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Case of mild Schmid-type metaphyseal chondrodysplasia with novel sequence variation involving an unusual mutational site of the COL10A1 gene
European Journal of Medical Genetics 58 (2015) 175e179
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Clinical report
Case of mild Schmid-type metaphyseal chondrodysplasia with novel sequence variation involving an unusual mutational site of the COL10A1 gene Hyunwoong Park a, Susie Hong a, Sung Im Cho a, Tae-Joon Cho b, In Ho Choi b, Dong-Kyu Jin c, Young Bae Sohn d, Sung Won Park c, Hyun-Hae Cho e, Jung-Eun Cheon e, So Yeon Kim f, Ji Yeon Kim g, Sung Sup Park a, g, Moon-Woo Seong a, * a
Department of Laboratory Medicine, Seoul National University Hospital, Seoul, South Korea Department of Orthopedic Surgery, Seoul National University Hospital, Seoul, South Korea c Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea d Department of Medical Genetics, Ajou University School of Medicine, Suwon, South Korea e Department of Radiology, Seoul National University Hospital, Seoul, South Korea f Department of Laboratory Medicine, National Medical Center, Seoul, South Korea g Biomedical Research Institute, Seoul National University Hospital, Seoul, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 March 2014 Accepted 4 December 2014 Available online 24 December 2014
Schmid-type metaphyseal chondrodysplasia (MCDS) is characterized by short stature with short legs, bowing of the long bones, coxa vara, and waddling gait. MCDS is a relatively common form of MCD. Most mutations that cause MCDS occur within the carboxyl-terminal non-collagenous domain (NC1) of the COL10A1 gene. We performed mutational analysis of the COL10A1 genes in 4 unrelated Korean patients with diagnosed MCDS. Mutational analysis of COL10A1 identified c.1904_1915delinsT (p.Gln635LeufsX10) and c.1969dupG (p.Ala657GlyfsX10), 2 novel frameshift mutations, and c.2030T>A (p.Val677Glu) and c.862G>C (p.Gly288Arg) at unusual mutational sites, which could be pathogenic. We present the first report of the molecular characteristics of MCDS in 4 Korean patients. Our findings suggest that a novel sequence variation involving an unusual mutational site of the COL10A1 gene can cause mild MCDS. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: COL10A1 Schmid metaphyseal chondrodysplasia Collagen type X
1. Introduction Schmid-type metaphyseal chondrodysplasia (MCDS; MIM #156500) is characterized by short stature with short legs, bowing of the long bones, coxa vara, and waddling gait [Bateman et al., 2005; Woelfle et al., 2011]. MCDS is a relatively common form of MCD. Radiographic findings of MCDS are metaphyseal abnormality, widening and irregularity of the growth plates, and sclerosis. Distal femoral and proximal tibial metaphyses are the most consistently and severely affected sites [Bateman et al., 2005; Makitie et al., 2005]. MCDS results from autosomal * Corresponding author. Department of Laboratory Medicine, Seoul National University Hospital, 101, Daehak-ro, Jongno-gu, Seoul 110-744, South Korea. Tel.: þ82 2 2072 4180; fax: þ82 2 747 0359. E-mail address: [email protected] (M.-W. Seong). http://dx.doi.org/10.1016/j.ejmg.2014.12.011 1769-7212/Ó 2014 Elsevier Masson SAS. All rights reserved.
dominant mutations in COL10A1, the gene encoding a1(X) chains of type X collagen molecules [Kirsch and von der Mark, 1991; Makitie et al., 2005; McIntosh et al., 1994, 1995; Wallis et al., 1994; Warman et al., 1993]. Type X collagen is a shortchain, non-fibrillar collagen, which consists of 3 identical a1(X) chains [Kirsch and von der Mark, 1991; Makitie et al., 2005; Schmid and Linsenmayer, 1985]. COL10A1 mutations disrupt growth plate function and may interfere with endochondral ossification [Wilson et al., 2005; Woelfle et al., 2011]. Most mutations that cause MCDS occur within the C-terminal noncollagenous domain (NC1) of the COL10A1 gene [Marks et al., 1999]. Only 2 MCDS mutations have been found in the Nterminal non-collagenous domain (NC2) [Ikegawa et al., 1997]. Here, we report the molecular and clinical features of 2 patients with mild MCDS with missense mutations in the COL10A1 gene.
