Available online at www.annclinlabsci.org

Mutation Screening in Candidate Genes in Four Chinese Brachydactyly FamiliesSufang Dong1, Yinghui Wang2, Shengxiang Tao3, and Fang Zheng1

1Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan, 2Department of Ultrasonography, Zhong-nan Hospital, Wuhan University, Wuhan, and 3Department of Orthopedic, Zhongnan Hospital, Wuhan University, Wuhan, China

Abstract. Autosomal dominant brachydactyly (BD) is a skeletal disorder with several subtypes, including brachydactyly type A1 (BDA1) and brachydactyly type B1 (BDB1). Mutations in Indian hedgehog (IHH) are usually associated with BDA1, whereas heterozygous mutations in receptor tyrosine kinase-like orphan receptor 2 (ROR2) are mainly responsible for BDB1. On the basis of the clinical phenotype identification, we screened IHH and ROR2 by the candidate gene approach using PCR direct sequencing. We found three known mutations of IHH (c.283_285delGAG, p.E95del; c.298 G>A, p.D100N; c.300C>G, p.D100E) in three Chinese families with BDA1, and a novel heterozygous nonsense mutation of ROR2 (c.2273C>A, p.S758X) in a BDB1 family. It was noted that c.300C>G mutation was a new nucleotide substitution compared to the reported c.300C>A, which led to the same amino acid change (p.D100E). The novel non-sense mutation p.S758X was verified by absence in the unaffected family members and the 100 randomly-selected controls. In this paper, we report three recurrent mutations with a new nucleotide substitution of IHH in three Chinese families with BDA1 and a novel nonsense mutation in BDB1 pedigree. We therefore recommend the approach of candidate gene screening as the first choice for genetic testing for BD.

Key words: BDA1, BDB1, IHH, and ROR2

Introduction

Brachydactyly (BD), a general term for short digits, refers to abnormal development of the phalanges and/or metacarpals. Heritable BDs have been clas-sified into the subtypes A-E according to their pat-terns of skeletal involvement [1].

Brachydactyly type A1 (BDA1) (Online Mendelian Inheritance in Man Identification [OMIM ID]: 112500) was first interpreted as an autosomal-dominant Mendelian inheritance disorder. It is characterized by shortened middle phalanges in the hands and feet, shortness of the metacarpals except for the first digit, short stature and occasional fu-sion of the middle and terminal phalanges [2,3]. Mutations in Indian hedgehog (IHH) (GenBank Gene Identification [GBG ID]: 3549) located on chromosome 2q35-q36 were reported to cause the dominant disorder, BDA1 [4,5]. IHH is a central

signaling molecule in mediating skeletal develop-ment including condensation, growth and differen-tiation of chondrocyte, and development of joints and bone formation [6-8].

However, autosomal dominant BD type B (BDB) is the most severe form of BD. It is readily distin-guished from other subtypes by the characteristic of a split distal phalanges of thumbs, shortening/hy-poplasia of the distal and middle phalanges, nail dysplasia, and variable degrees of distal and proxi-mal symphalangism, while the feet are similarly but less severely affected [9]. BDB1 (OMIM ID: 113000) is usually caused by heterozygous truncat-ing mutations in the cytoplasmic region of receptor tyrosine kinase-like orphan receptor 2 (ROR2) (GBG ID: 4920) on 9q22 [9]. Homozygous loss of func-tion mutations spread throughout ROR2 have been shown to cause recessive Robinow syndrome (RRS) (OMIM ID: 268310), which involves more exten-sive and serious damage including vertebral anoma-lies, brachymelia of the arms, and ventricular septal defect [10,11].

Annals of Clinical & Laboratory Science, vol. 45, no. 1, 2015

0091-7370/15/001-094. © 2015 by the Association of Clinical Scientists, Inc.

