American Journal of Medical Genetics 47:272-277 (1993)

Hydrops-Ectopic Calcification-Moth-Eaten Skeletal Dysplasia (Greenberg Dysplasia): Prenatal Diagnosis and Further Delineation of a Rare Genetic Disorder

David Chitayat, Helen Gruber, Brendan J. Mullen, David Pauzner, Teresa Costa, Ralph Lachman, and David L. Rimoin Department of Pediatrics, Division of Medical Genetics (D.C., T.C.), The Hospital for Sick Children and The Prenatal Diagnosis Program (D.C.), Toronto General Hospital, and Departments of Pathology (B.J.M.) and Obstetrics and Gynecology (D.P.), Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada; Department of Pediatrics, CedarSinai Medical Centre (H.G., D.L.R .) and Harbor-UCLA Medical Centre, Torrance (R.L.), Los Angeles, California

An offspring of consanguineous parents of East-Indian origin was found prenatally to have hydrops short limb dwarfism, polydactyly and chondro~osseous changes

with Greenberg hydrops-ectopic calcification-moth-eaten skeletal dysplasia (HEM) dysplasia. The radiological changes include platyspondyly with multiple extra os- sification centers, extraneous calcification in the ribs, sternum, pelvis, and epiphysis, and moth-eaten long bones. The histopathological changes included chondrocytes with dilated rough endoplasmic reticulum and inclusion bodies with homogeneous material of inter- mediate electron density.

These findings further delineate the spec- trum of this rare autosomal recessive skeletal dysplasia. o 1993 Wiley-Liss, Inc.

CLINICAL REPORT Fetal ultrasonography (US) done on a 24-year-old

G2P1 woman at 17 weeks of gestation showed a fetus with short limb dwarfism, hexadactyly of both hands, hydrops fetalis, and cystic hygroma (Fig. 1). There was no blood group incompatibility and complete hemo-

KEY WORDS fetal, skeletal dysplasia, lethal, inherited, polydactyly, cystic hygroma, autosomal recessive

INTRODUCTION The association of hydrops fetalis with ectopic cal-

cifications and “moth-eaten” skeletal dysplasia was first reported by Greenberg et al. [1988] who presented sibs born to a consanguineous couple of Greek origin; Spranger and Maroteaux [19901 reported the third case.

We report the fourth case of this rare skeletal dyspla- sia extending our knowledge of the spectrum and the ethnic diversity of this disorder.

Received for publication November 30, 1992; revision received March 23, 1993.

Address reprint requests to Dr. D. Chitayat, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 2C4. cystic hygroma.

Fig. 1. Fetal ultrasonography at 17 weeks of gestation showing

0 1993 Wiley-Liss, Inc.

HEM Dysplasia 273

Radiographic Findings Fetal skeletal survey documented deficient ossifica-

tion of the membranous bone of the skull. The base of the skull was extremely dense and the orbits were highly arched. There was severe midface hypoplasia with small maxilla and mandible. There were tracheal and hyoidl cricoid calcifications, platyspondyly with multiple extra ossification centers of the vertebral bodies, dense bones in the spinous processes, and pedicular and laminar maldevelopment. The chest had a barrel shape with 11 pairs of ribs and elongated clavicles. All ribs were short and showed ossification gaps. The iliac wings appeared moth eaten and the tubular bones were fragmented and had a mottled appearance. The epiphyses had irregular calcifications and the metaphyses and diaphysis were short and thick. There were brachydactyly, absent distal phalanges, and bilateral hexadactyly (Fig. 3). Metacar- pal and phalange bones were short. The tarsal bones had an abnormal shape with punctate calcifications.

MATERIALS AND METHODS Specimens of rib and long bones were fixed in 10%

neutral buffered formalin and embedded undecalcified

grams, including red blood cell indices and hemoglobin electrophoresis on parents, were normal. The father of this pregnancy was 30 years old; the couple’s first preg- nancy resulted in a neonatal death of a female fetus. No abnormality was detected on autopsy. The couple was of East-Indian descent and first cousins (their fathers were brothers).

