Osteopetrosis: a Single Centre Experience of Stem Cell Transplantation and Prenatal Diagnosis

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Bone Marrow Transplantation (2001) 27, 129–132  2001 Nature Publishing Group All rights reserved 0268–3369/01 $15.00 www.nature.com/bmt Osteopetrosis: a single centre experience of stem cell transplantation and prenatal diagnosis

J Kapelushnik1, C Shalev1, I Yaniv2, M Aker3, R Carmi4, Z Cohen1, A Mozer1, C Schulman5, G Stein2 and R Or3

1Hemato-Oncology Unit, 4Genetics Dept, and 5Radiology Institute, Soroka University Medical Centre, Beer-Sheva; 2Bone Marrow Transplantation Unit, Schneider Children’s Hospital; and 3Hadassah Hospital, Ein Kerem, Jerusalem, Israel

Summary: course of these patients with infantile osteopetrosis, who were all diagnosed at our center. Malignant osteopetrosis (MOP) is an autosomal recessive disease in which osteoclast dysfunction results in excessive bone deposition and early infant death. Thir- Patients and methods teen children suffering from MOP from four related families all belonging to one Bedouin tribe, were stud- We present a retrospective study of 13 infants (eight ied. The disease was diagnosed as early as at a few days females and 5 males) diagnosed with MOP at a mean age postnatal to 5 months. Nine children underwent BMT, of 41 days (range 1 day to five months). All the patients four of whom are still alive; one is blind and two have belong to four interrelated Bedouin families living in the markedly reduced vision. Four children who did not Negev region of Israel and all of them are children of con- undergo BMT died between 4 and 6 months of age. sanguineous parents. In four infants, the diagnosis was Recently, the gene for MOP has been mapped for this established soon after birth; in three, based on a family Bedouin tribe allowing prenatal diagnosis. Seven preg- history of a previously affected sibling, and in the fourth nancies were subsequently prenatally diagnosed and case, based on prenatal diagnosis. However, the parents two fetuses were found to be affected. Pregnancy was declined termination of pregnancy. electively terminated in one case. In the other case the parents refused and after establishing the diagnosis, the Initial symptoms newborn was transplanted at the age of 7 days. Bone Marrow Transplantation (2001) 27, 129–132. All patients were small for their gestational age with a birth Keywords: osteopetrosis; bone marrow transplantation; weight percentile smaller than that of their head circumfer- prenatal diagnosis ence. Bulging fontanelle and hepatosplenomegaly were the presenting clinical symptoms in 12 of the 13 infants (Table 1). In two patients, lumbar punctures were carried out and normal cerebro-fluid pressure was measured. CTs Osteopetrosis, a heterogeneous group of inherited diseases of the brain were performed on all infants; however, no of the skeleton, is known in three clinical variants namely: (1) infantile or malignant osteopetrosis, an autosomal recessive disorder; (2) the autosomal dominant type that Table 1 Patient characteristics pre-BMT clinically presents during early adolescence and is a more 1,2 benign form; and (3) a malfunction of the osteoclasts Patient Age at Hepato- WBC Platelets Hbg Ca PTH combined with renal tubular acidosis due to carbonic anhy- No. diagnosis splenomegaly ×108/l ×109/l g% mg/dl ng/l drase II deficiency.3,4 Malignant osteopetrosis (MOP) is (days) characterized by a lack of osteoclastic function which pre- vents bone re-absorption and leads to excessive bone min- 1 150 + 28 105 11 6.7 305 2 100 + 16 40 9.4 7.3 NA eralization and bone encroachment on the cranial cavity, 310− 8.2 74 17 7.1 501 foramina and marrow space. Afflicted patients die in early 443+ 25 59 13 7.9 NA childhood.5–8 In a group of 13 Bedouin