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2. Materials and methods 2.1. Subjects Four unrelated Korean patients with diagnosed MCDS were included in this study. Informed consent was obtained from all patients or their legal guardians to provide clinical information and blood samples. All patients underwent detailed clinical and physical examination and their radiologic findings were reviewed. 2.2. Mutational analysis Genomic DNA was extracted from peripheral blood using Gentra PureGene DNA isolation kits (Gentra Systems, Inc. Minneapolis, MN, USA). We analyzed all exons and their flanking regions of the COL10A1 gene via direct sequencing. The COL10A1 gene was amplified under the following polymerase chain reaction conditions: 94 C for 5 min, followed by 35 cycles of 94 C for 30 s, 60 C for 30 s, and 72 C for 1 min, and a final step at 72 C for 7 min. Amplified products were bidirectionally sequenced on an ABI Prism 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), and then analyzed using Sequencher software (Gene Codes Co, Ann Arbor, MI, USA) and Mutation Surveyor (Softgenetics, State College, PA, USA). 2.3. Investigation of clinical significance of a novel sequence variation To determine the pathogenicity of 2 novel missense variations, we have predicted their functional effects using the following information: the characteristics of the amino acids substituted, interspecies amino acid conservation, and protein structural information. We compared those with the prediction results of the programs MutationTaster [Schwarz et al., 2010], Polyphen [Ramensky et al., 2002], AGVGD [Mathe et al., 2006], and SIFT [Ng and Henikoff, 2001]. The mutational analysis included at least 94 healthy individuals who served as control for the 1% polymorphism criterion [Collins and Schwartz, 2002]. 3. Case study The clinical and radiological features of the 4 patients enrolled in this study are summarized in Table 1. 1) Patient 1, a 10-year-old male patient, appeared normal at birth, had no dysmorphic face, but presented with both bowed leg deformity and waddling gait at 1 year of age. There was no familial history. His height was 119.2 cm, which was <3 percentile (3.21 SDS), at the age of 10 years, 7 months. He received an Ilizarov apparatus at 6 years of age. He had varus tendency at walking. His radiological findings revealed genu valgum, epiphyseal widening and metaphyseal irregularity of the distal femur, proximal tibia and fibula (Fig. 1). We found a novel frameshift mutation, c.1904_1915delinsT (p.Gln635LeufsX10),
Fig. 1. Radiographs of patient 1. A, Epiphyseal widening and metaphyseal irregularity were shown at proximal tibia and fibula at 6 years of age. B, Epiphyseal widening and metaphyseal irregularity were shown at distal femur at 8 years of age.
Table 1 Clinical and radiological features in 4 patients with Schmid-type metaphyseal dysplasia. Patient
Age at diagnosis
Sex
Height (percentile)
Waddling gait
Deformities
Coxa magna
Coxa vara
Flaring of metaphyses
Wide and irregular metaphyses
Surgeries
1 2 3 4
10 4 9 9
M F M M
119.2 cm (<3 percentile) 90.3 cm (<3 percentile) 119.6 cm (<3 percentile) 123 cm (<10 percentile)
Yes Yes No No
Genu Genu Genu Genu
No Yes No No
No Yes Yes No
No Yes No No
Yes Yes Yes Yes
Ilizarov apparatus No No Corrective osteotomy of both tibia and fibula
valgum varus varus valgum
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Fig. 2. Radiographs of patient 2 showing metaphyseal irregularity and beaking and epiphyseal widening of proximal and distal femur and proximal and distal tibia and fibula at 21 months of age.
in the NC1 domain. We could not conduct a segregation analysis because family samples were not available. 2) Patient 2, a 4-year-old female patient, was normal at birth and had no dysmorphic face, but demonstrated both bowed leg deformity and waddling gait at 1 year of age. She was born to clinically normal parents. Her height was 90.3 cm, which was <3 percentile (3.44 SDS), at the age of 4 years, 7 months. Her radiological findings revealed genu varum, coxa vara metaphyseal irregularity, beaking, epiphyseal widening of proximal
and distal femur (Fig. 2). We identified a novel frameshift mutation, c.1969dupG (p.Ala657GlyfsX10), in the NC1 domain. We could not conduct a segregation analysis because family samples were not available. 3) Patient 3, a 9-year-old male patient, appeared normal at birth and had no dysmorphic face. He was born to clinically healthy parents. His height was 119.6 cm, which was <3 percentile (2.67 SDS), at the age of 9 years 9 months. He complained of knee pain and had a genu varus deformity. Radiological
Fig. 3. Radiographs of patient 3 showing coxa vara, a widened epiphyseal line of the proximal and distal femur and metaphyseal irregularity on proximal femur. Mild epiphyseal widening was also observed in proximal tibia and fibula at 7 years of age.
