Heterotopic ossification (HO) is a pathological condition in which lamellar bone forms at extraskeletal sites, such as skeletal muscle, tendons, ligaments, and fascia.1,2 Heterotopic ossification can be classified as nonhereditary HO (NHHO) or hereditary HO (HHO).2,3 Nonhereditary HO may occur after musculoskeletal trauma, following central nervous system injury, with certain arthropathies, or following injury or surgery that is often sustained in the context of age-related pathology.2
Hereditary HO includes 2 different forms: fibrodysplasia ossificans progressiva (FOP) and progressive osseous heteroplasia (POH).1 Patients with classic FOP present with progressive HO with characteristic anatomic patterns.1,4 Heterotopic bone formation in POH patients occurs in an asymmetric mosaic distribution and is mainly intramembranous.1 Each of these disorders is caused by mutations in a single (different) gene, thereby indicating that these disease-causing genes are critical for regulating bone tissue formation.1 Fibrodysplasia ossificans progressiva is caused by mutations in the ACVR1/ALK2 gene,1,4 whereas POH is caused by a mutation in the GNAS locus.1 Previous studies have demonstrated that the clinical diagnosis of FOP can be confirmed by DNA sequence analysis of the ACVR1 gene.5,6 DNA sequencing can also be used to evaluate suspected cases of atypical FOP or FOP variants.6 Because HO in POH is mainly intramembranous,1 which is distinguished from the affected individuals in this family, the current authors mainly focused this study on ACVR1 gene mutations, rather than GNAS.
In this study, the authors investigated the clinical manifestations of affected individuals in this family and performed pedigree analysis and ACVR1 gene mutational analysis of all the family members and 3 sporadic NHHO patients who did not belong to this family. All members of this family carry 2 homozygous silent mutations in the ACVR1 gene—c.270C>T and c.690G>A—which is inconsistent with previous reports of FOP, especially in Chinese FOP patients.7 As indicated by previous studies, FOP is an autosomal-dominant disorder; most cases arise from spontaneous new autosomal dominant mutations.8,9 There are few reported cases of affected multigenerational families with FOP, and most known cases occur de novo in families.8–12 Considering the available findings of clinical and radiographic features together in this current study, these results may suggest that the affected individuals of this family may not be diagnosed as having FOP.
To the authors' knowledge, this is the first well-documented case of 3 generations of a Chinese family with multifocal and bilateral HO involvement. This study may lead to the identification of a new form of HHO or extend understanding of the FOP-variant syndrome.
Materials and Methods
The detailed clinical features of each affected individual of this family, including a routine medical history and the results of a physical examination and clinically relevant photographic and radiographic analyses, were obtained. This 3-generation Chinese family includes 7 members, 3 males and 4 females, with an average age of 42.3 years. Three sporadic NHHO patients who did not belong to this family included 2 males and 1 female with an average age of 31.4 years. After receiving investigational review board approval, peripheral blood samples were obtained from each member of the family and 3 sporadic NHHO patients after informed consent was obtained.
Exon sequencing was performed on all members of this family as well as the 3 sporadic NHHO patients, who were used to compare the results with this family, to investigate mutations in the ACVR1 gene. Peripheral blood was collected after informed consent for DNA testing was obtained. Genomic DNA was extracted from the peripheral blood samples by using a commercial DNA extraction kit (QIAamp DNA Micro; Qiagen) according to the manufacturer's protocol.
The ACVR1 gene consists of 11 exons, of which the first 2 are noncoding. A total of 9 transcripts are produced from the gene. The information on exons provided in this article refers to transcript ENST00000263640 (Ensembl database). Amplification of the 11 exons of the ACVR1 gene was performed using a set of polymerase chain reaction reagents (CapitalBio Co, Ltd) and specifically designed primers, and this was followed by DNA sequencing on an ABI 3730XL automated sequencer (Applied Biosystems). The data files were imported into SMD software (Sequence Mutation Detector, CapitalBio Co, Ltd) for sequence analysis.
DNA sequence polymorphisms were searched for presence in the National Center for Biotechnology Information (NCBI) database ( http://www.ncbi.nlm.nih.gov/RefSeq) and the NCBI dbSNP database ( http://www.ncbi.nlm.nih.gov/projects/SNP).
The pedigree of the studied family is shown in Figure 1.