patients, all children 514+ 6.7 204 12.4 6.2 330 of consanguineous parents, the gene for MOP was recently 61+ 17.7 23 15 7.3 225 + localized to chromosome 11q12–13 by linkage analysis.9 790 23 31 8.1 5.2 190 845+ 38 30 10.4 7.3 NA In this report, we present the clinical phenotype and disease 912+ 24 11 61 9.6 211 10 75 + 32 51 8.9 8.8 223 11 60 + 11.3 221 16.7 5.9 174 12 35 + 22 34 9.1 8.8 NA + Correspondence: J Kapelushnik, Paediatric Hemato-Oncology, Soroka 13 1 34 157 15.8 8.7 NA Medical Centre and, Ben-Gurion University of the Negev, Beer-Sheva, Israel 84101 NA = not available; WBC = white blood cells; PTH = parathyroid hor- Received 2 May 2000; accepted 4 October 2000 mone (normal range 10–55 ng/l). Osteopetrosis and bone marrow transplantation J Kapelushnik et al 130 signs of hydrocephalus or increased intracranial pressure gestation age by either chorionic villous biopsy or amnioc- were detected. In three infants, ocular involvement and entesis. optic atrophy were diagnosed at the age of 2 and 3 months. One infant presented with hypocalcemic seizures at the age of 10 days. Bone marrow transplantation Nine of 13 patients underwent BMT at the age of 7 days Laboratory data at diagnosis to 6 months (mean 50 days). One of the infants diagnosed at birth was transplanted at the age of 7 days. Eight patients Peripheral blood smears of all patients, including those received HLA-A, B, DR-matched sibling donor marrow diagnosed soon after birth, showed a leukemoid reaction. and one was transplanted with bone marrow from his Two children were referred to our center with the diagnosis mother (5/6 HLA-matched to rejection vector). The pre- of congenital leukemia. Patient-specific data are listed in parative regimen is shown in Table 2. All patients received Table 1. Thrombocytopenia, less than 150 × 109/l was cyclosporin A as GVHD prophylaxis. Seven patients had detected in 10 patients. Anemia, with a hemoglobin level an abnormal visual response with various degrees of clini- less than 9 g% (range 8.9–6.1 g%) was found in four cal visual impairment at the time of BMT. Of the four patients at the age of 12 days to 4 months. In four patients, patients who were not transplanted (three due to the lack the serum calcium levels (less than 7 mg) (normal range of a compatible donor and one due to parental refusal) three 8.4–10.4 mg/dl) were identified. One patient with hypocal- died of infection and bone marrow failure and one of intra- cemic seizures had a serum calcium level of 5.2 mg/dl. cranial bleeding at the ages of 4 to 6 months. Serum parathyroid hormone levels was determined in eight patients, all of whom had increased levels. Plasma lactate dehydrogenase, bone alkaline phosphase and 1,25 vitamin Results D, were elevated in all patients. All patients, including the four cases diagnosed at birth, had radiological evidence of Bone marrow transplantation markedly increased bone density. A bone fracture was observed in one patient after birth. Seven of nine BMT patients engrafted at a median of 24 days (range 15–56 days for ANC of more than 1000/dl). Prenatal diagnosis One patient engrafted partially (200/dl ANC) and died of pulmonary haemorrhage. One patient died of ARDS with- Using linked micro-satellite markers in the region of chro- out engrafting, two patients died of alveolar hemorrhage mosome 11q12–13,9 prenatal diagnoses were performed for and two patients of severe ARDS. Four patients are alive, seven pregnancies with a 25% risk for fetuses affected with of whom three are 2.5–7 years post BMT; all four are full MOP. Prenatal diagnosis was performed at 10–16 weeks of donor chimeras. Patient characteristics of the surviving

Table 2 Bone marrow transplantation

Patient No. Age at BMT Conditions Engraftment Outcome

Ͼ1000 ANL (day) Ͼ20 000 platelets (day)