Address correspondence to Fang Zheng, Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan, 430071, China; fax: +86 27 67813233; e mail: zhengfang@whu.edu.cn

94

In the present study, we identified three known mutations including two missense mutations and one deletion mutation in IHH in BDA1 families and a novel nonsense mutation in ROR2 in a BDB1 pedigree by the candidate gene approach.

Materials and Methods

Clinical Description. Four Chinese BD pedigrees A, B, C and D (Figure 1) were recruited in Zhongnan Hospital (Wuhan, China). Written informed consents were ob-tained from all participating adults and the parents of children under 18 years old. Participants underwent clinical radiographic examination of the hands and feet to assess their BD phenotype. One hundred individuals of the Chinese Han population without diagnostic fea-tures of BD were recruited from the Examination Center of Zhongnan Hospital to serve as controls. This research was approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University and followed the tenets of the Declaration of Helsinki.

Mutation screening. DNA samples were isolated from venous blood samples by standard phenol/chloroform extraction. Mutation detection was performed through the candidate gene approach. IHH was screened in Families A, B, and C with BDA1, and ROR2 was screened in family D with BDB1. Both genes were ana-lyzed by PCR amplification followed by direct DNA se-quencing. The PCR primers were designed in the flank-ing intron sequences.

The PCR conditions included an initial denaturation for 5 min at 95°C, followed by 35 cycles for 30 sec at 95°C, 45 sec at annealing temperature, 45 sec at 72°C, and a final extension for 10 min at 72°C. The PCR products were sequenced by the ABI 3130 genetic analyzer (Life

Technologies Corporation, CA, USA) in the Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University. The sequencing results were analyzed using Chromas (version 2.0) and com-pared with the reference sequence in the NCBI database.

SSCP Analysis. Since the nonsense mutation discovered in family D was novel, single-strand conforma-tion polymorphism (SSCP) analy-sis was performed to determine whether the mutation was co-segre-gated with the disease in Family D and absent in 100 randomly chosen controls.

In brief, a specific pair of primers for SSCP analysis was designed. The PCR product of the segment containing the putative mutation in ROR2 was tested by SSCP as previously described [12]. All participating family members in Family D and some random controls were chosen to verify the SSCP results by DNA sequencing.

Results

Clinical Description. There were similar clinical phenotypes in affected individuals of Families A, B and C, such as the appearance of the hands and feet, height, and radiographic characteristics. In Family A, the proband AII-1 was 168 cm in height. Radiographic analysis of his hands showed bilateral shortening of the middle phalanges as well as broad epiphyses of metacarpals and proximal phalanges in digits 2-5, but the proximal phalange of digit 1 was unaffected and only showed shortened distal pha-langes (Figure 2A). Features of the proband’s feet were similar to those of his hands but more serious, with middle phalanges missing in toes 3-5 (Figure 2A). In Family B, the proband BII-1 was 155cm in height. Her radiographs showed more serious com-plications in the hands and feet than AII-1 (Figure 3). Her hands showed bilateral fused middle pha-langes in fingers 2,4, and 5, shortened middle pha-langes in finger 3, shortened metacarpals in fingers 3,4,5, and malformed epiphyses (Figure 2B). The feet showed similar signs, and all middle phalanges were fused with distal phalanges (Figure 2B). In Family C, the three-year-old proband (CII-1) dis-played shortened middle phalanges; neither the fu-sion of the phalanges nor the absence of the middle

Figure 1. Pedigrees of BD families. A-C. The pedigree of BDA1. D.The pedi-gree of BDB1. The arrow points to the proband. Asterisks sign indicates the in-dividual participating in this research.

Mutation identification of IHH and ROR2 in brachydactyly disease 95

phalanges were observed (Figure 2C). Other clini-cal features could not be identified because of the development of the bones. Families A, B and C were thus identified and characterized with a clini-cal diagnosis of BDA1. All individuals in the above three families participated in our study.

