Tracing the Genetic Origins of Osteonecrosis of the Femoral Head

Tracing the Genetic Origins of Osteonecrosis of the Femoral Head Wei-Ming Chen, MD,*,† Yu-Fen Liu, PhD,‡ and Shih-Feng Tsai, MD, PhD‡,§,储 Osteonecrosis of the femoral head (ONFH), occurring in children or adults, rarely shows familial aggregation. Recent reports from Taiwan and Japan, however, indicate that an inherited form of ONFH is associated with mutation of the type II collagen gene (COL2A1). These patients had not been exposed to known environmental risk factors, such as steroid medications, yet they presented with typical clinical and radiographic features of ONFH. Unlike other genetic disorders due to COL2A1 mutations, these ONFH cases have normal skeletal development before disease onset. The clinical manifestation of COL2A1-associated ONFH varies between pedigrees, and it appears that age of onset has a major effect on disease phenotype, reflecting different degrees of bone regeneration concurrent with bone damage in the hip joint. Although the inherited form of ONFH is rare, it provides an opportunity to study the natural history and pathogenetic mechanism of ONFH. Genetic approach for investigating ONFH is reviewed here, highlighting our current understanding of the many possible causes of ONFH, and at the end, casting a realistic projection on how genetics can advance the development for managing this debilitating disease. Semin Arthro 18:175-179 © 2007 Elsevier Inc. All rights reserved. KEYWORDS collagen, genetic, bone damage, bone regeneration

O

steonecrosis of femoral head (ONFH) results from a variety of etiologies but its pathophysiology has not been completely elucidated1 despite the existence of several theories regarding the causes of the disease.2 For osteonecrosis presenting as a late complication of femoral neck fracture, ie, traumatic osteonecrosis due to disrupted vascular supply as a consequence of damaged joint capsule during fracture, the incidence is not particularly different in Taiwan compared with the reports from other countries. However, from our observation, the prevalence of nontraumatic osteonecrosis as an indication for total hip arthroplasty (THA) seems higher in Taiwan as opposed to North American and European countries as it comprises more than 50% of the patients

*Department of Surgery and Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei. †Department of Orthopedics and Traumatology, Taipei Veterans General Hospital, Taipei. ‡Institute of Genome Sciences and Genome Research Center, National YangMing University, Taipei. §Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli. 储Institute of Molecular Medicine, National Tsing-Hua University, Hsinchu. Address reprint requests to Shih-Feng Tsai, MD, PhD, Division of Molecular and Genomic Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan Town, Miaoli County 350, Taiwan. E-mail: petsai@ nhri.org.tw

1045-4527/07/$-see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1053/j.sart.2007.06.003

receiving THA (hip replacement data, Taiwan National Health Insurance Bureau). Similarly, the proportion of THAs performed for nontraumatic osteonecrosis are also reported to be higher in neighboring Asian countries, including Japan and Korea. This is quite different from the situation in the non-Asian populations. In Sweden, data from the Hip Arthroplasty Register (www. jru.orthop.gu.se) in 2005 reveal that the majority of the patients received THA due to degenerative or traumatic arthritis (85%) as opposed to femoral head necrosis (2.2%). The disparity in indications for THA among different ethnic groups, particularly Asian, suggests that genetic factors may play an important role in the pathogenesis of nontraumatic osteonecrosis. A substantial proportion of patients with nontraumatic osteonecrosis are associated with known etiologic factors, for example, alcoholism or steroid medications. However, only a small portion of patients with alcoholism or receiving steroid treatment develop osteonecrosis. These findings suggest that nontraumatic ONFH may be influenced by genetic background and, if so, the disease could be heritable in rare occurence. On this basis, efforts have been made in our clinics to identify familial traits in patients with nontraumatic osteonecrosis. Also, we have sought to identify candidate genes associated with nontraumatic osteonecrosis in the sporadic 175

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W.-M. Chen, Y.-F. Liu, and S.-F. Tsai hematological disorders (see related paper in this issue by Glueck and coworkers). In this review, we focus on the recent findings by Liu and coworkers15 and Miyamoto and coworkers16 on the type II collagen variants in adult and pediatric ONFH.

COL2A1 and Inherited ONFH

Figure 1 Multiple ethiologies of osteonecrosis of the femoral head (ONFH). ONFH occurs when the bone regeneration process is insufficient to repair the bone damage caused by physical or metabolic stress. Both genetic and environmental factors contribute to the disease pathogenesis.

cases with different severities and etiologies. We envision the possibility of identifying marker genes that correlate with disease risk and predict disease outcome. Once the marker genes become available, screening can be implemented to identify high-risk individuals, enabling earlier proactive investigation and treatment.