1 6 months Cam, Bu, TT, Cyclo 32 50 Alive (7.5 y) Impaired vision Short stature 2 4 months Cam, Bu, TT, Cyclo 56 56 Alive (5.5 y) CGVHD (mild) Cataract Short stature 3 1 month ATG, Bu, Cyclo 24 50 Alive (2.5 y) Blind 4 7 days ATG, VP-16 Cyclo 15 24 Alive (1 y) Impaired vision 5 24 days Cam, Bu, Cyclo 26 Platelet dependent Died – AH (+ 28 days) 6 1 month Bu, Cyclo 20 39 Died – ARDS (+83 days) 7 10 days Bu, Cyclo — — Died – ARDS (+19 days) 8 30 days Bu, Cyclo 26 Platelet Died – ARDS dependent (+22 days) 9 20 days Bu, Cyclo (200 ANL) Platelet Died – AH dependent (+80 days)

CAM = campath-1G; Cyclo = cyclophosphamide; ARDS = acute respiratory distress syndrome; Bu = busulfan; CGVHD = chronic graft-versus-host disease; AH = alveolar hemorrhage; TT = thiotepa; ATA = anti-thymocyte globulin.

Bone Marrow Transplantation Osteopetrosis and bone marrow transplantation J Kapelushnik et al 131 children are listed in Table 2. All patients are failing to hypersensitivity to busulfan administered as part of the pre- thrive with their height and weight below the 5th percentile. parative regimen for BMT. However, we cannot exclude They all have impaired vision with two patients (6 months the possibility that, due to extramedullary hematopoiesis, and 2.5 years post BMT) being legally blind, although at macrophages and granulocytes are displaced. Following the time of transplantation they had normal vision. A third intensive chemotherapy these cells undergo apoptosis, patient has a decrease in visual acuity with mild chronic releasing cytokines at high levels. GVHD and bilateral cataracts. Normalization of bone Although osteopetrosis is genetically heterogeneous and resorption was evident by tabulation of long bones demon- there is probably more than one genetic locus and certainly strated by X-ray analysis. The intellectual development of more than one mutation even for the malignant form, it is all patients is normal. None of the patients developed likely that all our patients, being homozygotes by descent acute GVHD. from a common ancestor, bear the same gene defect. This allows a fairly accurate prenatal diagnosis even at the Prenatal diagnosis present stage where only linkage data are available. The recent cloning of the mouse osteopetrosis gene18 holds Seven fetuses (five males and two females) were prenatally promise for the future identification of the human homo- tested for MOP. Five were found to be unaffected and they logue. Since optimal therapy for MOP is available today were born at term with no evidence of the disease, whereas (including BMT, even at a very early stage of the disease), two fetuses were diagnosed with MOP. One pregnancy was cloning of the human MOP gene may eventually lead to electively terminated, whereas in the second case, termin- future gene therapy. For the present, however, prenatal stem ation was refused on religious grounds. This newborn’s cell transplantation may become a more realistic treatment MOP was verified soon after birth and he was subsequently option for this disease. transplanted at the age of 7 days. Currently, 6 months post BMT he has a normal blood count and is a 100% donor chimera. However, he is severely visually impaired. References 1 Sly W. Carbonic Anhydrase deficiency syndrome: osteopetrosis with renal tubular acidosis and cerebral calcifications. Discussion In: Scriver C, Beaudet A, Sly W, Valle D (eds). The Metabolic Basis of Inherited Disease. McGraw-Hill: New York, 1989, Osteopetrosis is a rare congenital autosomal recessive dis- pp 2857–2866. order characterized by the development of abnormally 2 Helfrich M, Aronson D, Everts V et al. Morphologic features dense bones, anemia and progressive deafness and blind- of bone in human osteopetrosis. Bone 1991; 12: 411–419. ness. 3 van Hul W, Bollerslev J, Gram J et al. Localization of a gene Although the clinical course of MOP is rapidly progress- for autosomal dominant osteopetrosis (Albers–Schonberg ive and in most of the affected