Severe Malignant Osteopetrosis Caused by a GL Gene Mutation


  • The authors have no conflict of interest


Infantile malignant autosomal recessive osteopetrosis is a genetically heterogeneous disease caused by the inability of OCLs to resorb and remodel bone, resulting in generalized osteosclerosis and obliteration of marrow spaces and cranial foramina. The classical clinical features are pathological fractures, visual impairment, and bone marrow failure.

Two human genes have been described as the cause of this form of osteopetrosis: the T-cell immune-regulator-1 (TCIRG1) gene, which is mutated in >50% of the patients, and the chloride channel 7 (ClCN7) gene, which accounts for ∼10% of cases. We report the clinical, radiographic, and histopathologic findings of the first human osteopetrosis case caused by a mutation in the grey-lethal (GL) gene. The patient, a 9-day-old male infant, presented with a very severe osteopetrotic phenotype including substantial hepatosplenomegaly since birth, cytopenia, and progressive major liver failure. Skeletal radiographs revealed a generalized increase in bone density with loss of corticomedullary differentiation. Histopathologic bone examination showed the typical osteopetrotic changes, with absence of resorptive activity, and osteoclasts, slightly decreased in number, with evident morphological alterations.


INFANTILE MALIGNANT AUTOSOMAL recessive osteopetrosis (ARO; MIM 259700) is a severe, rare bone disorder characterized by defective osteoclast (OCL) function resulting in decreased bone resorption and generalized osteosclerosis.(1) It generally presents in the first year of life, usually within the first 3 months.(1,2) Defective resorption leads to both the development of densely sclerotic fragile bones and progressive obliteration of the marrow spaces and cranial foramina. Marrow obliteration, typically associated with extramedullary hemopoiesis and hepatosplenomegaly, results in anemia and thrombocytopenia; nerve entrapment accounts for progressive blindness and hearing loss.(1) Other major manifestations are failure to thrive, pathological fractures, and increased infection rate caused by defective neutrophil superoxide function.(3)

The natural course of ARO is characterized by early mortality: only 30% of children are still alive at the age of 6 years, the mortality rate being higher in the first 2 years of life. The main causes of death are severe bone marrow failure and overwhelming infections.(1,2,4) Bone marrow transplantation (BMT) is at present the only treatment that significantly alters the course of the disease, and it should be offered as early as possible, preferably before 3 months of age; BMT both corrects the osseous and hematologic abnormalities and prevents the neurological complications caused by nerve entrapment.(5–8)

Two genes, T-cell immune-regulator-1 (TCIRG1; also called Atp6a3) and chloride channel 7 (ClCN7), both necessary for the ability of the OCL ruffled border to generate acidification in the extracellular environment, have been involved in human ARO. TCIRG1, encoding the OCL-specific α3 subunit of the vacuolar proton pump (VPP), which mediates the acidification of the bone/OCL interface, is mutated in >50% of patients.(9–12)ClCN-7, a chloride channel that acts in concert with VPP by providing an electrical shunt for the proton pump, is altered in ∼10% of patients;(13–15) it also accounts for some cases of autosomal dominant osteopetrosis (ADO; MIM:166600).

A spontaneous mouse model, the gl (grey-lethal) mutant, closely resembles human ARO and has proved great interest in its molecular and histopathological dissection.(16) A recent characterization of the gl mutation has shown that a mutation in the human GL gene, coding for a cytoplasmic protein involved in OCL functional activity, may underlie the development of a severe form of human ARO.(17) We report the clinical, histopathologic, and radiographic findings of the first case of human ARO caused by a GL gene mutation.


A 9-day-old male infant with severe hepatosplenomegaly and thrombocytopenia was referred to our institution. He had been born at term from the first gestation of apparently non-consanguineous parents. Delivery was by cesarean section for acute fetal distress, with Apgar scores of 1, 2, and 5 at 1, 5, and 10 minutes, respectively; in the first days of life, the patient underwent mechanical ventilation for acute respiratory failure. On the second day, he developed generalized seizures, for which pharmacologic treatment was started.

On admission, the infant was jaundiced, with generalized hypotonia. Physical examination revealed abdominal distension and marked hepatosplenomegaly: both liver and spleen were 6 cm below the costal margin. Lungs were clear, and the heart examination was normal. Oxygen saturation was good in an oxygen-enriched atmosphere.