Is ONFH Genetic? Like many common diseases, ONFH is multifactorial, and the development of the disease depends on the interactions of many genetic and environmental factors (Fig. 1). Several outstanding reviews are readily available and they have summarized our current thinking on the pathogenic mechanism(s) of ONFH.1,3 The concept of a multifactorial etiology of idiopathic osteonecrosis as the “accumulated cell stress,”4 and, in recent extrapolation, as “accumulated tissue stress”5 is similar to the theory explaining polygenic traits in human genetics. Under this theory, more than one factor might be involved and, as a result of multiple hits, ONFH develops when a critical threshold is reached. Taking a human genetic approach, critical factors for ONFH pathogenesis could be dissected using the state-of-art genomic technology, but it would require careful planning and execution on patient selection and study design and reasonably large amounts of resources. We reason that a Mendelian disorder of osteonecrosis, when displayed as a simple trait in families, will be useful in unlocking the biochemical and cellular basis underlying the pathogenesis of ONFH. Osteonecrosis results from many different etiologies. Therefore, a so-called “genetic heterogeneity” or “phenocopy” is likely to exist in ONFH, that is, multiple genes and different alleles can give rise to the same disease or diseases with similar phenotypes. In clinical practice, most cases do not have a family history. As shown in Table 1, a variety of medical conditions can lead to the development of ONFH, and multiple sequence changes have been identified in genes involved in the vascular system,6-12 steroid metabolism,13 alcohol metabolism,14 type II collagen structure,15,16 and other underlying diseases,17-19 including

Although the majority of idiopathic ONFH cases are sporadic, three pedigrees that showed an autosomal dominant mode of inheritance were identified in Taiwan.15,20 Initially, we focused on one of the two four-generation pedigrees. Among 75 subjects in the family, there are 5 males and 11 females affected with idiopathic ONFH, including twin sisters. In this pedigree, the mode of inheritance was autosomal dominant and the average age of onset in the 12 patients was 26 years (range: 15-48 years). Index cases and affected relatives from the family presented with symptoms of pain in the groin, and physical examination revealed that they had average height, normal appearance, and normal musculoskeletal system examination without any signs of chondrodysplasia. None of the affected individuals have any evidence of systemic lupus erythematosus, sickle cell disease, or Gaucher’s disease. All but one individual denied ethanol consumption. Subsequently, another family affected with ONFH was also identified, and the two pedigrees were not related to each other. The second family included 77 members in four generations. Among them, 16 (8 males and 8 females) were affected with ONFH, and the age of onset ranged from 12 to 37 years. Applying linkage analysis to the first four-generation pedigree, we excluded linkage between the index family and three genes related to thrombophilia and hypofibrinolysis: protein C, protein S, and plasminogen activator inhibitor. Furthermore, by genome-wide scan, a significant two-point logarithmic odds (LOD) score of 3.45 at ␪ ⫽ 0 was obtained between the index ONFH family and marker D12S85 on chromosome 12. High-resolution mapping was then conducted in the second ONFH family and replicated the linkage to D12S368 (Pedigree I: LOD score 2.47, ␪ ⫽ 0.05; Pedigree II: LOD score 2.81, ␪ ⫽ 0.10). Since the disease only appeared in adults, we also applied an age-dependent penetrance model and the combined multipoint LOD score achieved 6.43 between D12S1663 and D12S85. Thus, we could map the candidate gene for autosomal dominant ONFH to a 15-cM region between D12S1663 and D12S1632 on chromosome 12q13.20 To further define the genetic interval for ONFH in these families, we performed haplotype analysis. From within the 12q13 critical region, we selected several candidates and examined sequence variation in the type II collagen gene (COL2A1) in patients with the inherited form of ONFH. DNA sequence analysis revealed a glycineto-serine mutation in the G–X–Y repeat of type II collagen in all affected individuals in three pedigrees. In two large families, a 3665G⬎A mutation occurred in exon 50 of the COL2A1 gene and the substitution resulted in a Gly1170Ser codon change. Both pedigrees harbor the same mutation but the mutant alleles exist in different haplotype backgrounds.