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Table 2 Molecular features of 4 novel mutations in patients with Schmid-type metaphyseal dysplasia. Patient
1 2 3 4
Nucleotide change
c.1904_1915delinsT c.1969dupG c.2030T>A c.862G>C
Amino acid change
p.Gln635LeufsX10 p.Ala657GlyfsX10 p.V677E p.G288R
Mutation type
Frameshift Frameshift Missense Missense
Domain
NC1 NC1 NC1 Triple helix
In silico prediction MutationTaster
Polyphen
AGVGD
SIFT
Disease causing Disease causing
Possibly damaging Unknown
Medium Most likely
Affected Affected
Frequency in control individual
0/94 0/94
NC1, C-terminal non-collagenous.
examination showed coxa vara, a widened epiphyseal line of the proximal and distal femur and metaphyseal irregularity on proximal femur. Mild epiphyseal widening was also observed in proximal tibia and fibula (Fig. 3). We detected a novel missense variant, c.2030T>A (p.Val677Glu), in the NC1 domain. We could not conduct a segregation analysis because family samples were not available. The variant c.2030T>A (p.Val677Glu) was predicted to be pathogenic by 3 of the following programs: MutationTaster, PolyPhen, AGVGD, and SIFT, and was not identified in normal controls (Table 2). In addition, this novel variant was well conserved across species such as Bos taurus, Macaca mulatta, Pan troglodytes, Mus musculus, Rattus norvegicus, and Danio rerio (Fig. 4). Therefore, we regarded this novel variant as probable pathogenic mutation.
4) Patient 4, a 21-year-old male patient, appeared normal at birth and had no dysmorphic face. He had bilateral hip pain and abnormal gait at 7 years of age. His height was 123 cm, which was <10 percentile (1.53 SDS), at 9 years of age. He received corrective osteotomy of both tibia and fibula at few years ago. His height was 167.5 cm, which was <25 percentile (1.04 SDS), at 21 years of age. His radiological findings revealed genu valgum, metaphyseal irregularity of proximal and distal tibia and fibula (Fig. 5). We identified a novel missense variant, c.862G>C (p.Gly288Arg), in the triple-helical domain. Because the samples of family member were not available, segregation analysis was not possible. The variant c.862G>C (p.Gly288Arg) was predicted to be pathogenic by 3 of the following programs: MutationTaster, PolyPhen, AGVGD, and SIFT. This variant was not
Fig. 4. Multiple alignments using HomoloGene and amino acid conservation of 2 novel missense variations: c.2030T>A (p.Val677Glu) and c.862G>C (p.Gly288Arg) in COL10A1 across several species.
Fig. 5. Radiographs of patient 4 showing genu valgum, metaphyseal irregularity of proximal and distal tibia and fibula at 7 years of age.
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Fig. 6. Schematic representation of 4 novel sequence variants location in type X collagen chain. One possible pathogenic variant (p.Gly288Arg) located within helix. Two novel frameshift mutation (p.Gln635LeufsX10 and p.Ala657GlyfsX10) and one possible pathogenic missense variant (p.Val677Glu) located within NC2. S, signal peptide; NC2, noncollagenous domain 2; HELIX, triple helical domain; NC1, non-collagenous domain 1. Figure based on [Bateman et al., 2005].
found in normal controls (Table 2). In addition, this novel variant was highly conserved across species such as B. taurus, M. mulatta, P. troglodytes, M. musculus, R. norvegicus, and D. rerio (Fig. 4). Therefore, we regarded this novel variant as probable pathogenic mutation.
