European Journal of Medical Genetics 58 (2015) 105e110
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Clinical report
A novel CANT1 mutation in three Indian patients with Desbuquois dysplasia Kim type Ankur Singh a, Ok-Hwa Kim b, Aritoshi Iida c, Woong-Yang Park d, Shiro Ikegawa c, Seema Kapoor e, * a
Department of Pediatrics, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India Department of Radiology, Woorisoa Children’s Hospital, Seoul, Republic of Korea Laboratory for Bone and Joint Diseases, RIKEN Center for Integrated Medical Sciences, Tokyo, Japan d Translational Genomics Laboratory, Samsung Genome Institute, Samsung Medical Centre, Republic of Korea e Division of Genetics, MAMC Associated Lok Nayak Hospital, New Delhi, India b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 July 2014 Accepted 23 November 2014 Available online 5 December 2014
Desbuquois dysplasia (DBQD) is a rare skeletal dysplasia characterized by severe short stature, laxity, dislocation of multiple joints and developmental delay. DBQD is clinically heterogeneous. Distinct radiographic hand abnormalities such as the presence of extra-ossification distal to the second metacarpal or normal hand has led to its classification into types 1 and 2. Furthermore, the third type of DBQD, Kim type has been reported which is characterized by short metacarpals and elongated phalanges. However, DBQD Kim type has been exclusively reported in Japanese and Korean and its clinical characteristics remain to be delineated. Mutations in the calcium-activated nucleotidase 1 (CANT1) gene have been reported in all three types of DBQD. Previously reported patients with DBQD Kim type had a common mutation c.676G>A (p.Val226Met), which had a common founder between Japanese and Korean. Here, we report 3 Indian patients with DBQD, Kim type from 2 families which were unrelated to each other. We identified a novel mutation of CANT1, c.467C>T (p.Ser156Phe), in all the patients in the homozygous form. Our results show that DBQD Kim type is not exclusive to East Asians and also report a novel mutation from the Indian subcontinent. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Desbuquois dysplasia Kim type CANT1 Novel mutation
1. Introduction Desbuquois dysplasia (DBQD; MIM #251450) is a rare skeletal dysplasia included within the multiple joint dislocations group of the skeletal dysplasias [Warman et al., 2011]. Its clinical phenotypes include manifestations as micromelic short stature with flat midface, multiple joint dislocations, and progressive scoliosis. The skeletal findings are characterized by advanced carpal/tarsal bone ages, epi-metaphyseal dysplasia of the long bones and ‘Swedish key’ appearance of the proximal femora [Huber et al., 2009]. DBQD is clinically and radiographically heterogeneous. It has been classified into 2 types based on characteristic hand anomalies; i.e., whether or not typical hands with an extra ossification center distal to the second metacarpal and/or a delta phalanx of the thumb are present (type 1) or absent (type 2) [Faivre et al., 2004].
* Corresponding author. M-439, Ground Floor, Guruharkishan Nagar, Paschim Vihar, New Delhi, India. Tel.: þ91 23239417. E-mail address: [email protected] (S. Kapoor). http://dx.doi.org/10.1016/j.ejmg.2014.11.006 1769-7212/Ó 2014 Elsevier Masson SAS. All rights reserved.