Twelve individuals in Family D participated in our study, among which there were three affected indi-viduals (Figure 1D). The proband DII-5 exhibited classical manifestations including large flat thumbs, bilateral hypoplasia of the distal and middle pha-langes of fingers 2-5, and nail hypoplasia, or even the absence of nails on some fingers and toes. Radiographs of the proband showed split distal phalanges of the thumbs and hypoplasia of the dis-tal and middle phalanges. The metacarpals and metatarsals were of normal number, and the feet had a similar appearance (Figure 2D,E). DIII-3 had more serious signs, including no nails of many fingers and severe absence/hypoplasia of the distal/middle phalanges than the proband DII-5 (Figure 2E). It was noted that the bigger flat thumb of DIII-3 underwent plastic surgery. No other congre-gating medical problems were reported. Family D was thus identified and characterized with a clinical diagnosis of BDB1.

Mutation screening. Since IHH was the most com-mon candidate gene for autosomal dominant BDA1, while ROR2 was responsible for autosomal dominant BDB1, IHH was screened in all mem-bers of Families A, B and C while ROR2 was screened in the participants from Family D. In Family A, sequencing analysis of the PCR product

amplified from the affected showed a heterozygous deletion mutation (c.283_285delGAG; p. E95del) (Figure 3A). In Family B, a heterozygous missense mutation (c.300C>G; p. D100E) (Figure 3A) was identified in all affected members. In Family C, there was only one patient (C-II1), and he had a heterozygous missense mutation (c.298G>A; p. D100N) (Figure 3A), which we hypothesized was a spontaneous mutation since the parents didn’t carry it. All of the above mutations were located in exon 1 of IHH. ROR2 in all recruited Family D members was screened by PCR-based direct DNA sequencing. A new heterozygous C>A mutation (c.2273C>A; p.S758X) was identified. The muta-tion led to a stop codon in all three affected mem-bers (Figure 3B). There were no such mutations in the unaffected individuals in the four families.

SSCP results. The SSCP results showed that there was no p.S758X mutation in the unaffected family members or the 100 controls. According to the electrophoresis pattern, there were only two seg-ments in the lanes of samples from unaffected fam-ily members and controls. In contrast, three bands appeared in those from the affected individuals. It suggested that the c.2273C>A heterozygous muta-tion was co-segregated perfectly with the disease in Family B and was not present in the controls. The SSCP results of some randomly chosen controls were verified by DNA sequencing. Since the other three mutations in IHH were previously found, verified, and functionally studied in other BDA1 families, we regarded them as causative mutations for BDA1, and mutation verification in the control population was not carried out [4,13].

Figure 2. Phenotypes of hands and feet in four Chinese families. Hands and feet radiographs of AII-1 (A), BII-1 (B), CII-1 (C). (D) Radiographs of the DII-5’s hands and feet. (E) The DII-5’s hands (above) and the DIII-3’s hands (below). Red Arrow points shortened middle phalanges; white arrow points a split distal phalange of thumb.

Annals of Clinical & Laboratory Science, vol. 45, no. 1, 201596

Mutation identification of IHH and ROR2 in brachydactyly disease

Discussion

BDA1 or BDB1 phenotypes in four Chinese fami-lies were identified through clinical and radio-graphic examination of the hands and feet. All the affected individuals showed typical clinical features, such as bilateral and marked shortening of the mid-dle phalanges on hands and feet in the BDA1 fam-ily, or hypoplasia of middle/terminal phalanges on the hands and feet, nail hypoplasia, or missing as well as bifid thumbs in the BDB1 family. Though BDs can be classified to five subtypes (A-E) and there are commonly more than one candidate gene for each type, IHH and ROR2 have been reported as the common candidate genes for autosomal dominant BDA1 and BDB1, respectively. Mutation screening by the candidate gene approach was per-formed in four families, and the causative muta-tions were identified rapidly. These results indicate the candidate gene approach, based on comprehen-sive and thorough clinical characteristic analysis, is an efficient method for clinical genetic testing for BD.

