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Keywords:

  • autosomal dominant osteopetrosis;
  • chromosome 11;
  • LRP5;
  • TCIRG1

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. Acknowledgements
  5. REFERENCES

The osteopetroses are a heterogeneous group of genetic conditions characterized by increased bone density due to impaired bone resorption by osteoclasts. Within the autosomal dominant form of osteopetrosis, the radiological type I (ADOI) is characterized by a generalized osteosclerosis, most pronounced at the cranial vault. The patients are often asymptomatic but some suffer from pain and hearing loss. ADOI is the only type of osteopetrosis not associated with an increased fracture rate. Linkage analysis in two families with ADOI from Danish origin enabled us to assign the disease-causing gene to chromosome 11q12-13. A summated maximum lod score of +6.54 was obtained with marker D11S1889 and key recombinants allowed delineation of a candidate region of 6.6 cM between markers D11S1765 and D11S4113. Previously, genes causing other conditions with abnormal bone density have been identified from this chromosomal region. The TCIRG1gene was shown to underly autosomal recessive osteopetrosis (ARO), and, recently, mutations in the LRP5gene were found both in the osteoporosis-pseudoglioma syndrome and the high bone mass trait. Because both genes map within the candidate region for ADOI, it can not be excluded that ADOI is caused by mutations in either the TCIRG1or the LRP5gene.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. Acknowledgements
  5. REFERENCES

BONE IS a dynamic tissue that is remodeled constantly by osteoblastic bone formation and bone resorption by osteoclasts. Disturbance of the balance between these two processes can result in a broad spectrum of pathological conditions. One category of such conditions is the osteopetroses grouped by a shared underlying pathogenic mechanism, that is, impaired bone resorption.(1)

Different types of osteopetrosis are described both in humans and in animals.(2,3) Despite the fact that several genes have been implicated in either induced or spontaneous osteopetrotic animal models,(4) in humans, currently, only three genes have been associated with osteopetrosis. The gene coding for carbonic anhydrase II has been shown to cause a recessive form of osteopetrosis that is associated with renal tubular acidosis and cerebral calcification (MIM 259710).(5) Recently, the malignant recessive form (MIM 259700), in most cases lethal at early age, is proven to be caused by mutations in the TCIRG1 gene in a subset of cases(6,7) and in a few patients in the ClCN7 gene.(8,9)

The autosomal dominant form of osteopetrosis (ADO; MIM 166600) is characterized by bone pain, back pain, and fractures sometimes associated with complications such as osteomyelitis and cranial nerve compression causing facial nerve palsy and vision and hearing problems.(10) However, a high percentage of patients are asymptomatic and only diagnosed by incidental radiography. Based on radiological and clinical data, two subtypes (ADOI and ADOII) were recognized.(2,11) ADOII, the form originally described by Albers-Schönberg,(12) is characterized radiologically by a sandwich-like appearance of the spine because of thickening of the end plates of the vertebral bodies (“Rugger-Jersey spine”) and endobone structures. ADOI is characterized by a generalized diffuse osteosclerosis, which is most pronounced at the cranial vault (Figs. 1,2,3, and 4). This type presents as a fully penetrant disease, compared with type II where the penetrance has been estimated between 60 and 80%.(13) No increased fracture rate is present in these patients.

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Figure FIG. 1.. X-ray from individual III9 (family A) at the age of 34 years. Lateral radiograph of the skull shows uniform sclerosis of calvarium and the skull base with an enlargement of the cranial vault. There is obliteration of the frontal sinuses and mastoids.

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Figure FIG. 2.. Plain radiograph of the lumbar spine (lateral view) from the same individual as in Fig. 1. There is generalized increased radiopacity of the vertebral bodies and the posterior elements of the spine because of osteosclerosis.

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Figure FIG. 3.. Radiograph of the pelvis (AP view). Sclerosis is seen of the vertebral and pelvic bones, the sacrum, and proximal femora.