Another variant of DBQD has been described by Kim, which is characterized by advanced carpal bone age without an extra ossification at the metacarpal but short metacarpals with relative phalangeal elongation associated with subluxation/dislocations of the phalangeal joint that is different from types 1 and 2. This in addition is also associated with big great toes with wide sandal gap and normal intelligence [Kim et al., 2010]. Recently, mutations in the CANT1 gene have been reported in DBQD types 1 and 2 and include 10 nonsense mutations, 12 missense mutations, 1 intronic splice site, 1 splice acceptor site and 3 frameshift mutations in 33 individuals with DBQD types 1 and type 2 [Faden et al., 2010; Furuichi et al., 2011; Huber et al., 2009; Laccone et al., 2011; Nizon et al., 2012]. CANT1 mutations have also been found in eight individuals with DBQD Kim type, all being missense mutations [Furuichi et al., 2011; Nizon et al., 2012]. Interestingly, all DBQD Kim type patients (except one from Turkey) had the same mutation at least on one allele, c.676G>A (p.Val226Met) which is presumed to be a common founder mutation between Korean and Japanese. Recently a second gene (XYLT1) for DBQD has been identified which is mainly
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A. Singh et al. / European Journal of Medical Genetics 58 (2015) 105e110
responsible for DBQD type 2 [Bui et al., 2014]. Here we report on 3 Indian patients showing characteristic radiographic features of DBQD Kim type but with a novel mutation in the CANT1 gene. All patients were homozygotes for the mutation. 2. Clinical reports 2.1. Patient 1 An 8-year-old boy was referred to the genetic unit for evaluation of short-limbed short stature and scoliosis (Fig. 1). He was born as full term to a mating between first cousins. His development as assessed by standard tests demonstrated his IQ to be normal except his motor milestones. He had started walking independently at the age of 3 years and going upstairs at 5 years of age. Anthropometric parameters at age of eight years were a height of 106 cm (4.8 z scores) and weight of 22 kg (1.0 z score). Dysmorphic evaluation revealed epicanthic folds, depressed nasal bridge, flat facial profile, with everted nostrils (Fig. 2). The hands were short with short thumbs and relatively small fingers (Fig. 3). The great toes were short and broad and demonstrated a wide sandal gap. The second and third toes were elongated. Radiographs revealed advanced carpal bone age, shortness of metacarpals in comparison to elongated phalanges (Fig. 4). The rest of the skeletal survey showed prominent lesser
Fig. 2. Clinical photograph of patient 1, aged 8 years male, showing epicanthic folds, depressed nasal bridge, flat facial profile, with everted nostrils.
trochanter (Swedish key appearance), dysplastic epiphyses of the long bones, and lumbar scoliosis (Fig. 5). Echocardiography revealed dextrocardia but a structurally normal heart. Biochemical parameters were normal.
Fig. 1. Clinical photograph of patient 1 aged 8 years male, showing scoliosis.
Fig. 3. Clinical photograph of hand of patient 1, aged 8 years male, showing short hand with short thumb with relatively small fingers.
A. Singh et al. / European Journal of Medical Genetics 58 (2015) 105e110
107
Fig. 4. Radiograph at age 8 years shows markedly advanced carpal bone ages (approximately 13 years) and apparently short the third and fourth metacarpals. The proximal and middle phalanges are rather elongated but the distal phalanges are short. Note almost fused phalangeal epiphyses.
2.2. Patients 2 and 3 Two siblings, who were not related to patient 1, were referred for evaluation of disproportionate short stature and varus deformity of knees. Sibs were products of mating between first cousins, born through vaginal delivery at home with no birth records. Elder male sib was 14 years and 2 months of age and the younger female was 12 years 9 months at the time of examination. The anthropometric parameters of the elder male sib were height of 115 cm (5.8 z score) and weight of 25 kg (2 z score). He had a round face, wide set eyes, joint laxity at knees and fingers, severe genu vara, short thumbs and greater toes, wide sandal gap and flat feet (Fig. 6). The striking features were very elongated fingers with clubbed and crooked appearance. The third and fourth digits were specifically elongated (Fig. 7). Radiographs of hand showed elongated phalanges with relative shortening of metacarpals (Fig. 8). X-ray pelvis showed characteristic Swedish key deformity of proximal femora (Fig. 9). Younger sister had similar dysmorphism and radiographic findings (Figs. 6, 10e12). Her height was 111 cm (6.1 z score). Clinical and radiographic findings of patient 1 and both sibs (patient 2 and 3) are tabulated in Table 1.
