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foot malformation with long-bone deficiency and BHLHA9 duplication: Two cases and expansion of the phenotype to radial agenesis
European Journal of Medical Genetics 56 (2013) 88e92
Contents lists available at SciVerse ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Short clinical report
Split-hand/foot malformation with long-bone deficiency and BHLHA9 duplication: Two cases and expansion of the phenotype to radial agenesis Florence Petit a, b, *, Joris Andrieux c, Bénédicte Demeer d, Louis-Michel Collet e, Henri Copin f, g, i, Elise Boudry-Labis c, Fabienne Escande h, Sylvie Manouvrier-Hanu a, b, Michèle Mathieu-Dramard d, i,1 a
Service de Génétique Clinique, Hôpital Jeanne de Flandre, CHRU, Lille, France Université Lille Nord de France, France Laboratoire de Génétique Médicale, Hôpital Jeanne de Flandre, CHRU, Lille, France d Service de Génétique Clinique, Hôpital Nord, CHU, Amiens, France e Service de Chirurgie orthopédique pédiatrique, Hôpital Nord, CHU, Amiens, France f Service de Médecine et Biologie de la reproduction et de Cytogénétique, Hôpital Nord, CHU, Amiens, France g Université de Picardie Jules Verne, France h Laboratoire de Biochimie et Biologie Moléculaire, Centre de Biologie Pathologie, CHRU, Lille, France i Service de Gynécologie-Obstétrique, Centre multidisciplinaire de diagnostic prénatal, Hôpital Nord, CHU, Amiens, France b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 August 2012 Accepted 19 November 2012 Available online 30 November 2012
Split-hand/foot malformation (SHFM) with long-bone deficiency (SHFLD, MIM#119100) is a rare condition characterised by SHFM associated with long-bone malformation usually involving the tibia. Previous published data reported several unrelated patients with 17p13.3 duplication and SHFLD. Recently, BHLHA9 has been proposed to be the major candidate gene responsible for this limb malformation. Here we report two new patients affected with ectrodactyly harbouring a 17p13.3 duplication detected by array-CGH. Both duplications contain 3 genes including BHLHA9 and are inherited from an unaffected parent. One of the patients presents a complete radial agenesis, expanding the phenotype of SHFLD3. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: BHLHA9 Ectrodactyly SHFLD SHFM 17p13.3 duplication
1. Introduction Split-hand/foot malformation (SHFM) is a congenital limb malformation characterised by a defect of the central rays of hands and/or feet due to the failure of median apical ectodermal ridge activity [1]. This is a genetically heterogeneous group with 6 loci currently known in the non-syndromic forms (SHFM1-6). Among the syndromic SHFM, split-hand/foot malformation with long-bone deficiency (SHFLD, MIM#119100) is a rare condition characterised by the association of SHFM and a long-bone malformation being usually a tibial defect (tibial hemimelia, hypoplasia, aplasia or
* Corresponding author. Service de Génétique Clinique, Hôpital Jeanne de Flandre, CHRU, 59037 Lille Cedex, France. Tel.: þ33 3 20 44 49 11; fax: þ33 3 20 44 49 01. E-mail address: fl[email protected] (F. Petit). 1 These authors contributed equally to this work. 1769-7212/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmg.2012.11.002
dysplasia). Different modes of inheritance have been suggested: autosomal dominant with reduced penetrance [2e7], autosomal recessive [8e10] and digenic inheritance [11e13]. Clinical manifestations are highly variable among the same family, and pedigrees often show non-penetrant individuals. Few loci have been associated with SHFLD: loci on chromosomes 1q42.2eq43 and 6q14.1 have been suggested on genome wide linkage analysis of a large SHFLD family [11]; 2q14.2 locus has been reported after breakpoint mapping of a de novo translocation in a single patient [14]. A locus on 17p13.3 (MIM#612576) was first identified by linkage analysis of a large Brazilian family in which 9 individuals were affected by autosomal dominant split-hand/foot malformation and long-bone deficiency of variable expression and incomplete penetrance [15,16]. 17p13.3 duplications were then reported in unrelated families with SHFLD [6,17]. Klopocki et al. (2012) refined the minimal critical region analysing 17 families with such duplications and identified an 11.8 kb region encompassing a single gene, BHLHA9. This gene encodes a putative basic loop helix transcription
F. Petit et al. / European Journal of Medical Genetics 56 (2013) 88e92
factor expressed in the limb bud mesenchyme underlying the apical ectodermal ridge in mouse and zebrafish embryos. Knockdown of BHLHA9 in zebrafish results in shortening of the pectoral fins, confirming its role in limb development [6]. We report two new patients affected with ectrodactyly, both having a 17p13.3 duplication containing BHLHA9 inherited from an unaffected parent. One of them presented a complete radial agenesis, expanding the phenotype of SHFLD3. 2. Clinical report Patient 1 is a 16 months old boy, fourth child of healthy unrelated parents of French origin. Family history was unremarkable, except a maternal aunt with preaxial polydactyly. An anamnios and a single umbilical artery were diagnosed during the second trimester of pregnancy. The boy was born at the gestational age of 36 weeks by iterative caesarean section. His birth weight was