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Figure FIG. 4.. Plain radiograph of the right femur shows bone sclerosis with narrowing of the medullary canal.

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Because no gene or genetic localization for ADOI has been reported, we performed a linkage study on two extended families of Danish origin both originating from the county of Fyn in Denmark (Figs. 5 and 6). Blood samples were taken from 25 individuals in family A and from 8 individuals in family B after obtaining informed consent. Patients were diagnosed based on the presence of osteosclerosis on X-rays and for some individuals bone mineral density (BMD) measurements. The previously reported family A(11) is updated in this study with the diagnosis of ADOI in two more individuals. Individual III2 (II2 in previous study) was not radiologically examined before, but radiological evidence for ADOI was now obtained. The diagnosis of ADOI in one of his daughters already made him an obligate carrier of the disease gene. Individual IV6 (III6 before) was very young at the time of the previous study. X-rays taken now were still inconclusive because of the young age of the girl. Therefore, bone mineral content (BMC)/BMD measurements were performed. The proximal femoral BMD value was +3.6 SD above normal of the same age (Z score) and clearly indicates this individual as being affected. In comparison with a previous study, 4 extra individuals at risk were studied. Three of these individuals, a father and 2 daughters (III4, IV3, and IV4) clearly show the radiological signs of ADOI. The 4th individual (IV7) is a young boy at the age of 7 years. Because the X-rays taken from him were not completely conclusive, BMC/BMD measurements were performed. His age-adjusted BMD of the spine was high but within the normal range (Z score, +1,8), making a definite conclusion impossible.

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Figure FIG. 5.. Pedigree of the Danish ADOI family A and associated chromosome 11q12-13 haplotypes. Men are represented by squares, women are represented by circles. Blackened symbols represent affected individuals. The haplotypes segregating with the disease phenotype are indicated with blackened bars. The haplotypes between brackets are deduced from results obtained in close relatives.

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Figure FIG. 6.. Pedigree of the Danish ADOI family B with associated chromosome 11q12-13 haplotypes.

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Family B has not been described before. This four-generation pedigree (Fig. 6) also originates from the county of Fyn, Denmark. They were all asymptomatic but otoneurological examinations have shown mild entrapment of cranial nerves. The radiological pictures of the patients are very similar to those from patients of family A. Six patients were diagnosed in this family and 1 already deceased individual was reported to be affected. In total over the two families, 20 patients are diagnosed or reported to be affected including 9 men and 11 women. No evidence is available for nonpenetrance.

To perform genetic linkage studies, DNA was isolated from fresh leukocytes using standard procedures. Markers from the region of interest on chromosome 11 were selected from the Généthon Genetic Linkage Map.(14) One oligonucleotide from each pair of primers was labeled with T7 polynucleotide kinase before polymerase chain reaction (PCR). Amplified products were separated by size on a 6% polyacrylamide gel and visualized by autoradiography. Two point log of the odds (LOD) scores were calculated using MLINK version 5.1.(15) The disease frequency was set at 1/100,000 and an autosomal dominant mode of inheritance with 100% penetrance was assumed. Allele frequencies for the markers were set at 1/n with n being the number of alleles reported by Généthon.(14)

The chromosome 11q12-13 region is one of the priority regions analyzed in the two families because of the previous assignments of genes involved in bone-related diseases. As shown in Table 1, results obtained in family A provide clear evidence for linkage between ADOI and this chromosomal region. A maximum LOD score of +5.42 was obtained with markers D11S4113 and D11S4136 and LOD scores above +3 were obtained with five other markers. Analysis of the smaller family B confirmed this linkage for marker D11S4191 with a maximum LOD score above +2.00. The maximum summated LOD score over the two families is +6.54 for marker D11S1889 (Table 1).