Fig. 5. Radiograph of pelvis of patient 1, aged 8 years male, showed Swedish key deformity of bilateral proximal femora.
Fig. 6. Clinical photograph of patient 2 (male) and patient 3 (female), showing round face, wide set eyes, joint laxity at knees and fingers, severe genu vara, short thumbs and greater toes, wide sandal gap and flat feet.
3. Mutation analysis After obtaining informed consents, genomic DNA was isolated from the peripheral blood from both affected and unaffected members of families. Each exon and the flanking intronic sequences of CANT1 were amplified using primers based on UCSC reference gene sequence. After purification, the PCR samples were directly sequenced using an ABI 3100 semi-automated sequencing analyzer.
Fig. 7. Clinical photograph of patient 2, aged 14 years 2 months, showing elongated fingers with clubbed and crooked appearance. The third and fourth digits were specifically elongated.
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Fig. 10. Clinical photograph of patient 3, aged 12 years 9 months, showing elongated fingers with clubbed and crooked appearance. The third and fourth digits were specifically elongated.
Fig. 8. Radiograph of hand of patient 2, aged 14 years 2 months, shows short metacarpals, especially at the third metacarpal and markedly elongated proximal and middle phalanges. The distal phalanges are short. Dislocation of interphalangeal joint of the thumb is noted. There appears to be premature fusion of all interphalangeal joints. Precocious fusion of intercarpal joints is evident. Note the elongated distal ulna touching the carpal bones.
The DNA sequences were analyzed using Finch TV version 1.4.0 (Geospiza, Seattle, WA). To exclude the possibility of polymorphism, c.467C>T was examined for 96 ethnicity-matched controls using the invader assay coupled with PCR [Ohnishi et al., 2001]. The missense mutation was evaluated by public databases including dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/), 1000 Genome (http://www.1000genomes.org/variation-pattern-finder), PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http:// sift.jcvi.org/) and PROVEAN (http://provean.jcvi.org/genome_ submit.php).
Fig. 9. X-ray pelvis showed characteristic Swedish key deformity of proximal femora. The greater trochanter of the femora are elevated. Capital femoral epiphyseal dysplasia is noted. Premature degenerative arthritis of the hip joints with narrowing of the joint spaces.
4. Result The sequence analysis showed that three affected individuals had a missense variant, c.467C>T (p.Ser156Phe) in the homozygous state. The invader assay coupled with PCR showed that the parents were carriers for the mutation and that c.467C>T was absent in 96 ethnicity-matched controls. This variant was not found in dbSNP and 1000 Genome database. The amino acid 156 was in the topological domain, which was moderately conserved among CANT1 of diverse species and related apyrase domain [Furuichi et al., 2011]. P.Ser156Phe was predicted to have a damaging function against normal gene product by PolyPhen2 (prediction score 1.00), SIFT (0), and PROVEAN (5.44). 5. Discussion In this study, we describe three Indian individuals with DBQD Kim type who had homozygous CANT1 c.467C>T mutations. This is
Fig. 11. Radiograph of hand of patient 3, aged 12 years 9 months, shows short metacarpals, especially at the third metacarpal and markedly elongated proximal and middle phalanges. The distal phalanges are short.
A. Singh et al. / European Journal of Medical Genetics 58 (2015) 105e110
Fig. 12. X-ray pelvis showed characteristic Swedish key deformity of proximal femora. Premature degenerative osteoarthritis of hip joints with narrowing of the joint spaces are noted.