4. Discussion Type X collagen is a short-chain, non-fibrillar collagen, which consists of 3 identical a1(X) chains each comprised of a 463-amino acid Gly-X-Y collagenous domain (COL1) flanked by a 38-residue Nterminal non-collagenous NC2 and a 161-residue C-terminal noncollagenous NC1 domain [Bateman et al., 2005; Kirsch and von der Mark, 1991; Makitie et al., 2005]. The NC1 domain is the important region in MCDS and is different from other collagen diseases. Most mutations are found in the C-terminal globular domain (NC1 domain), with the exception of 2 missense mutations located at the N-terminal signal peptide cleavage site identified in Japanese cases [Bateman et al., 2004; Ikegawa et al., 1997]. We identified 4 novel sequence variations in COL10A1 in 4 Korean patients with MCDS, one of which is at an unusual site (Fig. 6). Patient 3 demonstrated short statue and knee pain and had a genu varus deformity. Patient 4 presented slightly short stature, hip pain, and abnormal gait. Missense mutations in the collagen X triple helix could also cause a dominant negative effect if the structure and stability of the helical domain is as critical to the function of collagen X as it is in case of fibrillar collagens [Bateman et al., 2005]. The clinical variability in severity of MCDS is greater than previously thought. This finding raises the question of whether mutations in the collagen X triple helical domain or NC2 domain can also cause MCDS [Bateman et al., 2004]. The novel missense variants c.2030T>A (p.Val677Glu) and c.862G>C (p.Gly288Arg) were not detected in 94 control subjects and the amino acid sequence was well conserved among various species. Consistent with the predictions of 4 in silico software programs utilized in this study, we could consider these novel variants to be possibly pathogenic, even though variant c.2030T>A (p.Val677Glu) was located in the immediate downstream region, where mutations were clustered in the NC1 domain, and variant c.862G>C (p.Gly288Arg) was located in the triple-helical domain [Tan et al., 2008]. But the pathogenicity of these two novel possibly pathogenic missense variants are not clear because of absence of familial segregation analysis and use of in silico prediction software. We present the first report of the molecular characteristics of MCDS in 4 Korean patients and the first case of a possibly pathogenic variant in the triple-helical domain. Our findings suggest that the novel sequence variation involving an unusual mutational site of the COL10A1 gene can cause mild MCDS. Finally, the results
presented in this work warrants future in depth screening of the COL10A1 gene in weak suspects of (late detected) MCDS. Genotypeephenotype relationship studies of the COL10A1 gene in MCDS patients with variable clinical severity are needed.
References Bateman JF, Freddi S, McNeil R, Thompson E, Hermanns P, Savarirayan R, et al. Identification of four novel COL10A1 missense mutations in Schmid metaphyseal chondrodysplasia: further evidence that collagen X NC1 mutations impair trimer assembly. Hum Mutat 2004;23:396. Bateman JF, Wilson R, Freddi S, Lamande SR, Savarirayan R. Mutations of COL10A1 in Schmid metaphyseal chondrodysplasia. Hum Mutat 2005;25:525e34. Collins JS, Schwartz CE. Detecting polymorphisms and mutations in candidate genes. Am J Hum Genet 2002;71:1251e2. Ikegawa S, Nakamura K, Nagano A, Haga N, Nakamura Y. Mutations in the N-terminal globular domain of the type X collagen gene (COL10A1) in patients with Schmid metaphyseal chondrodysplasia. Hum Mutat 1997;9:131e5. Kirsch T, von der Mark K. Isolation of human type X collagen and immunolocalization in fetal human cartilage. Eur J Biochem 1991;196:575e80. Makitie O, Susic M, Ward L, Barclay C, Glorieux FH, Cole WG. Schmid type of metaphyseal chondrodysplasia and COL10A1 mutations-findings in 10 patients. Am J Med Genet 2005;137:241e8. Marks DS, Gregory CA, Wallis GA, Brass A, Kadler KE, Boot-handford RP. Metaphyseal chondrodysplasia type Schmid mutations are predicted to occur in two distinct three-dimensional clusters within type X collagen NC1 domains that retain the ability to trimerize. J Biol Chem 1999;274:3632e41. Mathe E, Olivier M, Kato S, Ishioka C, Hainaut P, Tavtigian SV. Computational approaches for predicting the biological effect of p53 missense mutations: a comparison of three sequence analysis based methods. Nucleic Acids Res 2006;34:1317e25. McIntosh I, Abbott MH, Francomano CA. Concentration of mutations causing Schmid metaphyseal chondrodysplasia in the C-terminal noncollagenous domain of type X collagen. Hum Mutat 1995;5:121e5. McIntosh I, Abbott MH, Warman ML, Olsen BR, Francomano CA. Additional mutations of type X collagen confirm COL10A1 as the Schmid metaphyseal chondrodysplasia