In Family A, a three-nucleotide deletion (c.283_285delGAG; p. E95del) in IHH led to a deletion of Glu amino acid, which was reported by

Lodder in a three-generation Dutch family with BDA1 [14]. In Family B, the p.D100E mutation caused by c.300C>A had been reported [13], but we identified a new nucleotide substitution of c.300C>G leading to the same amino acid change. In Family C, the known mutation (c.298G>A; p. D100N) was consid-ered spontaneous, based on the ab-sence of the mutation in the parents. All three mutations presented here were in the highly-conserved N-terminal signaling domain of IHH, and might affect the interaction between IHH and receptor Patched (PTC) through changes in the polar-ity of the charged area, the local ter-tiary structure, or the temperature-sensitive and calcium-dependent stability of this domain, and thus cause abnormal bone development and formation [15].

In Family D, we found a novel nonsense mutation (c.2273C>A; p.S758X) resulting in an intracellular protein truncation of 186 amino acids located in serine/threonine-rich domain of the exon 9. This mutation co-segregated perfectly with the disease in this family, and was further confirmed as a muta-tion by its absence in 100 controls. To our knowl-edge, 11 mutations in ROR2 responsible for BDB1 had been reported (Table 1), and the twelfth muta-tion was identified in our study. Almost all were lo-cated in exons 8 and 9 of ROR2.

Mutations in ROR2 that led to BDB1 were almost heterozygote, were located in the cytoplasmic re-gion immediately before or after the tyrosine kinase (TK) domain of ROR2, and resulted in intracellular protein truncation of the C terminal region [16,17]. The p.S758X, without exception, also accorded with this heterozygous mutation located in serine-threonine rich (ST1) domain after the TK domain. Since ROR2 has an important role in normal chon-drocyte differentiation, and activation of ROR2 ki-nase requires the C terminal region for recruitment of the non-receptor kinase Src (v-src avian sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog) and was also mediated by Scr phosphorylation, the

Figure 3. A. Sequencing results in 4 BDA1 pedigrees. The wild-type nu-cleotide sequence in the unaffected (above left); 283_285delGAG muta-tion (the red line) in family A (above right); 300C>G mutation (red ar-row) in the family B (below left); 298 G>A mutation (red arrow) in family C (below right). B. Sequencing results in BDB1 pedigree. The wild-type nucleotide sequence in the unaffected (left); 2273C>A mutation (red ar-row) in family D (right).

97

p.S758X mutation yielded a receptor of partial pro-tein truncation in the cytoplasmic region. This re-ceptor, which was competent to bind Wnt (wing-less-type MMTV integration site family) ligand but unable to produce a productive tyrosine phosphor-ylation signal response, might be the reason for the defect in kinase activation via failure to recruit Src [17]. It was demonstrated that the distal mutations after the TK domain had a more severe phenotype than the proximal mutation before the TK domain [9,18]. Intriguingly, the p.S758X mutation belongs to the distal mutation, but it only cause a mild phe-notype in the proband and a moderately severe phenotype in his offspring according to Schwabe’s grouping criteria (Figure 2D,E)[9]. This issue could be confirmed or overturned by more samples and requires further functional studies for ROR2.

In summary, we reported the recurrence of three mutations in IHH leading to BDA1 and the identi-fication of a novel mutation in ROR2, associated with autosomal dominant BDB1. We recommend the candidate gene approach as a preferred method in genetic testing for BD.

Acknowledgements

We are grateful for the participation of the families in this re-search. This study was supported by the National Basic Research Program of China (973 Program, 2012CB720605) and the Fundamental Research Funds for the Central Universities (Grant No. 2012303020210).

References

1. Laboratory G, Bell J. The Treasury of Human Inheritance: On Hereditary Digital Anomalies. On Brachydactyly and Symphalangism. Cambridge University Press; 1951.

2. Bo G, Lin H. Answering a century old riddle: brachydactyly type A1. Cell research 2004; 14:179-187.

3. Lacombe D, Delrue MA, Rooryck C, Morice‐Picard F, Arveiler B, Maugey‐Laulom B, Mundlos S, Toutain A, Chateil JF. Brachydactyly type A1 with short humerus and associated skel-etal features. American Journal of Medical Genetics Part A 2010; 152:3016-3021.