the first report of the CANT1 mutation in DBDQ from the Indian subcontinent. In DBQD Kim type, only a recurrent allelic variant c.676G>A (p.Val226Met) was found in seven patients from Japan and Korea on at least one allele [Furuichi et al., 2011]. Recently,
109
c.909C>G (p.Ser303Arg) mutation was reported in patient of Turkish descent [Nizon et al., 2012]. We found high similarity in clinical and radiological features of the affected Indian individuals with the previously reported individuals with DBQD Kim variant. Predominant findings favoring the diagnosis of Kim variant are: elongated fingers, shortness of all or part of the metacarpals, club foot, short and broad great toe, elongated toes, wide sandal gap, flat feet and normal intelligence. Typical changes that further delineate Kim type are in hands and feet. Short metacarpals/ metatarsals with elongated middle and proximal phalanges and short distal phalanges were consistent radiological findings in the affected patients (Table 1). In our study, we detected a recurrent homozygous mutation, c.467C>T (p.Ser156Phe) that caused DBQD Kim type. This finding reveals that Kim variant is present in Indian population beyond Japan, Korea and Turkey. In conclusion, clinical and molecular data in our study further strengthen the involvement of CANT1 in the causation of DBQD Kim variant. This novel variant is present in coding region of the CANT1 gene. This report also delineates the wider distribution of DBQD Kim variant beyond East Asia region. Additional studies will be needed to understand the exact role of CANT1 in proteoglycan synthesis and the relation between the location of the mutation in CANT1 and phenotypic variation of DBQD subtypes.
Table 1 Clinical and radiological features of the three patients with Desbuquois dysplasia Kim variant.
A-Clinical Gender Parental consanguinity Birth weight Age at examination Height at examination (z scores) Intelligence Round face Wide set, protruding eyes Flat nose Thoracolumbar scoliosis Genu varum Joint laxity/knee Joint dislocation/finger Elongated finger Club foot Flat Foot Short and broad great toe Wide sandal gap Hypotonia Associated anomaly B-Radiological 1-Pelvis/Hip Swedish key appearance Elevated greater trochanter Proximal femoral epiphyseal flattening Coxa valga Joint space narrowing 2-Hands/Feet Advanced bone age Short metacarpal/metatarsal Phalangeal dislocations Precocious fusion of phalangeal epiphysis Narrowing of intercarpal/intertarsal bone space 3-Long bone Flat and dysplastic epiphysis Dislocation/subluxation 4-Spine Coronal cleft Scoliosis Disc space narrowing Degenerative spondylosis
Patient 1
Patient 2
Patient 3
Male þ Not known 8 years 106 cm (4.8) Normal þ þ þ þ þ þ þ þ þ Dextrocardia
Male þ Not known 14 years 2 months 115 (5.8) Normal þ þ þ þ þ þ þ þ þ þ þ
Female þ Not known 12 years 9 months 111 (6.1) Normal þ þ þ þ þ þ þ þ þ þ þ
þ þ þ
þ þ þ þ þ
þ þ þ þ þ
þ þ þ _
þ þ þ þ þ
þ þ þ þ þ
þ
þ Knee, elbow, shoulder
þ Knee, patella
þ þ
þ þ þ
þ
110
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Funding None.
Conflict of interest None.
References Bui C, Huber C, Tuysuz B, Alanay Y, Bole-Feysot C, Leroy JG, et al. XYLT1 mutations in Desbuquois dysplasia type 2. Am J Hum Genet 2014;94:405e14. Faden M, Al-Zahrani F, Arafah D, Alkuraya FS. Mutation of CANT1 causes Desbuquois dysplasia. Am J Med Genet A 2010;152:1157e60. Faivre L, Cormier-Daire V, Eliott AM, Field F, Munnich A, Maroteaux P, et al. Desbuquois dysplasia, a reevaluation with abnormal and “normal” hands: radiographic manifestations. Am J Med Genet A 2004;124:48e53.