locus. Hum Mol Genet 1994;3:303e7. Ng PC, Henikoff S. Predicting deleterious amino acid substitutions. Genome Res 2001;11:863e74. Ramensky V, Bork P, Sunyaev S. Human non-synonymous SNPs: server and survey. Nucleic Acids Res 2002;30:3894e900. Schmid TM, Linsenmayer TF. Immunohistochemical localization of short chain cartilage collagen (type X) in avian tissues. J Cell Biol 1985;100:598e605. Schwarz JM, Rödelsperger C, Schuelke M, Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods 2010;7:575e6. Tan JT, Kremer F, Freddi S, Bell KM, Baker NL, Lamande SR, et al. Competency for nonsense-mediated reduction in collagen X mRNA is specified by the 30 UTR and corresponds to the position of mutations in Schmid metaphyseal chondrodysplasia. Am J Hum Genet 2008;82:786e93. Wallis GA, Rash B, Sweetman WA, Thomas JT, Super M, Evans G, et al. Amino acid substitutions of conserved residues in the carboxyl-terminal domain of the alpha 1(X) chain of type X collagen occur in two unrelated families with metaphyseal chondrodysplasia type Schmid. Am J Hum Genet 1994;54:169e78. Warman ML, Abbott M, Apte SS, Hefferon T, McIntosh I, Cohn DH, et al. A type X collagen mutation causes Schmid metaphyseal chondrodysplasia. Nat Genet 1993;5:79e82. Wilson R, Freddi S, Chan D, Cheah KSE, Bateman JF. Misfolding of collagen X chains harboring Schmid metaphyseal chondrodysplasia mutations results in aberrant disulfide bond formation, intracellular retention, and activation of the unfolded protein response. J Biol Chem 2005;280:15544e52. Woelfle JV, Brenner RE, Zabel B, Reichel H, Nelitz M. Schmid-type metaphyseal chondrodysplasia as the result of a collagen type X defect due to a novel COL10A1 nonsense mutation. J Orthop Sci 2011;16:245e9.
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Clinical report
Case of mild Schmid-type metaphyseal chondrodysplasia with novel sequence variation involving an unusual mutational site of the COL10A1 gene Hyunwoong Park a, Susie Hong a, Sung Im Cho a, Tae-Joon Cho b, In Ho Choi b, Dong-Kyu Jin c, Young Bae Sohn d, Sung Won Park c, Hyun-Hae Cho e, Jung-Eun Cheon e, So Yeon Kim f, Ji Yeon Kim g, Sung Sup Park a, g, Moon-Woo Seong a, * a
Department of Laboratory Medicine, Seoul National University Hospital, Seoul, South Korea Department of Orthopedic Surgery, Seoul National University Hospital, Seoul, South Korea c Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea d Department of Medical Genetics, Ajou University School of Medicine, Suwon, South Korea e Department of Radiology, Seoul National University Hospital, Seoul, South Korea f Department of Laboratory Medicine, National Medical Center, Seoul, South Korea g Biomedical Research Institute, Seoul National University Hospital, Seoul, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 March 2014 Accepted 4 December 2014 Available online 24 December 2014
Schmid-type metaphyseal chondrodysplasia (MCDS) is characterized by short stature with short legs, bowing of the long bones, coxa vara, and waddling gait. MCDS is a relatively common form of MCD. Most mutations that cause MCDS occur within the carboxyl-terminal non-collagenous domain (NC1) of the COL10A1 gene. We performed mutational analysis of the COL10A1 genes in 4 unrelated Korean patients with diagnosed MCDS. Mutational analysis of COL10A1 identified c.1904_1915delinsT (p.Gln635LeufsX10) and c.1969dupG (p.Ala657GlyfsX10), 2 novel frameshift mutations, and c.2030T>A (p.Val677Glu) and c.862G>C (p.Gly288Arg) at unusual mutational sites, which could be pathogenic. We present the first report of the molecular characteristics of MCDS in 4 Korean patients. Our findings suggest that a novel sequence variation involving an unusual mutational site of the COL10A1 gene can cause mild MCDS. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: COL10A1 Schmid metaphyseal chondrodysplasia Collagen type X
1. Introduction Schmid-type metaphyseal chondrodysplasia (MCDS; MIM #156500) is characterized by short stature with short legs, bowing of the long bones, coxa vara, and waddling gait [Bateman et al., 2005; Woelfle et al., 2011]. MCDS is a relatively common form of MCD. Radiographic findings of MCDS are metaphyseal abnormality, widening and irregularity of the growth plates, and sclerosis. Distal femoral and proximal tibial metaphyses are the most consistently and severely affected sites [Bateman et al., 2005; Makitie et al., 2005]. MCDS results from autosomal * Corresponding author. Department of Laboratory Medicine, Seoul National University Hospital, 101, Daehak-ro, Jongno-gu, Seoul 110-744, South Korea. Tel.: þ82 2 2072 4180; fax: þ82 2 747 0359. E-mail address: [email protected] (M.-W. Seong). http://dx.doi.org/10.1016/j.ejmg.2014.12.011 1769-7212/Ó 2014 Elsevier Masson SAS. All rights reserved.