Tracing the genetic orgins of ONFH

177

Table 1 Genetic Etiology of Osteonecrosis of the Femoral Head Gene Vascular System MTHFRa FVa FIIa NOS3a HIF1␣a

Variant

Location

Codon Change

C677T G1691A G20210A 4a alleled ⴚ2755C>Ab ⴙ41224T>Cb ⴙ45319C>Tb CTCC haplotypec G1691A G455A A20T

Exon 5 Exon 10 3= UTR Intron 4 Promoter Intron 8 Exon 12

Ala222Val Arg506Gln

Exon 10 Promoter Exon 1

Arg506Gln

T389C

Exon 3

Leu129Pro

C3435T G2677T/A

Exon 26 Exon 21

A213G G1951A

Disorder

Reference

ONFH ONFH

Zalavras6 Bjorkman7

Nontraumatic ONFH Idiopathic ONFH in men

Koo8 Hong9

Legg-Perthes disease Legg-Perthes disease Sickle cell disease and ONFH

Glueck10 Dilley11 Milner12

Ile1142Ile Ala893Ser

Steroid-induced ONFH

Asano13

Exon 3 Exn 12

His48Arg Glu504Lys

Alcoholic ONFH

Chao14

G3665A G2306A G3508Ae

Exon 50 Exon 33 Exon 50

Gly1170Ser Gly717Ser Gly1170Ser

ONFH

Liu & Chen15

Legg-Perthes disease

Miyamoto16

A1226G

Exon 9

Asn370Ser

Kenet17

HFEa

G845A

Exon 4

Cys282Tyr

GLAa

C679T

Exon 5

Arg227X

Gaucher disease and Legg-Perthes disease Hemochromatosis (HC) and ONFH Fabry disease and ONFH

FVa FGBa HBBa HBA1a Steroid Metabolism ABCB1a Alcohol Metabolism ADH2a ALDH2a Type II Collagen COL2A1a

COL2A1a Other Conditions GBAa

Glu6Val

Rollot18 Lien19

Data were collected from NCBI [2007] and Human Gene Mutation Database (HGMD) (http://www.hgmd.cf.ac.uk/ac/index.php). aHIF1␣, hypoxia-inducible factor 1 alpha subunit gene; NOS3, nitric oxide synthase 3 (endothelial cell) gene; FV, coagulation factor V (proaccelerin, labile factor) gene; FII, coagulation factor II (thrombin) gene; MTHFR, 5,10-methylene-tetrahydrofolate reductase gene; FGB, fibrinogen beta chain gene; HBB, hemoglobin beta gene; HBA1, hemoglobin alpha 1 gene; ABCB1, ATP-binding cassette, sub-family B (MDR/TAP), member 1 gene; ADH2, alcohol dehydrogenase IB (class I), beta polypeptide gene; ALDH2, aldehyde dehydrogenase 2 family (mitochondrial) gene; COL2A12, collagen type II alpha 1 gene; GLA, galactosidase alpha gene; HFE, hemochromatosis gene; GBA, glucosidase beta; acid (includes glucosylceramidase) gene. bRef. seq.: GenBank accession number NM_001530 and contig NP_851397 (ⴙ1 denotes the first base of the translation start site). cCTCC haplotype constitutes from variant ⴚ2755C>A, ⴙ41224T>C, ⴙ45319C>T and ⴙ51610C>T (3= UTR). d27 base-repeat polymorphism. eRef. seq.: GenBank accession number NM_001844.3 (the A of the first ATG is denoted as ⴙ1).

In another pedigree, a 2306G⬎A mutation occurred in exon 33 of the gene, causing a glycine to serine change at codon 717. Taking together the results of genetic mapping and sequence analysis, we have concluded that COL2A1 mutation is associated with ONFH.15 Although we do not have direct evidence to support that the amino acid substation is the cause of the disease, the known structure and function of type II collagen favor this idea. Legg-Calve-Perthes disease (LCPD) is a common form of ischemic osteonecrosis of the immature femoral head in children.21 Most cases are sporadic, but Miyamoto and coworkers6 have recently reported a family that transmits typical LCPD symptoms with an autosomal dominant mode of inheritance. In this three-generation pedigree, variable phenotypes were observed in affected individuals. Some were associated with typical LCPD and cyclic changes, while others

presented mild involvement and weak regeneration in their femoral heads. Among 12 subjects in this family, 3 males and 2 females were diagnosed as LCPD or mild LCPD, and one affected female was associated with small femoral heads. In contrast to the Taiwanese pedigrees, the average age at diagnosis among the five affected individuals was 11.6 years (range: 5-15 years). None of the patients have any underlying risk factors, including steroid and alcohol use or autoimmune disease. All patients are apparently normal in their vision and hearing system. Instead of taking a genome-wide scan, linkage analysis was performed with five candidate genes, including COL2A1. A disease-associated haplotype was identified between microsatellite markers D12S85 and D12S368, and direct sequencing of the coding and intronic regions of COL2A1 detected a G to A mutation inside exon 50. Again, the substitution resulted in a Gly1170Ser codon