Laboratory data showed severe thrombocytopenia, direct hyperbilirubinemia, and high serum aspartate-aminotransferase (AST), alanine-aminotransferase (ALT), and lactate dehydrogenase (LDH) levels, a slight increase of alkaline phosphatase (ALP), and mild hypocalcemia. Serum sodium, potassium, phosphorus, creatinine, glucose, blood gases, hemoglobin, prothrombin activity, and partial thromboplastin time were normal. No serologic evidence of infection was detected.

Ultrasonographic examination of the abdomen confirmed the marked hepatosplenomegaly without ascites and revealed the normal appearance and blood flow of the inferior vena cava and hepatic and portal veins.

There were no echocardiographic and ophthalmologic abnormalities. A CT scan of the cranium and central nervous system revealed mild ventricular dilation and diffuse hypodensity of the white substance.

Skeletal radiographs revealed a generalized increase in bone density, with loss of corticomedullary differentiation and intense sclerosis of the skull and vertebrae. Skeletal, and particularly long bone, development was also harmonic in relation to neonatal age (Fig. 1).

Figure FIG. 1..

Skeletal radiographs. Generalized increase in bone density and loss of corticomedullary differentiation. (A) Predominant changes in the metaphyseal regions of the long bones of the limbs, with typical irregular and flaring aspect consistent with secondary rickets (metaphyseal maximum transversal diameter: distal femur = 2.2 cm; distal radius = 1 cm; proximal tibia = 1.8 cm). Béclard nucleus absent. (B) Ribs are uniformly sclerotic; both the vertebral bodies and the appendages are sclerotic and exaggerate the normal anterior vascular notches. (C) The frontal radiograph shows the typical “harlequin mask” or “space alien face” appearance, caused by the enlargement of calvaria and upward slating of the greater sphenoid alae. The skull's base is markedly sclerotic; the “prominent eyebrow” appearance is a consequence of sclerosis in the floor of the anterior cranial fossa. A milder thickening involves the calvaria. Mandible is hypoplastic and dental development is abnormal. The paranasal sinuses are poorly pneumatized.

The bone marrow was hypocellular; neither atypical cells nor elements suggesting a storage disease were found. Malignant ARO was diagnosed on these grounds.

During the subsequent 3 weeks, the organomegaly increased, and the infant developed anemia, respiratory infection, failure to thrive, and progressive hepatic insufficiency with ascites and coagulopathy.

He was treated with antibiotics, corticosteroids, calcium gluconate, calcitriol, vitamin K, and albumin, and received several plasma, platelet, and erythrocyte transfusions; total parenteral nutrition was necessary because of vomiting and gastrointestinal bleeding. The patient died on the 31st day of life because of multiorgan failure.

Mutation analysis and prenatal diagnosis

Sequence analysis of the patient's genomic DNA, extracted from peripheral blood lymphocytes, showed no mutations in the two genes known to be responsible for osteopetrosis (TCIRG1 and ClCN-7). A search for mutations in the GL gene, the human homolog of the murine gene inducing the gl phenotype, disclosed a homozygous mutation consisting of a G→A substitution at position +5 of the donor splice site of intron V (IVS5 +5 G→A), which leads to the skipping of exon 5 and produces two aberrant splicing transcripts, with consequent hypothetical aberrant proteins.(17) The mutation was ruled out as a polymorphism in 100N chromosomes. Parents' DNA was analyzed after informed consent; the parents were heterozygous for the mutation, and both asymptomatic, consistent with the recessive inheritance of the disease. Identification of the GL mutation also allowed prenatal diagnosis in a successive pregnancy; the fetus was found to be homozygous normal (Fig. 2).

Figure FIG. 2..

Prenatal diagnosis. Because the mutation in the GL gene creates a novel restriction site, the mutated allele is susceptible to restriction enzyme digestion and produces a new low molecular weight band. The patient is homozygous for the mutation; the fetus is homozygous for the wildtype allele, and both parents are heterozygous.