dominant mutations in COL10A1, the gene encoding a1(X) chains of type X collagen molecules [Kirsch and von der Mark, 1991; Makitie et al., 2005; McIntosh et al., 1994, 1995; Wallis et al., 1994; Warman et al., 1993]. Type X collagen is a shortchain, non-fibrillar collagen, which consists of 3 identical a1(X) chains [Kirsch and von der Mark, 1991; Makitie et al., 2005; Schmid and Linsenmayer, 1985]. COL10A1 mutations disrupt growth plate function and may interfere with endochondral ossification [Wilson et al., 2005; Woelfle et al., 2011]. Most mutations that cause MCDS occur within the C-terminal noncollagenous domain (NC1) of the COL10A1 gene [Marks et al., 1999]. Only 2 MCDS mutations have been found in the Nterminal non-collagenous domain (NC2) [Ikegawa et al., 1997]. Here, we report the molecular and clinical features of 2 patients with mild MCDS with missense mutations in the COL10A1 gene.
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2. Materials and methods 2.1. Subjects Four unrelated Korean patients with diagnosed MCDS were included in this study. Informed consent was obtained from all patients or their legal guardians to provide clinical information and blood samples. All patients underwent detailed clinical and physical examination and their radiologic findings were reviewed. 2.2. Mutational analysis Genomic DNA was extracted from peripheral blood using Gentra PureGene DNA isolation kits (Gentra Systems, Inc. Minneapolis, MN, USA). We analyzed all exons and their flanking regions of the COL10A1 gene via direct sequencing. The COL10A1 gene was amplified under the following polymerase chain reaction conditions: 94 C for 5 min, followed by 35 cycles of 94 C for 30 s, 60 C for 30 s, and 72 C for 1 min, and a final step at 72 C for 7 min. Amplified products were bidirectionally sequenced on an ABI Prism 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), and then analyzed using Sequencher software (Gene Codes Co, Ann Arbor, MI, USA) and Mutation Surveyor (Softgenetics, State College, PA, USA). 2.3. Investigation of clinical significance of a novel sequence variation To determine the pathogenicity of 2 novel missense variations, we have predicted their functional effects using the following information: the characteristics of the amino acids substituted, interspecies amino acid conservation, and protein structural information. We compared those with the prediction results of the programs MutationTaster [Schwarz et al., 2010], Polyphen [Ramensky et al., 2002], AGVGD [Mathe et al., 2006], and SIFT [Ng and Henikoff, 2001]. The mutational analysis included at least 94 healthy individuals who served as control for the 1% polymorphism criterion [Collins and Schwartz, 2002]. 3. Case study The clinical and radiological features of the 4 patients enrolled in this study are summarized in Table 1. 1) Patient 1, a 10-year-old male patient, appeared normal at birth, had no dysmorphic face, but presented with both bowed leg deformity and waddling gait at 1 year of age. There was no familial history. His height was 119.2 cm, which was <3 percentile (3.21 SDS), at the age of 10 years, 7 months. He received an Ilizarov apparatus at 6 years of age. He had varus tendency at walking. His radiological findings revealed genu valgum, epiphyseal widening and metaphyseal irregularity of the distal femur, proximal tibia and fibula (Fig. 1). We found a novel frameshift mutation, c.1904_1915delinsT (p.Gln635LeufsX10),
Fig. 1. Radiographs of patient 1. A, Epiphyseal widening and metaphyseal irregularity were shown at proximal tibia and fibula at 6 years of age. B, Epiphyseal widening and metaphyseal irregularity were shown at distal femur at 8 years of age.