Pedigree of the family examined in this study. Standard pedigree notation is used: circles and squares denote females and males, respectively; black symbols indicate affected individuals, which was confirmed by history, physical examination, and/or radiographic studies; and Roman numerals on the left indicate the generation. The age of each family member is presented on the right. An arrow is used to point to the index case (III.1).
The phenotypes of the 4 affected individuals in this family and comparisons with the 3 phenotypes of FOP are summarized in Table 1. Radiographs were obtained, and the findings are presented (Figures 2–4). All of the 4 affected individuals of this family presented normal appearance of the big toes and thumbs.
Comparison Among Patients in the Pedigree and the 3 Phenotypes of Fibrodysplasia Ossificans Progressiva
Presentations of the index patient (III.1). Anteroposterior radiograph of the pelvis. Massive ossification is seen around the right hip and accompanies the ankylosis of the hip joint. The anterior superior iliac spine and anterior posterior iliac spine of the left ilium are also affected (A). Three-dimensional reconstruction computed tomography of the pelvis demonstrating ossification within the anterior and medial of right hip joint, involved iliopsoas, and the medial thigh muscles, including pectineus, gracilis, adductor longus, adductor brevis, and adductor magnus (B). Lateral radiograph of the right elbow showing bony ankylosis formation, bridging the distal humerus to the proximal of radius (C). Anteroposterior radiograph of bilateral legs showing well-defined calcification formatted within the left interosseous membrane (D). Gross appearance of the biopsy specimen of the right hip showing trabecular bone is loosely arranged. There is a rim of calcification at the periphery (E).
Radiograph of the mother of the index patient (II.4). Extensive ossification affects bilateral hips and left iliopsoas muscle. The ossification is bridging from the anterior superior iliac spine to the lesser trochanter and the proximal lateral region of the left thigh. The massive ossification also presents around the right hip and accompanies the ankylosis of the right hip joint (A). Radiographs of the older uncle of the index patient (II.1). He has multifocal ossification affected bilateral hips and elbows, right psoas muscle, right proximal tibia, and right forearm interosseous membrane. He also has bony ankylosis of bilateral hip joints. He had no history of bilateral forearm fractures (B, C).
Radiographs of the younger uncle of the index patient (II.2). The multifocal heterotopic ossification also affected the proximal lateral region of the bilateral thigh and accompanies ankylosis of bilateral hip joints (A). The osteophytes are seen on the posterior aspect of the right distal humerus (B). It is notable that this affected individual experienced a nonunion fracture of the left proximal ulna for more than 20 years after open reduction and internal fixation (C).
History and Physical Examination
The index patient (III.1; 23 years old) was asymptomatic until 19 years old, when she presented with right hip pain after a sprain injury. Two weeks later, she began to experience a decrease in range of motion and function of the hip joint and was subsequently referred to the orthopedic clinic for further evaluation. After 1 month, she developed a limp due to fixation of the right hip. She remained ambulant (with the assistance of a walking stick) for the next 3 months. Her medical history was unremarkable. Physical examination revealed a 45° flexion contracture deformity of the right hip and limited range of motion in all directions. There were fixed palpable masses in the proximal anterior right thigh and right elbow (Figure 2A–D). The gross appearance of the biopsy specimen of the right hip is shown in Figure 2E.
The results of routine laboratory tests (including serum calcium, erythrocyte sedimentation rate [ESR], phosphorus, and alkaline phosphatase) were normal. Furthermore, the endocrine hormone test of the index patient, including triiodothyronine (T3), thyroxine (T4), free triiodothyronine (FT3), free thyroxine (FT4), thyroid-stimulating hormone (TSH), parathyroid hormone (PTH), and antithyroglobulin antibody (TGAB), was normal. There was no evidence of pseudohypoparathyroidism.
The index patient's mother (II.4; 47 years old) also presented with multifocal and bilateral HO and had been asymptomatic until she developed lower back and bilateral hip stiffness after trauma at 23 years old. Her bilateral hip joint also became fixed after trauma, and by the time she was 26 years old, she had developed severely limited ambulation (similarly to her brothers, II.1 and II.2). Eventually, she developed progressive ankylosis of the bilateral hip and HO of the left psoas muscle. Physical examination revealed a 15° flexion contracture deformity of the bilateral hip (Figure 3A).
The index patient's grandmother (I.2; 69 years old) also exhibited HO. At 22 years old, she developed ankylosis of the bilateral hip after trauma. In her 30s, she developed a succession of painless lumps on her back. Later, she required the aid of bilateral crutches to walk. She gradually became more disabled and was bed-bound for 34 years before her death due to pneumonia at 69 years old. Unfortunately, her medical records and radiographs are no longer available.