Termination of pregnancy was done by prostin induc- tion. This was a male fetus with weight of 124 g (normal 140-194 g), head circumference 13.4 cm (normal 12.5- 15.3 cm), and chest circumference 8.8 cm (normal 11.1- 12.6 cm). The crown-heel length was 12.5 cm and the crown-rump length 10.5 cm (normal 13.0-14.1 cm). The foot measured 1.6 cm (normal 2.1-2.8 cm). There was severe hydrops fetalis, cystic hygroma, rhizomelic and mesomelic shortness of the upper and lower limbs, and postaxial polydactyly on both hands (Fig. 2). No other congenital anomalies were detected. There was absence of hematopoietic marrow in long bones and marked ex- tramedullary hematopoiesis in the liver. The kidneys, gonads, and external genitalia were normal.

Chromosome analysis done on cultured fibroblasts was 46,XY.

Fig. 2. The fetus at 17 weeks gestation (A, B). Note the mesomelic and rhizomelic shortness of the limbs, hydrops fetalis, and cystic hygroma.

274 Chitayat et al.

Fig. 3. Skeletal films. Note the deficient skull ossification, laryngeal and tracheal calcifications (A), and moth-eaten long bones (B).

in either glycerol or methyl methacrylate for light mi- croscopy [Gruber, 19921 or in Spurr’s standard resin for transmission electron microscopy. Specimens for light microscopy were sectioned with a Reichert-Jung 2050 Supercut microtome at 2-5 micra and stained with a variety of stains. Thin sections for transmission ultra- structural studies were cut with an LKB ultra- microtome, transferred to Formvar-coated one-slot grids, stained with uranyl acetate and lead citrate, and examined in a Zeiss 902 electron microscope. Specimens for scanning electron microscopy were rinsed in buffer and immersed for one hour in a solution of 5% sodium hypochlorite to remove organic noncalcified matrix (in- cluding cells). Specimens were then dehydrated, dried to the critical point, coated with gold palladium, and exam-

ined in a Hitachi S405 A12 scanning electron micro- scope.

Histopathological Findings Chondro-osseous morphology. Light microscopic

examination of ribs, tibiae, and femora findings were very similar to those previously reported by Greenberg et al. [1988] and Spranger and Maroteaux [19901.

Chondro-osseous development was severely disor- dered. Growth plates were irregular and undulating and showed no normal cartilage column formation (Fig. 4). There was an abrupt transition from cartilage to abnor- mal bone (Fig. 5) . Calcified cartilage extended into the primary spongiosa. In some regions of the growth plate, a mesenchymal-like tissue component was present be-

HEM Dysplasia 275

Fig. 4. Light micrograph of a section of the rib showing unusual growth plate organization. Note the lack of columns of chondrocytes (A) and the unusual bone cartilage transition (B). Goldner’s stain; A, x 40; B, x170.

tween bone and cartilage (Fig. 6). In these areas, there was striking disorganization of cartilage, calcified car- tilage, and bone matrix. Advancing zones of cartilage ossification were present in inappropriate sites and were associated with the presence of abnormal, hypercellular bone. In resting cartilage, abnormal nodular deposits of calcification were present.

Chondrocytes examined with transmission electron microscopy were found to contain regions of dilated rough endoplasmic reticulum and inclusion bodies which held homogeneous material of intermediate elec- tron density (Fig. 7). Matrix contained sparse amounts of collagen and proteoglycan. In some regions, collagen fibrils were stringy and clumped. Regions of abnormal cartilage calcification showed 2 types of mineralization. In one type dense calcospherites were present near chon- drocytes (Fig. 8). Other mineralization sites were larger and more irregular, and were often associated with de- bris from degenerating chondrocytes (Fig. 9). Such ab- normal mineralization sites were similar to those previ- ously reported by Gruber et al. [19901.

DISCUSSION Hydrops ectopic calcification-moth-eaten skeletal

dysplasia (HEM) [Taybi and Lachman, 19901 was the

acronym given to a rare skeletal dysplasia reported first by Greenberg et al. [19881. The authors reported male and female sibs born to a consanguineous Greek couple suggesting autosomal recessive inheritance. Their first patient was a male who was born at 30 weeks gestation with marked hydrops fetalis and rhizomelic shortness of all limbs. In a subsequent pregnancy the US showed at 20 weeks fetus with markedly short limbs but without hydrops fetalis. In both cases no other external or inter- nal abnormalities were noted. The third case was re- ported by Spranger and Maroteaux 119901 but no infor- mation was given regarding the parental origin and family history.