178 change in type II collagen, within the Gly–X–Y repeat domain. Although Miyamoto and coworkers16 cited the nucleotide substation at position 3508 (based on the first ATG as ⫹ 1, equivalent to nucleotide 3665 in our numbering system), the mutation is identical to one of the two COL2A1 genetic variants reported by Liu and coworkers.15

Pathogenetic Implications We were fortunate to identify the COL2A1 mutation as the cause for the three Taiwanese pedigrees of adult-onset ONFH through a positional candidate gene approach.22 With this unusual occurrence, we have gained an opportunity to investigate the natural history and pathogenic mechanism of ONFH. DNA testing allowed us to identify, among the family members, individuals at risk for developing the disease. In fact, two adults originally assigned as normal were found to be carriers of the mutant gene and they subsequently complained about groin pain and a radiograph confirmed the presence of ONFH. Moreover, several carriers of the COL2A1 mutant allele were still young and asymptomatic. These individuals are valuable for us to study the clinical course of the disease, starting from a presymptomatic stage, even before MRI can detect any change. The report by Miyamato and coworkers6 on LCPD in a Japanese kindred is an additional reward of our original finding,15 and it has given new insight about the pathogenesis of osteonecrosis. Although the mutation (G1170S) is the same as in two Taiwanese pedigrees, the Japanese cases present the disease at much younger age when bone growth is still active, and they display as typical pediatric ONFH. Moreover, there appears to be phenotypic different expression of the disease among affected individuals in the Japanese pedigree, suggesting that other factors might modify the clinical presentation of ONFH. Type II collagen is an abundant protein in the human body, and the genetic disorders of the gene, known as type II collagen disorders, are manifold. The clinical spectrum of type II collagen disorders include spondyloepiphyseal dysplasia (SED) with variable severity, Stickler dysplasia type I (STD-I), and Kniest dysplasia (KND).23 An interesting genetic question about the inherited form of ONFH is the origin of the mutations, ie, are these pedigrees related? We have approached this issue by tracing the family name, geographical location, and the use of a genetic detective procedure called “haplotyping.” By applying the DNA barcode, we concluded that the two large Taiwanese pedigrees with the G1170S mutation are not of the same recent ancestry, and they presumably are distinct from the Japanese pedigree in genetic origin. At present, we do not know what other factor(s) might account for the different disease phenotypes seen in our cases15 and those reported by Miyamato and coworkers.16 The fact that in most ONFH the disease will not develop until adulthood suggests that the disease might result from accumulated tissue damage. We propose that a balance of bone damage and repair is essential for maintaining normal hip joint function (Fig. 1), and, as a bipedal animal, body weight can cause constant microtrauma to the femoral head,

W.-M. Chen, Y.-F. Liu, and S.-F. Tsai thus tipping the balance when collagen structure and function are compromised by a glycine-to-serine substitution in the G–X–Y repeat domain. Structural alteration at a different repeat position might have a distinctly different effect on triple helix formation or extracellular matrix interaction, and this can explain the diverse disease manifestation of the type II collagen disorder. The relevance of the COL2A1 gene to noninherited ONFH remains to be examined. We have shown that the disease in familial cases tends to develop at a younger age and it typically involves both hips.15 Could other subtle changes in the COL2A1 gene play a role in the sporadic cases of ONFH? We have sequenced the exonic sequences of the gene for 65 patients without a family history, and we did not find any sequence variation in the coding region that could be of apparent functional consequence.15 Nevertheless, we have not completely ruled out the possibility that dysfunction of type II collagen might occur through regulation of gene expression or other interacting proteins.

Toward Gene-Based Management of ONFH A major benefit brought about by the Human Genome Project is the potential for improving patient management through early detection, prevention, and treatment tailored to individual genetic difference.24 It is well known that the outcome of a patient with osteonecrosis depends on the stage of the lesion,25 and the prognosis of the patient would be more favorable if the diagnosis can be made early. In the three pedigrees we have analyzed, all adults who carry a COL2A1 mutant allele showed radiographic evidence of hip joint collapse, and most of them already had clinical symptoms of groin pain and limping gait. Thus, in genetic terms, the penetrance (defined as the proportion of individuals carrying a mutation that develop the disease) at age older than 30 years is nearly 100%. On the other hand, we can detect carriers of the mutant allele by DNA testing among children in the two large pedigrees who are currently free from any symptoms, but they will eventually develop the disease. The issue is when we should inform the affected individuals (or their parents) and, more importantly, what do we have to offer for these individuals? Regular follow-up in the outpatient department to monitor the onset of ONFH is important. Magnetic resonance imaging of the femoral head is the choice for imaging the hip joint as it is nonradiographic and a proven modality for early detection of the disease. The management of early-stage ONFH, however, remains difficult because of a lack of a universally accepted, effective protocol. The treatment options include protected weight bearing, nonsteroid antiinflammatory drugs, and femoral head sparing surgery, such as core decompression or Sugioka’s osteotomy of proximal femur. One can reasonably expect that, by detecting the disease early and by optimizing the treatment, surgical intervention with prosthetic replacement can be delayed. Thus, further development of treatment for early ONFH is well justified from a