Furuichi T, Dai J, Cho TJ, Sakazume S, Ikema M, Matsui Y, et al. CANT1 mutation is also responsible for Desbuquois dysplasia, type 2 and Kim variant. J Med Genet 2011;48:32e7. Huber C, Oules B, Bertoli M, Chami M, Fradin M, Alanay Y, et al. Identification of CANT1 mutations in Desbuquois dysplasia. Am J Hum Genet 2009;85:706e10. Kim OH, Nishimura G, Song HR, Matsui Y, Sakazume S, Yamada M, et al. A variant of Desbuquois dysplasia characterized by advanced carpal bone age, short metacarpals, and elongated phalanges: report of seven cases. Am J Med Genet A 2010;152:875e85. Laccone F, Schoner K, Krabichler B, Kluge B, Schwerdtfeger R, Schulze B, et al. Desbuquois dysplasia type I and fetal hydrops due to novel mutations in the CANT1 gene. Eur J Hum Genet 2011;19:1133e7. Nizon M, Huber C, De Leonardis F, Merrina R, Forlino A, Fradin M, et al. Further delineation of CANT1 phenotypic spectrum and demonstration of its role in proteoglycan synthesis. Hum Mutat 2012;33:1261e6. Ohnishi Y, Tanaka T, Ozaki K, Yamada R, Suzuki H, Nakamura Y. A high-throughput SNP typing system for genome-wide association studies. J Hum Genet 2001;46: 471e7. Warman ML, Cormier-Daire V, Hall C, Krakow D, Lachman R, LeMerrer M, et al. Nosology and classification of genetic skeletal disorders: 2010 revision. Am J Med Genet A 2011;155:943e68.
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Clinical report
A novel CANT1 mutation in three Indian patients with Desbuquois dysplasia Kim type Ankur Singh a, Ok-Hwa Kim b, Aritoshi Iida c, Woong-Yang Park d, Shiro Ikegawa c, Seema Kapoor e, * a
Department of Pediatrics, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India Department of Radiology, Woorisoa Children’s Hospital, Seoul, Republic of Korea Laboratory for Bone and Joint Diseases, RIKEN Center for Integrated Medical Sciences, Tokyo, Japan d Translational Genomics Laboratory, Samsung Genome Institute, Samsung Medical Centre, Republic of Korea e Division of Genetics, MAMC Associated Lok Nayak Hospital, New Delhi, India b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 July 2014 Accepted 23 November 2014 Available online 5 December 2014
Desbuquois dysplasia (DBQD) is a rare skeletal dysplasia characterized by severe short stature, laxity, dislocation of multiple joints and developmental delay. DBQD is clinically heterogeneous. Distinct radiographic hand abnormalities such as the presence of extra-ossification distal to the second metacarpal or normal hand has led to its classification into types 1 and 2. Furthermore, the third type of DBQD, Kim type has been reported which is characterized by short metacarpals and elongated phalanges. However, DBQD Kim type has been exclusively reported in Japanese and Korean and its clinical characteristics remain to be delineated. Mutations in the calcium-activated nucleotidase 1 (CANT1) gene have been reported in all three types of DBQD. Previously reported patients with DBQD Kim type had a common mutation c.676G>A (p.Val226Met), which had a common founder between Japanese and Korean. Here, we report 3 Indian patients with DBQD, Kim type from 2 families which were unrelated to each other. We identified a novel mutation of CANT1, c.467C>T (p.Ser156Phe), in all the patients in the homozygous form. Our results show that DBQD Kim type is not exclusive to East Asians and also report a novel mutation from the Indian subcontinent. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Desbuquois dysplasia Kim type CANT1 Novel mutation
1. Introduction Desbuquois dysplasia (DBQD; MIM #251450) is a rare skeletal dysplasia included within the multiple joint dislocations group of the skeletal dysplasias [Warman et al., 2011]. Its clinical phenotypes include manifestations as micromelic short stature with flat midface, multiple joint dislocations, and progressive scoliosis. The skeletal findings are characterized by advanced carpal/tarsal bone ages, epi-metaphyseal dysplasia of the long bones and ‘Swedish key’ appearance of the proximal femora [Huber et al., 2009]. DBQD is clinically and radiographically heterogeneous. It has been classified into 2 types based on characteristic hand anomalies; i.e., whether or not typical hands with an extra ossification center distal to the second metacarpal and/or a delta phalanx of the thumb are present (type 1) or absent (type 2) [Faivre et al., 2004].