Two uncles of the index patient also exhibited onset of HO with multifocal and bilateral involvement at 22 years old. The older uncle (II.1; 54 years old) presented with a 10° flexion contracture deformity of the bilateral hip and elbow (Figure 3B–C). The younger uncle (II.2; 52 years old) first experienced pain and stiffness of the bilateral hip joints at 21 years old. After 3 years, he experienced painful limitation of the movement of both hips, but especially the right hip. His radiographs showed extensive HO at the bilateral hip (Figure 4A) and the posterior aspect of the right elbow (Figure 4B). Interestingly, he experienced a left proximal ulnar fracture and underwent open reduction and internal fixation at 31 years old. However, the fracture remained nonunion for more than 20 years. Surprisingly, a radiograph of the left elbow showed no evidence of HO at the fracture site (Figure 4C).
Mutations in ACVR1 Exons
The results of DNA sequencing revealed several mutations in ACVR1 exons in this family and 3 sporadic NHHO patients (Table 2). The classical mutation (c.617GNA; R206H) that occurs in the majority of FOP patients was not identified in DNA extracted from all blood samples of this study, nor were any other of 12 heterozygous ACVR1 missense mutations and 1 3-bp deletion (elimination of 2 amino acids and insertion of a different amino acid) of ACVR1 variant mutation found. Furthermore, no previous studies have demonstrated that all of the 4 mutations in ACVR1 gene are associated with HO.
Mutational Analysis of ACVR1 Exons
The entire family, except for the father of the index case, who did not belong to the maternal family, carried 2 hereditary homozygous silent mutations in the ACVR1 gene: c.270C>T (A90A) in exon 4 and c.690G>A (E230E) in exon 7 (Figure 5). Additionally, the ACVR1 c.47 C>T nucleotide variant found in the index case's mother is not reported in SNP databases ( http://www.ncbi.nlm.nih.gov/SNP; http://www.ensembl.org/Homo_sapiens/genesnpview). Thus, the c.*47C>T mutation should be considered an individual variation.
Sequence analysis of ACVR1 exons in the family pedigree of the index case. The bases shown in the chromatogram are the reverse complementary bases of the corresponding mutation sites, because sequencing in these 2 regions was performed on the antisense strand. Exon sequencing results detected no constitutively activating mutation related to fibrodysplasia ossificans progressiva, and the genotype of the ACVR1 gene exhibited no differences among pedigree members. All members carried 2 homozygous silent mutations: c.270C>T (A90A) in exon 4 and c.690G>A (E230E) in exon 7.
Furthermore, 3 sporadic cases of NHHO carried a common mutation, c.*686T>C, whereas none of the members of this family exhibited mutation at the c.686T site (Figure 6), thus indicating inconsistency in the ACVR1 genotype between the sporadic NHHO cases and this family.
Comparison of mutations between the index case and 3 sporadic heterotopic ossification patients. None of the members of the maternal pedigree exhibited the c.*686T>C mutation in the ACVR1 gene. Heterotopic ossification patients 1 and 2 carried a heterozygous c.*686T>C mutation, and heterotopic ossification patient 3 carried a homozygous c.*686T>C mutation.
This study reported the clinical manifestations and the results of pedigree analysis and mutational analysis of the ACVR1 gene for 4 affected individuals in a 3-generation Chinese family of HO with multifocal and bilateral involvement. The results for this family are not compatible with 3 phenotypes of FOP and may therefore suggest a new form of HHO.
Until recently, there were approximately 800 known patients with FOP.8 The diagnosis of FOP is made on the basis of typical clinical and radiological findings, and mutational analysis of the ACVR1 gene is performed to confirm the results. Kaplan et al4 and Shore and Kaplan13 have recently classified FOP syndromes into 3 phenotypes according to clinical criteria: classic FOP, FOP-plus, and FOP-variant. The classic phenotype FOP involves progressive HO and malformed big toes and is associated with the c.617 G>A mutation; the FOP-plus phenotype HO involves malformed big toes and unique additional features, associated with either a c.617 G>A mutation or another ACVR1 mutation; and individuals affected by FOP-variant exhibit major variations in 1 or both classic defining features of FOP.