Table 1 Clinical and radiological features in 4 patients with Schmid-type metaphyseal dysplasia. Patient
Age at diagnosis
Sex
Height (percentile)
Waddling gait
Deformities
Coxa magna
Coxa vara
Flaring of metaphyses
Wide and irregular metaphyses
Surgeries
1 2 3 4
10 4 9 9
M F M M
119.2 cm (<3 percentile) 90.3 cm (<3 percentile) 119.6 cm (<3 percentile) 123 cm (<10 percentile)
Yes Yes No No
Genu Genu Genu Genu
No Yes No No
No Yes Yes No
No Yes No No
Yes Yes Yes Yes
Ilizarov apparatus No No Corrective osteotomy of both tibia and fibula
valgum varus varus valgum
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Fig. 2. Radiographs of patient 2 showing metaphyseal irregularity and beaking and epiphyseal widening of proximal and distal femur and proximal and distal tibia and fibula at 21 months of age.
in the NC1 domain. We could not conduct a segregation analysis because family samples were not available. 2) Patient 2, a 4-year-old female patient, was normal at birth and had no dysmorphic face, but demonstrated both bowed leg deformity and waddling gait at 1 year of age. She was born to clinically normal parents. Her height was 90.3 cm, which was <3 percentile (3.44 SDS), at the age of 4 years, 7 months. Her radiological findings revealed genu varum, coxa vara metaphyseal irregularity, beaking, epiphyseal widening of proximal
and distal femur (Fig. 2). We identified a novel frameshift mutation, c.1969dupG (p.Ala657GlyfsX10), in the NC1 domain. We could not conduct a segregation analysis because family samples were not available. 3) Patient 3, a 9-year-old male patient, appeared normal at birth and had no dysmorphic face. He was born to clinically healthy parents. His height was 119.6 cm, which was <3 percentile (2.67 SDS), at the age of 9 years 9 months. He complained of knee pain and had a genu varus deformity. Radiological
Fig. 3. Radiographs of patient 3 showing coxa vara, a widened epiphyseal line of the proximal and distal femur and metaphyseal irregularity on proximal femur. Mild epiphyseal widening was also observed in proximal tibia and fibula at 7 years of age.
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Table 2 Molecular features of 4 novel mutations in patients with Schmid-type metaphyseal dysplasia. Patient
1 2 3 4
Nucleotide change
c.1904_1915delinsT c.1969dupG c.2030T>A c.862G>C
Amino acid change
p.Gln635LeufsX10 p.Ala657GlyfsX10 p.V677E p.G288R
Mutation type
Frameshift Frameshift Missense Missense
Domain
NC1 NC1 NC1 Triple helix
In silico prediction MutationTaster
Polyphen
AGVGD
SIFT
Disease causing Disease causing
Possibly damaging Unknown
Medium Most likely
Affected Affected
Frequency in control individual
0/94 0/94
NC1, C-terminal non-collagenous.
examination showed coxa vara, a widened epiphyseal line of the proximal and distal femur and metaphyseal irregularity on proximal femur. Mild epiphyseal widening was also observed in proximal tibia and fibula (Fig. 3). We detected a novel missense variant, c.2030T>A (p.Val677Glu), in the NC1 domain. We could not conduct a segregation analysis because family samples were not available. The variant c.2030T>A (p.Val677Glu) was predicted to be pathogenic by 3 of the following programs: MutationTaster, PolyPhen, AGVGD, and SIFT, and was not identified in normal controls (Table 2). In addition, this novel variant was well conserved across species such as Bos taurus, Macaca mulatta, Pan troglodytes, Mus musculus, Rattus norvegicus, and Danio rerio (Fig. 4). Therefore, we regarded this novel variant as probable pathogenic mutation.
