structures within virtually all ance. This research was supported by a grant from the Shriners chondrocytes in the patient's cartilage, compared with Hospitals for Crippled Children and by National Institute of only occasional such staining in control cartilage. The Arthritis, Musculoskeletal and Skin Diseases grant AR37272. distribution and intensity of staining strongly suggests Electron-microscope facilities were provided in part by the accumulation of type II collagen within chondrocytes, Fred Meyer and R. Blaine Bramble Charitable Trust Founda- and the smooth, rounded profiles observed suggest a tions. vacuolar localization. The most straightforward interpretation is intracellular accumulation of type II collagen within the prominently dilated RER vacuoles of References the chondrocytes. We have made a number of attempts Borochowitz, Z., R. Lachman, G. E. Adomian, G. Spear, to test this interpretation by EM immunolocalization K. Jones, and D. L. Rimoin. 1988. Achondrogenesis type studies of plastic-embedded, fixed cartilage samples, I: delineation of further heterogeneity and identification but, owing to technical difficulties, we have been un- of two distinct subgroups. J. Pediatr. 112:23-31. able to demonstrate localization of antibody to the Borochowitz, Z., A. Ornoy, R. Lachman, and D. L. Rimoin. cisternae of the RER (or in the matrix). Therefore, the 1986. Achondrogenesis II-hypochondrogenesis: variabil- interpretation of intracellular accumulation of type II ity versus heterogeneity. Am. J. Med. Genet. 24:273-288. RER Bueno, M., F. Toledo, J. Toledo, T. Villegas, S. Lopez, J. Remi- collagen within vesicles, although highly proba- rez, and G. Garcia-Julian. 1980. Acondrogenesis, tipos I ble, must be considered tentative until definitive EM y II e hipocondrogenesis. An. Esp. Pediatr. 13:889-900. immunolocalization is demonstrated. Burgeson, R. E., and N. P. Morris. 1987. The collagen fam- In comparison with normal cartilage, the patient's ily of proteins. Pp. 3-28 in J. Uitto and A. J. Perejda, eds. matrix exhibited variably and sometimes severely di- Connective tissue disease: molecular pathology of the ex- minished amounts of type II collagen, as judged by intracellular matrix. Marcel Dekker, New York. tensity ofimmunofluorescent staining. Presumably this Chen, H., C. T. Liu, and S. S. Yang. 1981. Achondrogenesis: Achondrogenesis-Hypochondrogenesis 903

a review with special consideration ofachondrogenesis type Murray, L. W.,J. Bautista, P. James, and D. L. Rimoin. 1987. II (Langer-Saldino). Am. J. Med. Genet. 10:379-394. Normal type II collagen is not detected in cartilage of pa- Eyre, D. R., M. P. Upton, F. D. Shapiro, R. H. Wilkinson, tients with achondrogenesis II-hypochondrogenesis. Am. and G. F. Vawter. 1986. Nonexpression of cartilage type J. Hum. Genet. 41[Suppl]:A12. II collagen in a case of Langer-Saldino achondrogenesis. Poole, A. R., I. Pidoux, A. Reiner, L.-H. Tang, H. Choi, and Am. J. Hum. Genet. 39:52-67. L. Rosenberg. 1980. Localization of proteoglycan mono- Goard, K. E., and K. Kozlowski. 1973. Thanatophoric mer and link protein in the matrix of bovine articular car- dwarfism II. Pediatr. Radiol. 1:8-11. tilage: an immunohistochemical study. J. Histochem. Godfrey, M., and D. W. Hollister. 1988. Type II achondro- Cytochem. 28:621-635. genesis-hypochondrogenesis: identification of abnormal Rimoin, D. L., D. W. Hollister, R. S. Lachman, R. L. Kauf- type II collagen. Am. J. Hum. Genet. 