Tracing the genetic orgins of ONFH socioeconomic point of view. Our current understanding of the disease indicates that there are likely to be multiple genetic factors involved and identifying the individuals at risk and classifying the disease based on genetic profiles, therefore, is a high priority for a breakthrough in developing novel therapeutics using the cell or gene therapy approach.24

Acknowledgments The authors thank Dr. Shiro Ikegawa for sharing his findings on LCPD before publication and Drs. Yang and Hong, K. Kuo, and S. Ikegawa for critical reading and valuable comments on this manuscript. The study of ONFH is supported by a grant from the National Research Program for Genomic Medicine of the National Science Council (91-3112-B-075003 to W.-M.C.) and intramural funds from the National Health Research Institutes (to S.-F.T.) and Taipei Veterans General Hospital (to W.-M.C.).

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179 9. Hong JM, Kim TH, Chae SC, et al: Association study of hypoxia inducible factor 1alpha (HIF1alpha) with osteonecrosis of femoral head in a Korean population. Osteoarthritis Cartilage 2007 10. Glueck CJ, Brandt G, Gruppo R, et al: Resistance to activated protein C and Legg-Perthes disease. Clin Orthop Relat Res 338:139-152, 1997 11. Dilley A, Hooper WC, Austin H, et al: The beta fibrinogen gene G-455-A polymorphism is a risk factor for Legg-Perthes disease. J Thromb Haemost 1:2317-2321, 2003 12. Milner PF, Kraus AP, Sebes JI, et al: Sickle cell disease as a cause of osteonecrosis of the femoral head. N Engl J Med 325:1476-1481, 1991 13. Asano T, Takahashi KA, Fujioka M, et al: ABCB1, C3435T, and G2677T/A polymorphism decreased the risk for steroid-induced osteonecrosis of the femoral head after kidney transplantation. Pharmacogenetics 13:675-682, 2003 14. Chao YC, Wang SJ, Chu HC, et al: Investigation of alcohol metabolizing enzyme genes in chinese alcoholics with avascular necrosis of hip joint, pancreatitis and cirrhosis of the liver. Alcohol Alcohol 38:431-436, 2003 15. Liu YF, Chen WM, Lin YF, et al: Type II collagen gene variants and inherited osteonecrosis of the femoral head. N Engl J Med 352:22942301, 2005 16. Miyamoto Y, Matsuda T, Kitoh H, et al: A recurrent mutation in type II collagen gene causes Legg-Calve-Perthes disease in a Japanese family. Hum Genet 2007 17. Kenet G, et al: The 1226G (N370S) Gaucher mutation among patients with Legg-Calve-Perthes disease. Blood Cells Mol Dis 31:72-74, 2003 18. Rollot F, et al: Hemochromatosis and femoral head aseptic osteonecrosis: A nonfortuitous association? J Rheumatol 32:376-378, 2005 19. Lien YH, Lai LW: Bilateral femoral head and distal tibial osteonecrosis in a patient with Fabry disease. Am J Orthop 34:192-194, 2005 20. Chen WM, Liu YF, Lin MW, et al: Autosomal dominant avascular necrosis of femoral head in two Taiwanese pedigrees and linkage to chromosome 12q13. Am J Hum Genet 75:310-317, 2004 21. Thompson GH, Price CT, Roy D, et al: Legg-Calve-Perthes disease: Current concepts. Instr Course Lect 51:367-384, 2002 22. Prockop DJ: Type II collagen and avascular necrosis of the femoral head. N Engl J Med 352:2268-2270, 2005 23. Nishimura G, Haga N, Kitoh H, et al: The phenotypic spectrum of COL2A1 mutations. Hum Mutat 26:36-43, 2005 24. Guttmacher AE, Collins FS: Welcome to the genomic era. N Engl J Med 349:996-998, 2003 25. Hernigou P, Poignard A, Nogier A, et al: Fate of very small asymptomatic stage-I osteonecrotic lesions of the hip. J Bone Joint Surg Am 86A:2589-2593, 2004