Table Table 1.. Pairwise LOD Scores Between ADOI and Markers From Chromosome 11q12-13
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By haplotyping the markers on the pedigrees (Figs. 5 and 6), cosegregation between this chromosomal region and the disease is visualized. Moreover, recombinants enable us to delineate the candidate region. In family A, a recombination event on the proximal side can be deduced for patient II1 and is transmitted to his descendants. The recombination expands proximal of marker D11S1765 and marker D11S4205 possibly is involved (Fig. 5). Another recombinant on the proximal side only involving markers D11S4102, D11S905, and D11S1763 can be seen in individual III9 and her 3 children. On the distal side, individual IV4 recombines for markers D11S1314 and D11S4207. In family B, markers D11S4102 and D11S905 recombine in individual III1 and on the distal side recombinants are seen for all markers starting with D11S4113 in individuals III3 and III6 and their descendants. Lack of informativity makes a conclusion regarding the involvement or markers D11S913 and D11S4178 impossible (Fig. 6).

Altogether, the key recombinants from both families delineate a candidate region of 6.6 cM flanked by D11S1765 on the proximal side and by D11S4113 on the distal side (Fig. 7). Comparison of the haplotypes cosegregating with the disease in both families indicate that both families are related because a shared haplotype for the complete affected chromosomal region analyzed can be deduced for individuals I1 in both pedigrees (Figs. 5 and 6).

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Figure FIG. 7.. Delineation of the candidate region for ADOI and relative position of the TCIRG1 gene and the LRP5 gene. Idiogram of chromosome 11 and position of the markers used are shown. Intermarker distances in centimorgans were obtained from the Généthon linkage map.(14) In the right panel, the black bars indicate the delineated candidate region for ADOI and the position of both genes that are considered to be candidate genes for ADOI.

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The reason for testing the candidacy of this chromosomal region for harboring the ADOI gene was based on the fact that genes for several conditions associated with an abnormal bone density are identified from this chromosomal region. More precisely, the TCIRG1 gene underlying the malignant autosomal recessive form of osteopetrosis (ARO)(6,7) and the LRP5 gene causing both the osteoporosis-pseudoglioma syndrome (OPS), a recessive condition with juvenile-onset osteoporosis,(16,17) and a high bone mass (HBM) phenotype.(18) Both genes map close to each other within the region that could not be excluded for harboring the ADOI gene. D11S4178 is a marker flanking the LRP5 gene(18) but is not informative in the key recombination event in our family (Fig. 5). Therefore, we analyzed three other markers intragenic or close to LRP5 (D11S1917, D11S4087, and D11S1337) but none of these was informative for the key recombination event (data not shown). This implies that currently both the TCIRG1 gene and the LRP5 gene can be considered candidate genes for ADOI. In the first case, this would be a second example of an autosomal recessive osteopetrosis being allelic with a dominant form after our recently reported evidence that mutations in the ClCN7 gene underlie ADOII.(9) Alternatively, ADOI could be caused by mutations in the LRP5 gene making it allelic to OPS and HBM. The radiological features of ADOI and HBM are strikingly similar but clinically HBM patients do not have any complaints and are fully asymptomatic(19) whereas at least some ADOI patients suffer from severe pain.(10) Whether a shared pathogenic mechanism can underlie both conditions is at this point difficult to conclude. ADOI is caused by an impaired bone resorption with a markedly reduced number and size of osteoclasts(20) and no such data currently is available for the HBM phenotype. Mutation analysis of both the TCIRG1 gene and the LRP5 gene will reveal whether mutations in any of both are underlying ADOI or whether another, still unidentified, gene might be involved.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. Acknowledgements
  5. REFERENCES

This research was supported by a concerted-action grant from the University of Antwerp (to W.V.H.) and a grant (G.0404.00) from the “Fonds voor Wetenschappelijk onderzoek” (F.W.O. to W.V.H.) and an Interuniversity Attraction Pole grant to W.V.H. Dr. Van Wesenbeeck and Dr. Mathysen hold a predoctoral research position with the “Vlaams instituut voor de bevordering van het wetenschappelijk-technologisch onderzoek in de industrie.”

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. Acknowledgements
  5. REFERENCES
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