* Corresponding author. M-439, Ground Floor, Guruharkishan Nagar, Paschim Vihar, New Delhi, India. Tel.: þ91 23239417. E-mail address: [email protected] (S. Kapoor). http://dx.doi.org/10.1016/j.ejmg.2014.11.006 1769-7212/Ó 2014 Elsevier Masson SAS. All rights reserved.
Another variant of DBQD has been described by Kim, which is characterized by advanced carpal bone age without an extra ossification at the metacarpal but short metacarpals with relative phalangeal elongation associated with subluxation/dislocations of the phalangeal joint that is different from types 1 and 2. This in addition is also associated with big great toes with wide sandal gap and normal intelligence [Kim et al., 2010]. Recently, mutations in the CANT1 gene have been reported in DBQD types 1 and 2 and include 10 nonsense mutations, 12 missense mutations, 1 intronic splice site, 1 splice acceptor site and 3 frameshift mutations in 33 individuals with DBQD types 1 and type 2 [Faden et al., 2010; Furuichi et al., 2011; Huber et al., 2009; Laccone et al., 2011; Nizon et al., 2012]. CANT1 mutations have also been found in eight individuals with DBQD Kim type, all being missense mutations [Furuichi et al., 2011; Nizon et al., 2012]. Interestingly, all DBQD Kim type patients (except one from Turkey) had the same mutation at least on one allele, c.676G>A (p.Val226Met) which is presumed to be a common founder mutation between Korean and Japanese. Recently a second gene (XYLT1) for DBQD has been identified which is mainly
106
A. Singh et al. / European Journal of Medical Genetics 58 (2015) 105e110
responsible for DBQD type 2 [Bui et al., 2014]. Here we report on 3 Indian patients showing characteristic radiographic features of DBQD Kim type but with a novel mutation in the CANT1 gene. All patients were homozygotes for the mutation. 2. Clinical reports 2.1. Patient 1 An 8-year-old boy was referred to the genetic unit for evaluation of short-limbed short stature and scoliosis (Fig. 1). He was born as full term to a mating between first cousins. His development as assessed by standard tests demonstrated his IQ to be normal except his motor milestones. He had started walking independently at the age of 3 years and going upstairs at 5 years of age. Anthropometric parameters at age of eight years were a height of 106 cm (4.8 z scores) and weight of 22 kg (1.0 z score). Dysmorphic evaluation revealed epicanthic folds, depressed nasal bridge, flat facial profile, with everted nostrils (Fig. 2). The hands were short with short thumbs and relatively small fingers (Fig. 3). The great toes were short and broad and demonstrated a wide sandal gap. The second and third toes were elongated. Radiographs revealed advanced carpal bone age, shortness of metacarpals in comparison to elongated phalanges (Fig. 4). The rest of the skeletal survey showed prominent lesser
Fig. 2. Clinical photograph of patient 1, aged 8 years male, showing epicanthic folds, depressed nasal bridge, flat facial profile, with everted nostrils.
trochanter (Swedish key appearance), dysplastic epiphyses of the long bones, and lumbar scoliosis (Fig. 5). Echocardiography revealed dextrocardia but a structurally normal heart. Biochemical parameters were normal.
Fig. 1. Clinical photograph of patient 1 aged 8 years male, showing scoliosis.
Fig. 3. Clinical photograph of hand of patient 1, aged 8 years male, showing short hand with short thumb with relatively small fingers.
A. Singh et al. / European Journal of Medical Genetics 58 (2015) 105e110
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Fig. 4. Radiograph at age 8 years shows markedly advanced carpal bone ages (approximately 13 years) and apparently short the third and fourth metacarpals. The proximal and middle phalanges are rather elongated but the distal phalanges are short. Note almost fused phalangeal epiphyses.