43:904-913. man, W. H. McAlister, R. E. Rosenthal, and G. N. F. Graham, R. C., and M. J. Karnovsky. 1966. The early stages Hughes. 1974. Histologic studies in the chondrodys- ofabsorption ofinjected horseradish peroxidase in the prox- trophies. Birth Defects 10:274-295. imal tubules of mouse kidney: ultrastructural cytochemis- Saldino, R. M. 1971. Lethal short-limbed dwarfism: achon- try by a new technique. J. Histochem. Cytochem. 14: drogenesis and thanatophoric dwarfism. Am. J. Roentgenol. 291-302. Radium. Ther. Nucl. Med. 112:185-197. Hendrickx, G., F. Hoefsloot, P. Kramer, and U. Van Haelst. Sheehan, D. C., and B. B. Hrapchak. 1980. Theory and prac- 1983. Hypochondrogenesis: an additional case. Eur. J. Pedi- tice of histotechnology. Mosby, St. Louis. atr. 140:278-281. Sillence, D. O., W. A. Horton, and D. L. Rimoin. 1979. Mor- Hollister, D. W., L. Y. Sakai, N. P. Morris, L. H. Shimono, phologic studies in the skeletal dysplasias. Am. J. Pathol. and R. E. Burgeson. 1982. Production and characteriza- 96:813-869. tion of hybridoma antibody to native human type II colla- Spranger,J. 1985. Pattern recognition in bone dysplasias. Pp. gen. Coll. Relat. Res. 2:197-210. 315-342 in C. J. Papadatos and C. S. Bartsocas eds. Endo- Horton, W. A. 1984. Histochemistry: a valuable tool in con- crine genetics and genetics of growth. Alan R. Liss, New nective tissue research. Coll. Relat. Res. 4:231-237. York. Horton, W. A., M. A. Machado, J. W. Chou, and D. Camp- Spranger, J. W., L. 0. Langer, and H.-R. Wiedemann. 1974. bell. 1987. Achondrogenesis type II: abnormalities of ex- Bone dysplasias: an atlas ofconstitutional disorders ofskele- tracellular matrix. Pediatr. Res. 22:324. tal development. W. B. Saunders, Philadelphia. Horton, W. A., D. L. Rimoin, D. W. Hollister, and R. S. Lach- Stanescu, V., R. Stanescu, and P. Maroteaux. 1977. Etude man. 1979. Further heterogeneity within lethal neonatal morphologique et biochimique du cartilage de croissance short-limbed dwarfism: the platyspondylic types. J. Pedi- dans les osteochondrodysplasies. Arch. Fr. Pediatr. 34 atr. 94:736-742. [Suppl. 3]: I-LXXVII. Jimenez, R. B., L. B. Holmes, J. S. Kaiser, and A. L. Weber. Whitley, C. B., and R. J. Gorlin. 1983. Achondrogenesis: new 1973. Achondrogenesis. Pediatrics 51:1087-1090. nosology with evidence of genetic heterogeneity. Radiol- Karnovsky, M. J. 1965. A formaldehyde-gluteraldehyde fixa- ogy 148:693-698. tive of high osmolality for use in electron microscopy. J. Wiedemann, H.-R., W. Remagen, H. A. Hienz, R. J. Gorlin, Cell. Biol. 27:137A. and P. Maroteaux. 1974. Achondrogenesis within the scope Keene, D. R., L. Y. Sakai, H. P. Bachinger, and R. E. Burge- ofconnately manifested generalized skeletal dysplasias. Z. son. 1987. Type III collagen can be present on banded col- Kinderheilk. 116:223-251. lagen fibrils regardless of fibril diameter. J. Cell. Biol. Xanthakos, U. F., and M. M. Rejent. 1973. Achondrogene- 105:2393-2402. sis: case report and review of the literature. J. Pediatr. Maroteaux, P., V. Stanescu, and R. Stanescu. 1976. The le- 82:658-663. thal chondrodysplasias. Clin. Orthop. 114:31-45. Yang, S. S., A. J. Brough, G. S. Garewal, and J. Bernstein. . 1983. Hypochondrogenesis. Eur. J. Pediatr. 141: 1974. Two types of heritable achondrogenesis. J. Pediatr. 14-22. 85:796-801.