89
2710 g (p10), height 48 cm (p25) and occipito-frontal circumference 34 cm (p50). Examination at birth revealed bilateral congenital malformation of the upper limb (Fig. 1). On the right side, there was a complete radial agenesis with hypoplastic bowed ulna and oligodactyly (3 fingers). On the left side, there was an oligodactyly with 3 fingers and 4 metacarpals, and the radius appeared hypoplastic. He had no lower-limb malformation and no facial dysmorphism. Cardiac ultrasound showed a small atrial septal defect. Abdominoerenal ultrasounds, cervicoethoracoelumbar radiographs and audition tests were normal. Walking was achieved at 13 months. At 16 months old, at last examination, the patient had normal psychomotor development. Patient 2 is a 3 years old girl, second child of healthy unrelated parents of French origin. Family history was unremarkable regarding limb malformation. The mother had surgery for ventricular septal defect during childhood. A bilateral ectrodactyly of the upper limbs was diagnosed by ultrasounds at 20 weeks of
Fig. 1. Clinical and radiological findings in Patient 1. A: Right upper limb: ectrodactyly associated with radial deviation of the wrist and hypoplastic forearm. B: Right upper limb Xray at birth: radial agenesis and hypoplastic bowed ulna. C: Right hand X-ray at 23 months: absence of 1st and 3rd rays. Malposition of the 2nd digit with hypoplastic metacarpal. Fused metacarpals are observed in central ray, most likely corresponding to 3rd and 4th metacarpals. D: Left forearm: ectrodactyly with presence of 3 rays. Middle digit and nail are broad, consistent with fusion of 3rd and 4th rays. E: Left upper limb X-ray at birth: hypoplastic radius. F: Left hand X-ray at 23 months: absence of 1st and 3rd digits. Malposition of the 2nd digit with hypoplastic middle phalange. Broad middle digit consistent with fusion of 3rd and 4th rays. Hypoplasia of 3rd metacarpal. Only one carpal bone visible, consistent with delayed ossification or carpal anomalies.
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gestation. Foetal karyotype on amniotic fluid was normal at standard resolution 46,XX. She was born at term with weight 2760 g (p9e25), height 49 cm (p25e50) and occipito-frontal circumference 33 cm (p9). Examination at birth confirmed a bilateral asymmetric ectrodactyly, with absence of the third digit on the left hand and two digits on the right hand. These two digits comprised three phalanges. Considering the large metacarpals, we imply that these rays may correspond to a fusion of rays IeII and IVeV, but the interpretation is difficult (Fig. 2). There were neither forearm nor lower-limb malformation, and no facial dysmorphism. She also presented a small umbilical hernia and an atrial septal defect which closed spontaneously. At 3 years old, at the last examination, the patient had normal psychomotor and growth development. 2.1. Genomic rearrangement In both patients, array-CGH analysis using the 60K array AgilentÒ revealed 17p13.3 duplication (Fig. 3), confirmed by qPCR using primers in the BHLHA9 and ABR genes. No additional aberration has been identified by array-CGH. For Patient 1 a standard metaphase karyotype performed on leukocytes was normal 46,XY. TBX5 sequencing performed in the
hypothesis of HolteOram syndrome revealed no mutation for the proband. Array-CGH revealed a 17p13.3 duplication arr[hg18] 17p13.3(908,011 2,933,334e1,131,284 31,195,508 2) spanning 198 kbe287 kb (flanking non duplicated probes located at 908,011 bp and 1,195,508 bp from 17p telomere). This imbalance was confirmed by quantitative PCR performed for the proband and his parents, showing that the duplication was inherited from the unaffected father. Patient 2 has a slightly larger duplication arr[hg18] 17p13.3(849,124 2,879,373e1,131,284 31,195,508 2) spanning 252 kbe346 kb (flanking non duplicated probes located at 849,124 bp and 1,195,508 bp from 17p telomere). This imbalance was confirmed by qPCR performed for the proband and his parents, showing that the duplication was inherited from the unaffected father. Overlapping duplications identified in both patients involved 3 genes: the entire BHLHA9, part of ABR and TUSC5 genes (Fig. 3). 3. Discussion 17p13.3 duplications encompassing BHLHA9 gene have been recently identified as the most common aetiology of SHFLD [6]. We
Fig. 2. Clinical and radiological findings in Patient 2. A,E: Right upper limb: ectrodactyly with only two rays present. B,F: Left upper limb: ectrodactyly with absence of 3rd ray and hypoplastic 1st and 5th digits. C,D: Right and left upper limbs X-rays at birth: no anomaly of forearm bones. G: Right hand X-ray at 7 months: presence of only two rays with short metacarpals and three phalanges. H: Left hand X-ray at 7 months: absence of 3rd ray. Hypoplasia of 2nd and 5th middle phalanges. Two carpal bones visible, which is consistent with the age of the patient.