In patients with FOP, HO begins in soft tissues after birth, and the first event typically occurs in children before 5 years old.14–16 Progressive postnatal HO is a common feature shared by all patients with FOP. Over time, ectopic bone formation in FOP is progressive, cumulative, and extensive, and it bridges the joints of the axial and appendicular skeleton and causes nearly complete immobilization of the body.14,15
As shown in Table 1, the patients in this Chinese family presented distinctly different clinical and radiological features, including an unusually late onset of HO, normal appearance of the big toes and thumbs, and HO with multifocal and bilateral involvement. Therefore, the presentation of the 4 affected members in the current study was not consistent with the clinical diagnosis criteria for the 3 phenotypes of FOP.
Fibrodysplasia ossificans progressiva and POH are caused by mutations in a single disease-causing gene.10 A mutation in the ACVR1 gene (MIM 102576) was first identified as a genetic cause of FOP by Kaplan et al4 and Shore et al.10 Progressive osseous heteroplasia is caused by a mutation at the GNAS locus.10 Because HO formation in POH is mainly intramembranous,1 which is distinguished from the affected individuals of this family, the current authors mainly focused their study on ACVR1 gene mutations. Meanwhile, based on personal communication with Frederick S. Kaplan, MD (email, March 1, 2014), the authors did not perform GNAS exon sequencing analysis.
Fibrodysplasia ossificans progressiva is associated with a c.617G>A (p.R206H) mutation in approximately 97% of patients.17,18 Atypical FOP patients, such as those with FOP-plus and FOP-variant, tend to exhibit mutations other than the common p.R206H mutation. To date, a total of 13 heterozygous ACVR1 missense mutations and 1 3-bp deletion (elimination of 2 amino acids and insertion of a different amino acid) at highly conserved positions have been described in FOP patients.19
The most important finding of the current study was that all of the affected members of this family carried 2 homozygous silent mutations in the ACVR1 gene—c.270C>T and c.690G>A—a result inconsistent with previously described symptoms of FOP, especially in Chinese patients.7 In 2013, Zhang et al7 reported on 72 patients with confirmed FOP in China, in the largest ethnically homogeneous population of FOP patients in the world. All of the patients carried a c.617G>A (p.R206H) mutation in the ACVR1 gene, except for 2 patients who exhibited variant mutations of c.774G>C (p.R258S) and c.1067G>A (p.G356D). None of the 98 unaffected controls, including parents and siblings, had mutations in ACVR1. The difference between the current family and those in previous studies indicated that the 2 mutations observed in the ACVR1 gene (c.270C>T and c.690G>A) were probably not the disease-causing mutations in the affected patients in this family.
Additionally, all of the family members, including those who were unaffected (the index patient's aunt and her sister), carried the same mutations (c.270C>T and c.690G>A) in the ACVR1 gene, thus revealing that these 2 mutations in ACVR1 maybe were not disease-causing mutations, and it is possible that the disease-causing mutations of this Chinese pedigree are not located in the ACVR1 gene.
As indicated by previous studies, FOP is an autosomal-dominant disorder, but the etiology of most cases is a de novo mutation that is not inherited from a patient's parents.6 Most cases arise from spontaneous new autosomal-dominant mutations. There are few reported cases of affected multigenerational families with FOP, and most known cases occur de novo in families.8–12 Fewer than 10 families with autosomal-dominant inheritance of FOP have been reported.10 Inheritance can be from mothers or fathers.9,20 Maternal mosaicism can occur,21 and a paternal age effect has been reported.22 Together with the analysis of the affected individuals' clinical and radiographic features in the current study, these results indicate that the form of HO from which affected this family may not be diagnosed as FOP.
There has been controversy about whether patients in this 3-generation pedigree should be considered to exhibit a separate syndrome.23 The presentation of the 4 affected individuals in the family described is clearly not compatible with the 3 phenotypes of FOP, thus suggesting that the individuals included in this study may have a separate syndrome. Therefore, the authors designated the form of HO found in this family inherited multifocal and bilateral HO (IMBHO).
Additionally, the sequence analysis demonstrated the presence of a c.*686T>C mutation in 3 sporadic NHHO cases. However, this mutation was not detected in the affected family members. Therefore, IMBHO may also differ from NHHO. Considering the available findings together, the authors speculate that IMBHO is probably a new form of HHO. Although no research on IMBHO has been reported previously, the identification of disease-causing mutations in the genes of patients with this new disease has significant diagnostic and therapeutic implications. Nevertheless, further studies are needed to clarify this hypothesis.