2.2. Patients 2 and 3 Two siblings, who were not related to patient 1, were referred for evaluation of disproportionate short stature and varus deformity of knees. Sibs were products of mating between first cousins, born through vaginal delivery at home with no birth records. Elder male sib was 14 years and 2 months of age and the younger female was 12 years 9 months at the time of examination. The anthropometric parameters of the elder male sib were height of 115 cm (5.8 z score) and weight of 25 kg (2 z score). He had a round face, wide set eyes, joint laxity at knees and fingers, severe genu vara, short thumbs and greater toes, wide sandal gap and flat feet (Fig. 6). The striking features were very elongated fingers with clubbed and crooked appearance. The third and fourth digits were specifically elongated (Fig. 7). Radiographs of hand showed elongated phalanges with relative shortening of metacarpals (Fig. 8). X-ray pelvis showed characteristic Swedish key deformity of proximal femora (Fig. 9). Younger sister had similar dysmorphism and radiographic findings (Figs. 6, 10e12). Her height was 111 cm (6.1 z score). Clinical and radiographic findings of patient 1 and both sibs (patient 2 and 3) are tabulated in Table 1.
Fig. 5. Radiograph of pelvis of patient 1, aged 8 years male, showed Swedish key deformity of bilateral proximal femora.
Fig. 6. Clinical photograph of patient 2 (male) and patient 3 (female), showing round face, wide set eyes, joint laxity at knees and fingers, severe genu vara, short thumbs and greater toes, wide sandal gap and flat feet.
3. Mutation analysis After obtaining informed consents, genomic DNA was isolated from the peripheral blood from both affected and unaffected members of families. Each exon and the flanking intronic sequences of CANT1 were amplified using primers based on UCSC reference gene sequence. After purification, the PCR samples were directly sequenced using an ABI 3100 semi-automated sequencing analyzer.
Fig. 7. Clinical photograph of patient 2, aged 14 years 2 months, showing elongated fingers with clubbed and crooked appearance. The third and fourth digits were specifically elongated.
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Fig. 10. Clinical photograph of patient 3, aged 12 years 9 months, showing elongated fingers with clubbed and crooked appearance. The third and fourth digits were specifically elongated.
Fig. 8. Radiograph of hand of patient 2, aged 14 years 2 months, shows short metacarpals, especially at the third metacarpal and markedly elongated proximal and middle phalanges. The distal phalanges are short. Dislocation of interphalangeal joint of the thumb is noted. There appears to be premature fusion of all interphalangeal joints. Precocious fusion of intercarpal joints is evident. Note the elongated distal ulna touching the carpal bones.
The DNA sequences were analyzed using Finch TV version 1.4.0 (Geospiza, Seattle, WA). To exclude the possibility of polymorphism, c.467C>T was examined for 96 ethnicity-matched controls using the invader assay coupled with PCR [Ohnishi et al., 2001]. The missense mutation was evaluated by public databases including dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/), 1000 Genome (http://www.1000genomes.org/variation-pattern-finder), PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http:// sift.jcvi.org/) and PROVEAN (http://provean.jcvi.org/genome_ submit.php).
Fig. 9. X-ray pelvis showed characteristic Swedish key deformity of proximal femora. The greater trochanter of the femora are elevated. Capital femoral epiphyseal dysplasia is noted. Premature degenerative arthritis of the hip joints with narrowing of the joint spaces.
4. Result The sequence analysis showed that three affected individuals had a missense variant, c.467C>T (p.Ser156Phe) in the homozygous state. The invader assay coupled with PCR showed that the parents were carriers for the mutation and that c.467C>T was absent in 96 ethnicity-matched controls. This variant was not found in dbSNP and 1000 Genome database. The amino acid 156 was in the topological domain, which was moderately conserved among CANT1 of diverse species and related apyrase domain [Furuichi et al., 2011]. P.Ser156Phe was predicted to have a damaging function against normal gene product by PolyPhen2 (prediction score 1.00), SIFT (0), and PROVEAN (5.44). 5. Discussion In this study, we describe three Indian individuals with DBQD Kim type who had homozygous CANT1 c.467C>T mutations. This is
Fig. 11. Radiograph of hand of patient 3, aged 12 years 9 months, shows short metacarpals, especially at the third metacarpal and markedly elongated proximal and middle phalanges. The distal phalanges are short.