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Fig. 3. Schematic overview of the 17p13.3 duplications identified in Patients 1 and 2 showing annotated genes. The minimal and maximal duplicated regions are indicated in blue by large and narrow bars respectively (hg18). The minimal critical region (MCR) described by Klopocki et al., 2012 is indicated in light blue.
report herein two new patients presenting with bilateral asymmetric ectrodactyly and a 17p13.3 duplication containing ABR, BHLHA9 and TUSC5. High phenotypic variability is observed in patients with SHFLD harbouring a 17p13.3 duplication, and tibial involvement has been described in approximately 60% of affected 17p duplication carriers [6]. For the 2 patients reported here, only the upper limbs were involved, with asymmetrical ectrodactyly. Patient 1 has a complete unilateral radial agenesis and a hypoplastic bowed ulna. In the hypothesis of a HolteOram syndrome, we sequenced TBX5 and did not find a mutation in Patient 1. This is the first description of forearm malformation associated with a 17p13.3 duplication, expanding the phenotype of SHFLD3. Refining the minimal 17p13.3 critical region in 17 SHFLD families with 17p13.3 duplication Klopocki et al. (2012) identified BHLHA9 as the major candidate gene. It encodes a putative basic helixeloopehelix transcription factor expressed in the limb bud mesenchyme underlying the apical ectodermal ridge in mouse and zebrafish embryos. Knock-down of BHLHA9 in zebrafish by antisense morpholino oligonucleotides results in shortening of the pectoral fins, confirming its role in limb development in a dosage dependant manner. Whole mount in situ hybridisation in mice embryos revealed BHLHA9 expression in both forelimb and hindlimb, with no other major expression domain [6]. To our knowledge, Bhlha9 knock-down in mice has not been performed yet. At present, little is known about this gene function. A role in the regulation of survival and/or proliferation of the cells in the progress zone of the developing bud is suggested by Klopocki et al. (2012), but the molecular pathway has not been studied. About the two other genes concerned by the duplication in our patients, no involvement in limb development is currently known. TUSC5 expression might play a role in the pathogenesis of lung cancer, acting as a tumour suppressor [18]. ABR (active BCR-related) gene encodes a protein that is similar to the protein encoded by the BCR (breakpoint cluster region) gene located on chromosome 22 and involved in reciprocal translocations in Philadelphia chromosome-positive chronic myelogenous leukaemia [19]. It contains a GTPase-activating protein domain, a domain found in members of the Rho family of GTP-binding proteins. Functional studies in mice determined that this protein plays a role in vestibular morphogenesis [20]. Adult double null mutant for ABR and BCR genes displays abnormal saccule and utricule, responsible for impaired balance and motor coordination. Cochlea and semicircular canals are not affected and therefore no deafness is observed. No limb malformations were reported in these knock-out mice. In our two patients presenting ABR duplication, no vestibular impairment is reported. None of our patients had neurodevelopmental problems at the age of last examination, consistent with the fact that YWHAE gene
was not concerned by the duplication. This gene has been associated with autistic features and other behavioural symptoms, speech and motor delay, in patients sharing more proximal or larger 17p13.3 duplications [21]. The reason why the reported larger duplications involving BHLHA9 are not associated with limb malformation is unknown. This could be explained either by nonpenetrance or by the disruption of BHLHA9 regulatory elements in smaller duplications [17]. Incomplete penetrance has already been described in families reported by Armour et al. (2011) and Klopocki et al. (2012), only 30e50% of duplication carriers being affected with SHFLD [6,17]. The two patients reported herein inherited the duplication from their unaffected father. Thus, a second molecular event is