Histopathological findings

General postmortem examination showed a thin malnourished newborn. Abdomen was enlarged and resulted in a frog-like (batrachian) appearance. A copious ascitic effusion, partially hemorrhagic, was present in the abdominal cavity. Liver and spleen were markedly enlarged: liver weight was 281 g and spleen weight was 144 g. Liver had a deep green color; external surface was smooth and cut section of the green parenchyma was mottled with red-brown areas. Spleen had a smooth surface with intact capsule; the cut section was reddish-brown with abundant diffluent red pulp. No white pulp was apparent. No significant alterations of thoracic organs were detected. Scalp, skull, and meninx were regular. Brain weight was 328 g. Cerebral cortex showed regular gyration. The brain was fixed “in toto” and cut after fixation; no gross alterations were detected.

Microscopic examination showed profound disarray of the liver structure: the portal spaces were barely detectable and ductular metaplasia was present at their periphery. Liver cell plates were diffusely replaced by hemopoietic cells, mostly of the erythropoietic lineage, whereas myeloid cells were present in the portal areas. Diffuse fibrosis replaced the liver cell plates (Fig. 3).

Figure FIG. 3..

Liver microscopic examination. A portal tract is barely recognizable. Hematopoietic cells infiltrate the portal area and diffusely replace the parenchyma interrupting the liver cells plates. Ductular proliferation is evident. Hematoxylin-eosin staining.

Very striking microscopic alterations were present, as expected, in bones. Cartilage was of normal appearance, but endochondral ossification was abnormal: a persistent cartilaginous matrix was surrounded by osseous deposition of woven bone. No morphological signs of cartilage and bone resorption were detected; no resorption lacunae were present. OCLs were decreased in number and presented marked morphological alterations. First, their shape was unusual: they were irregularly elongated and devoid of the classic round outline; next, they presented anomalous localization near capillaries of the residual marrow spaces instead of in close contact to bone.

The alterations were less striking in membranous ossification, but in both types of bone formation, an abnormal thickness of trabeculae with no signs of active bone remodeling and resorption were seen. The abnormal bone deposition dramatically reduced bone marrow spaces (Figs. 4A–4C).

Figure FIG. 4..

Bone microscopic examination. (A) Ossification center in the sternum shows irregular seams of calcified cartilage with scalloped outlines embedded in rimmed by osseous depositions. No maturations of bone is present in central and more aged portions of the nucleus (hematoxylin-eosin staining of undecalcified tissue). (B) Trabecular bone. Cartilage is still present and occasional clusters of osteoclasts are detected, mainly in a pericapillary position and far from bone surface. The shape of osteoclasts is abnormally elongated. No activity of bone reabsorption is noted (hematoxylin-eosin staining). (C) Normal osteoclasts from a control case. (D) Interface between trabecular and compact bone (thoracic vertebral body): the limit between the two types of bone is very sharp, with artifactual clefts (arrows).

Compact bone at surface of bone segment was increased in thickness, with tapering toward the epiphysis. Interface between trabecular and compact bone was very sharp with occasional cleft. These clefts probably are handling artifacts but reflect a fragile region in the bone segment.

A defect of bone remodeling is the most probable explanation of these peculiar effects of sudden transition from compact to trabecular bone.

In compact bone, few OCLs were present, with only an occasional aspect of “tunneling”; this indicates very limited and localized bone remodeling and reabsorption (Fig. 4D).

Histopathologic examination of the brain showed normal laminar disposition of neurons in the cortex and no signs of neuronal storage disease. Conversely, the white matter presented increased cellularity, with astrocytes that had enlarged eosinophilic cytoplasm singularly scattered in the parenchyma, or with vague perivascular aggregation. Myelinization was diffusely decreased, as shown by faint staining with Luxol fast blue. No alterations were detected in cerebellum and brain stem (Fig. 5).

Figure FIG. 5..

Brain white matter microscopic examination. A slight increase in cellularity is noted with prominent reactive astrocytes with a vague pericapillary clustering. Astrocytes have bright eosinophilic cytoplasm and few cellular processes. Hematoxylin-eosin staining.


Infantile ARO is a rare, genetically heterogeneous disorder of bone metabolism, caused by the failure of OCL to resorb immature bones and characterized by a wide spectrum of clinical manifestations, including pathological fractures, marrow failure, and immunologic and neurologic deficits.

We describe the clinical, radiologic, and histopathologic findings in the first case of ARO associated with a GL gene mutation. The patient showed a homozygous point mutation causing a skipping of exon 5; both parents were heterozygous for the mutation. A thorough analysis of the parents' descent showed that they could indeed be consanguineous, because they came from a small nomad community of Italian carousel operators, where inbreeding is frequent.