The fetus reported by us was born to first cousins of East-Indian descent. This supports the assumption that HEM dysplasia is an autosomal recessive trait. Further- more, the different ethnic origins show that the muta- tion causing this disorder exists in different populations and is not a result of a founder effect. Both interfamilial and intrafamilial variability are found in this disorder. In the sibs reported by Greenberg et al. [19881 the first fetus had hydrops fetalis at 30 weeks of gestation while his sib was not hydropic at 20 weeks of gestation. The case reported by Spranger and Maroteaux [19901 was a stillborn with nonimmune hydrops fetalis. The gesta-

276 Chitayat et al.

Fig. 6. Light micrograph illustrating unusual mixture of bone, mesenchymal-like tissue, and bone in the rib. Goldner’s stain, X 170. Fig. 5. Scanning electron micrograph showing abrupt bone car-

tilage transition. x 200.

Fig. 7. Transmission electron micrograph illustrating dilatations of rough endoplasmic reticulum within a chondrocyte (arrows) and sparse Fig. 8. Transmission electron micrograph of abnormal cal- matrix. x 6,650. cospherite mineralizations near a resting chondrocyte. x 10,250.

HEM Dysplasia 277

anatomical abnormalities apart from the skeletal dys- plasia and hydrops fetalis. The fetus reported by us had cystic hygroma, 11 pairs of ribs, and polydactyly. Cystic hygroma was reported in other skeletal dysplasias, among them achondroplasia and achondrogenesis type I1 [Edwards and Graham, 19901. This is the first re- ported case of cystic hygroma in HEM dysplasia. The finding of postaxial polydactyly on both hands in our case was also not reported previously.

The histopathological findings in this disorder in- clude complete disorganization of calcified cartilage and of bone with brittle and hard focal calcium deposits in the cartilage. Islands of cartilage were surrounded by plates of lamellar bone. Cartilage cells were not ar- ranged in columns and there was ingrowth of mesenchy- ma1 tissue into the growth plate. The present case shows marked histological and ultrastructural similarities to the cartilage and boney abnormalities previously ob- served in the 2 sibs reported by Greenberg et a1 [19881. In addition, in the present fetus we have extended the findings with a more detailed cytological study of chon- drocytes which identified the presence of dilatations of rough endoplasmic reticulum and inclusion body mate- rial. The dilatation of the rough endoplasmic reticulum and the inclusion body material suggests the possibility of abnormal production of type I1 collagen. It will be important in future studies to obtain frozen cartilage and/or live chondrocytes to further define this possible molecular defect in this disorder.

REFERENCES Edwards MJ, Graham JM (1990): Posterior nuchal hygroma. Clin Peri-

natol 17:611-640. Greenberg CR, Rimoin DL, Gruber HE, DeSa DJB, Reed M, Lachman

RSA (1988): A new autosomal recessive lethal chondrodystrophy with congenital hydrops. Am J Med Genet 29:623-632.

Gruber HE (1992): Adaptations of Goldner’s Masson trichrome stain for the study of undecalcified plastic embedded bone. Biotechnic Histo- chem 67:30-34.

Gruber HE, Greenberg CR, Lachman RS, Rimoin DL (1990): Ultra- structural abnormalities in bone and calcifying cartilage in two siblings with a newly described recessive lethal chondrodysplasia. Ultrastruct Pathol 14:343-355.

Spranger J, Maroteaux P (1990): The lethal osteochondrodysplasias. Adv Hum Genet 19:ll-12.

Taybi H, Lachman RS (1990): “Radiology of Syndromes, Metabolic Disorders, and Skeletal Dysplasias.” Chicago: Year Book Medical Publishers, p 733.

Fig. 9. lkansmission electron micrograph of an abnormal site of mineralization occurring near cell debris. x 15,200.

tional age and the sex are not known. The fetus reported by us presented with nonimmune hydrops fetalis at 17 weeks gestation and also had cystic hygroma. Further- more, like the case presented by Greenberg et al. [19881 we found excessive extramedullary erythropoiesis in the liver with obliterated bone marrow spaces. Thus, the hydrops is most probably the result of severe anemia and/or hypoalbuminemia. Greenberg et al. [19881 sug- gested that the differences in severity between their 2 cases is a result of the difference in gestational age; however, our case presented with the most severe abnor- malities at 17 weeks gestation. Thus, the hydrops fetalis in this condition is not necessarily a time-dependent phenomenon and, in some cases, can be found as early as 17 weeks gestation.

The other 3 cases reported previously had no other