A. Singh et al. / European Journal of Medical Genetics 58 (2015) 105e110
Fig. 12. X-ray pelvis showed characteristic Swedish key deformity of proximal femora. Premature degenerative osteoarthritis of hip joints with narrowing of the joint spaces are noted.
the first report of the CANT1 mutation in DBDQ from the Indian subcontinent. In DBQD Kim type, only a recurrent allelic variant c.676G>A (p.Val226Met) was found in seven patients from Japan and Korea on at least one allele [Furuichi et al., 2011]. Recently,
109
c.909C>G (p.Ser303Arg) mutation was reported in patient of Turkish descent [Nizon et al., 2012]. We found high similarity in clinical and radiological features of the affected Indian individuals with the previously reported individuals with DBQD Kim variant. Predominant findings favoring the diagnosis of Kim variant are: elongated fingers, shortness of all or part of the metacarpals, club foot, short and broad great toe, elongated toes, wide sandal gap, flat feet and normal intelligence. Typical changes that further delineate Kim type are in hands and feet. Short metacarpals/ metatarsals with elongated middle and proximal phalanges and short distal phalanges were consistent radiological findings in the affected patients (Table 1). In our study, we detected a recurrent homozygous mutation, c.467C>T (p.Ser156Phe) that caused DBQD Kim type. This finding reveals that Kim variant is present in Indian population beyond Japan, Korea and Turkey. In conclusion, clinical and molecular data in our study further strengthen the involvement of CANT1 in the causation of DBQD Kim variant. This novel variant is present in coding region of the CANT1 gene. This report also delineates the wider distribution of DBQD Kim variant beyond East Asia region. Additional studies will be needed to understand the exact role of CANT1 in proteoglycan synthesis and the relation between the location of the mutation in CANT1 and phenotypic variation of DBQD subtypes.
Table 1 Clinical and radiological features of the three patients with Desbuquois dysplasia Kim variant.
A-Clinical Gender Parental consanguinity Birth weight Age at examination Height at examination (z scores) Intelligence Round face Wide set, protruding eyes Flat nose Thoracolumbar scoliosis Genu varum Joint laxity/knee Joint dislocation/finger Elongated finger Club foot Flat Foot Short and broad great toe Wide sandal gap Hypotonia Associated anomaly B-Radiological 1-Pelvis/Hip Swedish key appearance Elevated greater trochanter Proximal femoral epiphyseal flattening Coxa valga Joint space narrowing 2-Hands/Feet Advanced bone age Short metacarpal/metatarsal Phalangeal dislocations Precocious fusion of phalangeal epiphysis Narrowing of intercarpal/intertarsal bone space 3-Long bone Flat and dysplastic epiphysis Dislocation/subluxation 4-Spine Coronal cleft Scoliosis Disc space narrowing Degenerative spondylosis
Patient 1
Patient 2
Patient 3
Male þ Not known 8 years 106 cm (4.8) Normal þ þ þ þ þ þ þ þ þ Dextrocardia
Male þ Not known 14 years 2 months 115 (5.8) Normal þ þ þ þ þ þ þ þ þ þ þ
Female þ Not known 12 years 9 months 111 (6.1) Normal þ þ þ þ þ þ þ þ þ þ þ
þ þ þ
þ þ þ þ þ
þ þ þ þ þ
þ þ þ _
þ þ þ þ þ
þ þ þ þ þ
þ
þ Knee, elbow, shoulder
þ Knee, patella
þ þ
þ þ þ
þ
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Funding None.
Conflict of interest None.
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