The patient presented a very severe osteopetrotic phenotype, including substantial hepatosplenomegaly since birth, cytopenia, and progressive major liver failure. Radiographic scanning of the skeleton in the second week of life revealed a generalized increase in bone density with loss of corticomedullary differentiation. Histopathologic evaluation of bone showed, as expected, the osteopetrotic changes, with absence of morphological signs of resorptive activity, lack of Howship's lacunae, and strikingly reduced marrow spaces. OCLs were slightly decreased in number but showed evident morphological alterations. Particularly interesting is their elongated shape and the preferential pericapillary localization. Elongated OCLs have been observed in “in vitro” osteoclastogenesis tests from gl mice; gl OCLs have shown a defect in cytoskeletal reorganization, with lack of podosome formation and defective formation of actin rings and underdevelopment of the ruffled border.(16) We suggest that the human GL gene may also play a role in cytoskeletal function: its mutation would therefore explain the atypical OCL shape, and possibly, the migration defect with prevalent perivascular localization, even if anomalous recruitment and attachment to the bone surface are not present in the mouse model.

Particularly interesting is also the sharp transition from the compact to the trabecular component in the growing bone. Several factors may be considered: the lack of reabsorption of trabecular bone may impinge on cortical bone deposition, and cortical bone itself appeared of abnormal thickness and with very limited remodeling signs. This collision may also contribute to bone deformation as it is seen in older patients.

Furthermore, the interface is a “locus minoris resistantiae” in the abnormally thick bone and may itself contribute to bone segment deformity and possibly to pathological fracture. The persistence of cartilaginous seams embedded in trabecular bone may not be caused by an inability of cartilage catabolism but by the deposition of trabecular bone in the surface, and the lack of remodeling may protect cartilage from degradation.

Liver histopathologic alterations were dramatic, with brisk extramedullary hemopoiesis, marked interstitial fibrosis, and reduction of liver cell plates, thus leading to liver failure and ascitic fluid formation. Hepatic dysfunction was identified as the primary cause of death.

Generalized neurologic impairment in children with osteopetrosis ranges from mild central nervous system abnormalities to severe psychomotor retardation. Pathogenesis of the neurological damage is often unclear, and it is difficult to distinguish primary neurological changes directly related to the genetic defect and secondary effects caused by skull abnormalities.(14) Several manifestations, such as blindness and deafness, are usually attributed to nerve and vein compression, but primary retinal degeneration also causes visual impairment.(1,18) Neuronal storage disease in some osteopetrotic patients characterizes a subgroup who develop infantile neuroaxonal dystrophy (MIM 600392).(19–21)

Our patient developed generalized hypotonia and seizures, which were not attributable to his mild hypocalcemia, in the first days of life. No obliteration of cranial foramina was detected by radiological evaluation. Postmortem histopathologic analysis did not reveal either accumulation of neuronal ceroid lipofuscin or formation of axonal spheroids; a generalized white matter gliosis was detected, possibly because of pre-perinatal hypoxic injury. Anemia was not present at birth, and thus prenatal anemic insult could be excluded as the cause of hypoxia; an obstetrical complication may account for an acute brain hypoperfusion with secondary white matter damage, even if neither prematurity nor other evident obstetric risk factors were present. A metabolic cause of the brain damage caused by liver insufficiency cannot be ruled out, but seems to be improbable because no similar alterations were found in other conditions characterized by massive extramedullary hemopoiesis, such as severe neonatal hemolytic anemias.

Because the GL protein is highly expressed in human brain tissue (J Vacher, unpublished data, 2003), the GL gene defect may directly account for the neurologic damage in our patient. Identification of further patients with ARO associated with GL alterations will result in better definition of the clinical aspects of this form of malignant osteopetrosis. Further studies are required to clarify the pathogenesis of neurological damage. The implications of this issue for therapy are clear, because BMT, while successful in correcting the osseous and hematological abnormalities, would not influence the course of a primary neurodegeneration.(5)


This study was partially supported by MIUR cofin 2003 grant to UR, Canadian Institutes of Health Research Grant MOP440790 to JV, Project FIRB RBNE019J9W, Progetto Nazionale sulle Cellule Staminali (conv CS3 to PV), and Cofin 2003 “Malattie ereditarie dell'osso: basi genetiche e terapia cellulare.” Dr E Viora is thanked for villus sampling.