4) Patient 4, a 21-year-old male patient, appeared normal at birth and had no dysmorphic face. He had bilateral hip pain and abnormal gait at 7 years of age. His height was 123 cm, which was <10 percentile (1.53 SDS), at 9 years of age. He received corrective osteotomy of both tibia and fibula at few years ago. His height was 167.5 cm, which was <25 percentile (1.04 SDS), at 21 years of age. His radiological findings revealed genu valgum, metaphyseal irregularity of proximal and distal tibia and fibula (Fig. 5). We identified a novel missense variant, c.862G>C (p.Gly288Arg), in the triple-helical domain. Because the samples of family member were not available, segregation analysis was not possible. The variant c.862G>C (p.Gly288Arg) was predicted to be pathogenic by 3 of the following programs: MutationTaster, PolyPhen, AGVGD, and SIFT. This variant was not
Fig. 4. Multiple alignments using HomoloGene and amino acid conservation of 2 novel missense variations: c.2030T>A (p.Val677Glu) and c.862G>C (p.Gly288Arg) in COL10A1 across several species.
Fig. 5. Radiographs of patient 4 showing genu valgum, metaphyseal irregularity of proximal and distal tibia and fibula at 7 years of age.
H. Park et al. / European Journal of Medical Genetics 58 (2015) 175e179
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Fig. 6. Schematic representation of 4 novel sequence variants location in type X collagen chain. One possible pathogenic variant (p.Gly288Arg) located within helix. Two novel frameshift mutation (p.Gln635LeufsX10 and p.Ala657GlyfsX10) and one possible pathogenic missense variant (p.Val677Glu) located within NC2. S, signal peptide; NC2, noncollagenous domain 2; HELIX, triple helical domain; NC1, non-collagenous domain 1. Figure based on [Bateman et al., 2005].
found in normal controls (Table 2). In addition, this novel variant was highly conserved across species such as B. taurus, M. mulatta, P. troglodytes, M. musculus, R. norvegicus, and D. rerio (Fig. 4). Therefore, we regarded this novel variant as probable pathogenic mutation.
4. Discussion Type X collagen is a short-chain, non-fibrillar collagen, which consists of 3 identical a1(X) chains each comprised of a 463-amino acid Gly-X-Y collagenous domain (COL1) flanked by a 38-residue Nterminal non-collagenous NC2 and a 161-residue C-terminal noncollagenous NC1 domain [Bateman et al., 2005; Kirsch and von der Mark, 1991; Makitie et al., 2005]. The NC1 domain is the important region in MCDS and is different from other collagen diseases. Most mutations are found in the C-terminal globular domain (NC1 domain), with the exception of 2 missense mutations located at the N-terminal signal peptide cleavage site identified in Japanese cases [Bateman et al., 2004; Ikegawa et al., 1997]. We identified 4 novel sequence variations in COL10A1 in 4 Korean patients with MCDS, one of which is at an unusual site (Fig. 6). Patient 3 demonstrated short statue and knee pain and had a genu varus deformity. Patient 4 presented slightly short stature, hip pain, and abnormal gait. Missense mutations in the collagen X triple helix could also cause a dominant negative effect if the structure and stability of the helical domain is as critical to the function of collagen X as it is in case of fibrillar collagens [Bateman et al., 2005]. The clinical variability in severity of MCDS is greater than previously thought. This finding raises the question of whether mutations in the collagen X triple helical domain or NC2 domain can also cause MCDS [Bateman et al., 2004]. The novel missense variants c.2030T>A (p.Val677Glu) and c.862G>C (p.Gly288Arg) were not detected in 94 control subjects and the amino acid sequence was well conserved among various species. Consistent with the predictions of 4 in silico software programs utilized in this study, we could consider these novel variants to be possibly pathogenic, even though variant c.2030T>A (p.Val677Glu) was located in the immediate downstream region, where mutations were clustered in the NC1 domain, and variant c.862G>C (p.Gly288Arg) was located in the triple-helical domain [Tan et al., 2008]. But the pathogenicity of these two novel possibly pathogenic missense variants are not clear because of absence of familial segregation analysis and use of in silico prediction software. We present the first report of the molecular characteristics of MCDS in 4 Korean patients and the first case of a possibly pathogenic variant in the triple-helical domain. Our findings suggest that the novel sequence variation involving an unusual mutational site of the COL10A1 gene can cause mild MCDS. Finally, the results
presented in this work warrants future in depth screening of the COL10A1 gene in weak suspects of (late detected) MCDS. Genotypeephenotype relationship studies of the COL10A1 gene in MCDS patients with variable clinical severity are needed.
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