4. Byrnes AM, Racacho L, Grimsey A, Hudgins L, Kwan AC, Sangalli M, Kidd A, Yaron Y, Lau Y-L, Nikkel SM. Brachydactyly A-1 mutations restricted to the central region of the N-terminal active fragment of Indian Hedgehog. European Journal of Human Genetics 2009; 17:1112-1120.

5. Stattin E-L, Lindén B, Lönnerholm T, Schuster J, Dahl N. Brachydactyly type A1 associated with unusual radiological findings and a novel Arg158Cys mutation in the Indian hedge-hog (< i> IHH</i>) gene. European journal of medical genetics 2009; 52:297-302.

6. Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996; 273:613-622.

Table 1. Mutations in ROR2 identified in BDB1.

Nucleotide change Amino acid change Exon/intron Domaina Mutation nature Reference

1386+3_1386+5del3ins19 Complicated changeb Intron 8 before TK Heterozygous [9]1321_1325 Del CGGCG R441fsX15 Exon 8 before TK Homozygous [9]1324 C>Tc R441fsX15 Exon 8 before TK Heterozygous [19]1366_1367 insC L456fsX1 Exon 8 before TK Heterozygous [20]1398_1399 insA E467fsX57 Exon 9 before TK Heterozygous [9,21]2243 delC W748fsX24 Exon 9 after TK Heterozygous [22]2246 G>A W749X Exon 9 after TK Heterozygous [23]2247 G>A W749X Exon 9 after TK Heterozygous [24]2249 delG G750fsX23 Exon 9 after TK Heterozygous [23]2265 C>A Y755X Exon 9 after TK (ST1) Heterozygous [18,23]2273 C>A S758X Exon 9 after TK (ST1) Heterozygous Present study2278 C>T Q760X Exon 9 after TK (ST1) Heterozygous [9,25]

aBefore Tyrosine Kinase (TK) domain was same as N-terminal or proximal region; after TK domain and in ST1 domain was same as C-terminal or distal region. bComplicated change contained a 3-bp (CTC) deletion, which was replaced by a 19-bp fragment insertion (TACATTGTGTAAGCACAAA) at the 5’ (donor) splice site of intron 8, and resulted in an insertion of three amino acids, a frameshift and a premature stop codon. cThe patient carried the p.R441X mutation showed combined features of RRS and severe BDB1.

Annals of Clinical & Laboratory Science, vol. 45, no. 1, 201598

Mutation identification of IHH and ROR2 in brachydactyly disease

7. Long F, Zhang XM, Karp S, Yang Y, McMahon AP. Genetic manipulation of hedgehog signaling in the endochondral skel-eton reveals a direct role in the regulation of chondrocyte pro-liferation. Development 2001; 128:5099-5108.

8. Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, Karperien M, Defize LH, Ho C, Mulligan RC. PTH/PTHrP Receptor in Early Development and Indian Hedgehog--Regulated Bone Growth. Science 1996; 273:663-666.

9. Schwabe GC, Tinschert S, Buschow C, Meinecke P, Wolff G, Gillessen-Kaesbach G, Oldridge M, Wilkie AO, Kömec R, Mundlos S. Distinct Mutations in the Receptor Tyrosine Kinase Gene< i> ROR2</i> Cause Brachydactyly Type B. The American Journal of Human Genetics 2000; 67:822-831.

10. Afzal AR, Rajab A, Fenske CD, Oldridge M, Elanko N, Ternes-Pereira E, Tüysüz B, Murday VA, Patton MA, Wilkie AO. Recessive Robinow syndrome, allelic to dominant brachy-dactyly type B, is caused by mutation of ROR2. Nat Genet 2000; 25:419-422.