infants, lethal at a very disease) to chromosome 1p2. Am J Hum Genet 1997; 61: young age, some may survive for more than 6 years and a 363–369. minority of patients may live into their second or third dec- 4 Seiff CA, Chessels JM, Levinsky R. Allogenic bone-marrow 5–7 transplantation in infantile malignant osteopetrosis. Lancet ade. Clinical presentation and disease development vary 1983; 1: 437–441. among patients. In our group of patients, symptoms and 5 Gerritsen EJ, Vossen JM, Loo IHv et al. Autosomal recessive signs of the disease appeared very shortly after birth osteopetrosis: variability of findings at diagnosis and during included bulging fontanelles and evidence of extramedul- the natural course. Pediatrics 1994; 93: 247–253. lary hematopoiesis. Visual impairment and other neurologi- 6 Horton WA, Schimk RN, Lyama T. Osteopetrosis: further het- cal symptoms have been reported in most MOP patients in erogeneity. J Pediatr 1980; 97: 580–585. the first 3 months of life due to cranial nerve encroachment 7 Bollerslev J. Autosomal dominant osteopetrosis: bone metab- or primary retinal degeneration and neurodegeneration,10–12 olism and epidemiological, clinical, and hormonal aspects. which have persisted and even proceeded after BMT.13 All Endocr Rev 1989; 10: 45–67. our surviving patients also have visual impairment. One girl 8 Banco R, Seifert M, Marks S, McGuire J. Rickets and osteopetrosis: the osteosclerotic (oc) mice. Clin Orthop 1985; 201: who had no pre-BMT ophthalmological problems, 238–246. developed severe visual impairment due to optic nerve 9 Heaney C, Shalev H, Elbedour K et al. Human autosomal compression 1 year post BMT. This process proceeded recessive osteopetrosis maps to 11q13, a position predicted by despite 100% donor stem cell engraftment. comparative mapping of the murine osteosclerosis (oc) MOP has been treated with parathyroid hormone, high- mutation. Hum Mol Genet 1998; 7: 1407–1410. dose calcitriol and prednisone and recently with recombi- 10 Abinun M, Newson T, Rowe PW et al. Importance of neuro- nant human interferon gamma. However, the only curative logical assessment before bone marrow transplantation for approach known today for osteopetrosis is allogenic osteopetrosis. Arch Dis Child 1990; 80: 273–274. BMT.13–18 Only four of the nine patients who underwent 11 Ruben JB, Morris RJ, Judisch GF. Chorioretinal degeneration BMT in our group survived although eight of the nine in infantile malignant osteopetrosis. Am J Ophthalmol 1990; 110: 1–5. patients achieved primary engraftment. According to our 12 Al-Mefty O, Fox JL, Al-Rodhan N, Dew JH. Optic nerve experience and as previously described, the major trans- decompression in osteopetrosis. J Neurosurg 1988; 68: 80–84. plant-related complications are interstitial pneumonitis and 13 Eapen M, Davies SM, Ramsay NKC, Orchard PJ. Hematopo- alveolar hemorrhage, leading to respiratory failure and ietic stem cell transplantation for infantile osteopetrosis. Bone death.4,6,13 These complications could well be attributed to Marrow Transplant 1998; 22: 941–946.

Bone Marrow Transplantation Osteopetrosis and bone marrow transplantation J Kapelushnik et al 132 14 Coccia PF, Krivit W, Cervenka J et al. Successful bone mar- 17 Key LL, Ries WL, Rodriguiz RM, Hatcher HC. Recombinant row transplantation for infantile malignant osteopetrosis. New human interferon gamma therapy for osteopetrosis. J Pediatr Engl J Med 1980; 302: 701–708. 1992; 121: 119–124. 15 Key L, Carnes D, Cole S et al. Treatment of congenital osteo- 18 Li YP, Chen W, Liang Y et al. Atp6i-deﬁcient mice exhibit petrosis with high-dose calcitrol. New Engl J Med 1984; 310: severe osteopetrosis due to loss of osteoclast-mediated extra- 409–415. cellular acidiﬁcation. Nat Genet 1999; 23: 447–451. 16 Aarskog D, Asknes L, Haneberg B, Julshamn K. Acute response of parathyroid hormone in congenital osteopetrosis. Acta Paediatr Scand 1979; Suppl. 277: 75–80.

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