This study had 3 main limitations. First, it is possible that this peculiar combination of the 2 mutations (c.270C>T and c.690G>A) in the ACVR1 gene can be nonpenetrant in some individuals. Second, based on the autosomal-dominant model of the inheritance of HO, the limited available information about this family concerning the transmission of HHO makes genome-wide identification much more challenging. Finally, but most importantly, there is limited literature regarding gene mutations of HO in the Chinese population, mainly focused on the posterior longitudinal ligament.24 Until now, there have been no data or literature to show the frequency of these silent mutations of ACVR1 in Chinese populations without HO. The authors will next examine the frequency of silent mutation of the ACVR1 gene in the Chinese population without HO through a large study.
To the best of the authors' knowledge, this is the first report to document 3 generations of a family with multifocal and bilateral HO involvement. This study may lead to the identification of a new form of HHO or extend understanding of the FOP-variant syndrome.
- Shore EM, Kaplan FS. Inherited human diseases of heterotopic bone formation. Nat Rev Rheumatol. 2010;6(9):518–527. doi:10.1038/nrrheum.2010.122 [CrossRef] PMID:20703219
- Foley KL, Hebela N, Keenan MA, Pignolo RJ. Histopathology of periarticular non-hereditary heterotopic ossification. Bone. 2018;109:65–70. doi:10.1016/j.bone.2017.12.006 [CrossRef] PMID:29225159
- Shehab D, Elgazzar AH, Collier BD. Heterotopic ossification. J Nucl Med. 2002;43(3):346–353. PMID:11884494
- Kaplan FS, Xu M, Seemann P, et al. Classic and atypical fibrodysplasia ossificans progressiva (FOP) phenotypes are caused by mutations in the bone morphogenetic protein (BMP) type I receptor ACVR1. Hum Mutat. 2009;30(3):379–390. doi:10.1002/humu.20868 [CrossRef] PMID:19085907
- Kaplan FS, Xu M, Glaser DL, et al. Early diagnosis of fibrodysplasia ossificans progressiva. Pediatrics. 2008;121(5):e1295–e1300. doi:10.1542/peds.2007-1980 [CrossRef] PMID:18450872
- Kaplan FS, Chakkalakal SA, Shore EM. Fibrodysplasia ossificans progressiva: mechanisms and models of skeletal metamorphosis. Dis Model Mech. 2012;5(6):756–762. doi:10.1242/dmm.010280 [CrossRef] PMID:23115204
- Zhang W, Zhang K, Song L, et al. The phenotype and genotype of fibrodysplasia ossificans progressiva in China: a report of 72 cases. Bone. 2013;57(2):386–391. doi:10.1016/j.bone.2013.09.002 [CrossRef] PMID:24051199
- Kaplan FS, Kobori JA, Orellana C, et al. Multi-system involvement in a severe variant of fibrodysplasia ossificans progressiva (ACVR1 c.772G>A; R258G): a report of two patients. Am J Med Genet A. 2015;167A (10):2265–2271. doi:10.1002/ajmg.a.37205 [CrossRef] PMID:26097044
- Shore EM, Feldman GJ, Xu M, Kaplan FS. The genetics of fibrodysplasia ossificans progressiva. Clin Rev Bone Miner Metab. 2005;3(3–4):201–204. doi:10.1385/BMM:3:3-4:201 [CrossRef]
- Shore EM, Xu M, Feldman GJ, et al. A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva. Nat Genet. 2006;38(5):525–527. doi:10.1038/ng1783 [CrossRef] PMID:16642017
- Feldman G, Li M, Martin S, et al. Fibrodysplasia ossificans progressiva, a heritable disorder of severe heterotopic ossification, maps to human chromosome 4q27-31. Am J Hum Genet. 2000;66(1):128–135. doi:10.1086/302724 [CrossRef] PMID:10631143
- Duncan E, Brown M, Shore EM. The revolution in human monogenic disease mapping. Genes (Basel). 2014;5(3):792–803. doi:10.3390/genes5030792 [CrossRef] PMID:25198531
- Shore EM, Kaplan FS. Insights from a rare genetic disorder of extra-skeletal bone formation, fibrodysplasia ossificans progressiva (FOP). Bone. 2008;43(3):427–433. doi:10.1016/j.bone.2008.05.013 [CrossRef] PMID:18590993
- Shore EM, Kaplan FS. Role of altered signal transduction in heterotopic ossification and fibrodysplasia ossificans progressiva. Curr Osteoporos Rep. 2011;9(2):83–88. doi:10.1007/s11914-011-0046-3 [CrossRef] PMID:21340697
- Kaplan FS, Glaser DL, Shore EM, et al. The phenotype of fibrodysplasia ossificans progressiva. Clin Rev Bone Miner Metab. 2005;3(3–4):183–188. doi:10.1385/BMM:3:3-4:183 [CrossRef]
- Kaplan FS, Groppe JC, Seemann P, Pignolo RJ, Shore EM. Fibrodysplasia ossificans progressiva: developmental implications of a novel metamorphogene. In Bronner F, Farach-Carson M, Roach H, eds. Bone and Development, vol. 6. Springer-Verlag; 2010:233–249.