11. van Bokhoven H, Celli J, Kayserili H, van Beusekom E, Balci S, Brussel W, Skovby F, Kerr B, Percin EF, Akarsu N. Mutation of the gene encoding the ROR2 tyrosine kinase causes autoso-mal recessive Robinow syndrome. Nat Genet 2000; 25:423-426.

12. Cheng X, Ding J, Zheng F, Zhou X, Xiong C. Two mutations in LDLR gene were found in two Chinese families with famil-ial hypercholesterolemia. Molecular biology reports 2009; 36:2053-2057.

13. Gao B, Guo J, She C, Shu A, Yang M, Tan Z, Yang X, Guo S, Feng G, He L. Mutations in IHH, encoding Indian hedgehog, cause brachydactyly type A-1. Nature genetics 2001; 28:386-388.

14. Lodder E, Hoogeboom A, Coert J, de Graaff E. Deletion of 1 amino acid in Indian hedgehog leads to brachydactylyA1. American Journal of Medical Genetics Part A 2008; 146:2152-2154.

15. Ma G, Yu J, Xiao Y, Chan D, Gao B, Hu J, He Y, Guo S, Zhou J, Zhang L. Indian hedgehog mutations causing brachydactyly type A1 impair Hedgehog signal transduction at multiple lev-els. Cell research 2011; 21:1343-1357.

16. Afzal AR, Jeffery S. One gene, two phenotypes: ROR2 muta-tions in autosomal recessive Robinow syndrome and autosomal dominant brachydactyly type B. Hum Mutat 2003; 22:1-11.

17. Akbarzadeh S, Wheldon LM, Sweet SM, Talma S, Mardakheh FK, Heath JK. The deleted in brachydactyly B domain of ROR2 is required for receptor activation by recruitment of Src. PLoS One 2008; 3:e1873.

18. Hamamy H, Saleh N, Oldridge M, Al‐Hadidy A, Ajlouni K. Brachydactyly type B1: report of a family with de novo ROR2 mutation. Clin Genet 2006; 70:538-540.

19. Schwarzer W, Witte F, Rajab A, Mundlos S, Stricker S. A gra-dient of ROR2 protein stability and membrane localization confers brachydactyly type B or Robinow syndrome pheno-types. Hum Mol Genet 2009; 18:4013-4021.

20. Kjaer KW, Tiner M, Cingoz S, Karatosun V, Tommerup N, Mundlos S, Gunal I. A novel subtype of distal symphalangism affecting only the 4th finger. American Journal of Medical Genetics Part A 2009; 149:1571-1573.

21. Yang W, Tan F, Sun M, Zeng X, Liu J, Liu G, Luo H, Zhang X. [Identification of a recurrent mutation in the ROR2 gene in a Chinese family with brachydactyly type B]. Zhonghua yi xue yi chuan xue za zhi= Zhonghua yixue yichuanxue zazhi= Chinese journal of medical genetics 2004; 21:61-63.

22. Lv D, Luo Y, Yang W, Cao L, Wen Y, Zhao X, Sun M, Lo WH, Zhang X. A novel single-base deletion in ROR2 causes atypical brachydactyly type B1 with cutaneous syndactyly in a large Chinese family. J Hum Genet 2009; 54:422-425.

23. Oldridge M, Ana M, Maringa M, Propping P, Mansour S, Pollitt C, DeChiara TM, Kimble RB, Valenzuela DM, Yancopoulos GD. Dominant mutations in ROR2, encoding an orphan receptor tyrosine kinase, cause brachydactyly type B. Nat Genet 2000; 24:275-278.

24. Bacchelli C, Wilson L, Cook J, Winter R, Goodman F. ROR2 is mutated in hereditary brachydactyly with nail dysplasia, but not in Sorsby syndrome. Clin Genet 2003; 64:263-265.

25. Habib R, Ahmad W. A nonsense mutation in the gene ROR2 underlying autosomal dominant brachydactyly type B. Clin Dysmorphol 2013; 22:47-50.

99