- Kaplan FS, Pignolo RJ, Shore EM. The FOP metamorphogene encodes a novel type I receptor that dysregulates BMP signaling. Cytokine Growth Factor Rev. 2009;20(5–6):399–407. doi:10.1016/j.cytogfr.2009.10.006 [CrossRef] PMID:19896889
- Kaplan FS, Lounev VY, Wang H, Pignolo RJ, Shore EM. Fibrodysplasia ossificans progressiva: a blueprint for metamorphosis. Ann N Y Acad Sci. 2011;1237(1):5–10. doi:10.1111/j.1749-6632.2011.06195.x [CrossRef] PMID:22082359
- Pacifici M, Shore EM. Common mutations in ALK2/ACVR1, a multi-faceted receptor, have roles in distinct pediatric musculoskeletal and neural orphan disorders. Cytokine Growth Factor Rev. 2016;27:93–104. doi:10.1016/j.cytogfr.2015.12.007 [CrossRef] PMID:26776312
- Kaplan FS, McCluskey W, Hahn G, Tabas JA, Muenke M, Zasloff MA. Genetic transmission of fibrodysplasia ossificans progressive: report of a family. J Bone Joint Surg Am.1993;75(8):1214–1220. doi:10.2106/00004623-199308000-00011 [CrossRef] PMID:8354680
- Janoff HB, Muenke M, Johnson LO, et al. Fibrodysplasia ossificans progressiva in two half-sisters: evidence for maternal mosaicism. Am J Med Genet. 1996;61(4):320–324. doi:10.1002/(SICI)1096-8628(19960202)61:4<320::AIDAJMG4>3.0.CO;2-Y [CrossRef] PMID:8834042
- Rogers JG, Chase GA. Paternal age effect in fibrodysplasia ossificans progressiva. J Med Genet. 1979;16(2):147–148. doi:10.1136/jmg.16.2.147 [CrossRef] PMID:287808
- Hennekam RC. What to call a syndrome. Am J Med Genet A. 2007;143A(10):1021–1024. doi:10.1002/ajmg.a.31674 [CrossRef] PMID:17366584
- Wei W, He HL, Chen CY, et al. Whole exome sequencing implicates PTCH1 and COL17A1 genes in ossification of the posterior longitudinal ligament of the cervical spine in Chinese patients. Genet Mol Res. 2014;13(1):1794–1804. doi:10.4238/2014.March.17.7 [CrossRef] PMID:24668667
Comparison Among Patients in the Pedigree and the 3 Phenotypes of Fibrodysplasia Ossificans Progressiva
|Variable||Pedigree patients||Classic FOP||FOP-plus||FOP-variant|
|ACVR1 mutation||c.270C>T and c.690G>A||c.617 G>A||c.617 G>A||Variable|
|Codon change||A90A and E230E||R206H||R206H||Variable|
|Sex||M (n=2), F (n=2)||M, F||M, F||M, F|
|Age range of onset of ossification, y||19–23||1–10||1–10||Variable|
|Classic FOP features|
| Progressive HO||100%||100%||100%|
| Malformed great toe (hallux valgus)||100%||100%|
| Severe or mild digit reduction deficits||No||No||No||100%|
| Unique/unusual FOP-associated clinical feature(s)||No||No||100%||Variable|
Mutational Analysis of ACVR1 Exons
| II.1 (maternal uncle)||TT||AA|
| II.2 (maternal uncle)||TT||AA|
| II.4 (mother)||TT||AA||C/T|
| III.1 (index case)||TT||AA|
| II.3 (maternal aunt)||TT||AA|
| II.5 (father)a||C/T||G/A||C/T|
| III.2 (sister)||TT||AA|
|Sporadic heterotopic ossification patients|
| Patient 1||TT||AA||C/T|
| Patient 2||TT||AA||C/T|
| Patient 3||C/T||AA||CC|