likely to explain this observation and further studies are necessary to identify these modifiers in order to decode the pathogenesis of SHFLD3. More striking is the asymmetry observed in some patients having only one limb affected, suggesting the involvement of stochastic factors as well. Acknowledgements The authors thank Guillaume Jedraszak and Richard Gouron for their contribution to improve the manuscript. This work was partially supported by the French national clinical research program (Programme Hospitalier de Recherche Clinique). References [1] P.H. Duijf, H. van Bokhoven, H.G. Brunner, Pathogenesis of split-hand/splitfoot malformation, Human Molecular Genetics 12 (Spec No 1) (2003) R51e R60. [2] H.E. Hoyme, K.L. Jones, W.L. Nyhan, R.M. Pauli, M. Robinow, Autosomal dominant ectrodactyly and absence of long bones of upper or lower limbs: further clinical delineation, The Journal of Pediatrics 111 (1987) 538e543. [3] R.N. Sener, E. Isikan, H.B. Diren, B.S. Sayli, F. Sener, Bilateral split-hand with bilateral tibial aplasia, Pediatric Radiology 19 (1989) 258e260. [4] F. Majewski, W. Kuster, B. ter Haar, T. Goecke, Aplasia of tibia with split-hand/ split-foot deformity. Report of six families with 35 cases and considerations about variability and penetrance, Human Genetics 70 (1985) 136e147. [5] E. Majewski, T. Goecke, P. Meinecke, Ectrodactyly and absence (hypoplasia) of the tibia: are there dominant and recessive types? American Journal of Medical Genetics 63 (1996) 185e189. [6] E. Klopocki, S. Lohan, S.C. Doelken, S. Stricker, C.W. Ockeloen, R. Soares Thiele de Aguiar, K. Lezirovitz, R.C. Mingroni Netto, A. Jamsheer, H. Shah, I. Kurth, R. Habenicht, M. Warman, K. Devriendt, U. Kordass, M. Hempel, A. Rajab, O. Makitie, M. Naveed, U. Radhakrishna, S.E. Antonarakis, D. Horn, S. Mundlos, Duplications of BHLHA9 are associated with ectrodactyly and tibia hemimelia inherited in non-Mendelian fashion, Journal of Medical Genetics 49 (2012) 119e125. [7] V.M.D. Kaloustian, W.A. Mnaymneh, Bilateral tibial aplasia with lobster-claw hands. A rare genetic entity, Acta Paediatrica Scandinavica 62 (1973) 77e78. [8] I. Witters, K. Devriendt, P. Moerman, J. Caudron, C. Van Hole, J.P. Fryns, Bilateral tibial agenesis with ectrodactyly (OMIM 119100): further evidence for autosomal recessive inheritance, American Journal of Medical Genetics 104 (2001) 209e213.
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[9] G. Kohn, R. el Shawwa, M. Grunebaum, Aplasia of the tibia with bifurcation of the femur and ectrodactyly: evidence for an autosomal recessive type, American Journal of Medical Genetics 33 (1989) 172e175. [10] M. McKay, S.K. Clarren, R. Zorn, Isolated tibial hemimelia in sibs: an autosomal-recessive disorder? American Journal of Medical Genetics 17 (1984) 603e607. [11] M. Naveed, S.K. Nath, M. Gaines, M.T. Al-Ali, N. Al-Khaja, D. Hutchings, J. Golla, S. Deutsch, A. Bottani, S.E. Antonarakis, U. Ratnamala, U. Radhakrishna, Genomewide linkage scan for split-hand/foot malformation with long-bone deficiency in a large Arab family identifies two novel susceptibility loci on chromosomes 1q42.2eq43 and 6q14.1, American Journal of Human Genetics 80 (2007) 105e111. [12] J. Zlotogora, On the inheritance of the split hand/split foot malformation, American Journal of Medical Genetics 53 (1994) 29e32. [13] J. Zlotogora, N. Nubani, Is there an autosomal recessive form of the split hand and split foot malformation? Journal of Medical Genetics 26 (1989) 138e140. [14] C. Babbs, R. Heller, D.B. Everman, M. Crocker, S.R. Twigg, C.E. Schwartz, H. Giele, A.O. Wilkie, A new locus for split hand/foot malformation with long bone deficiency (SHFLD) at 2q14.2 identified from a chromosome translocation, Human Genetics 122 (2007) 191e199. [15] K. Lezirovitz, S.R. Maestrelli, N.H. Cotrim, P.A. Otto, P.L. Pearson, R.C. MingroniNetto, A novel locus for split-hand/foot malformation associated with tibial hemimelia (SHFLD syndrome) maps to chromosome region 17p13.1e17p13.3, Human Genetics 123 (2008) 625e631. [16] A. Richieri-Costa, D. Brunoni, J. Laredo Filho, S. Kasinski, Tibial aplasiaectrodactyly as variant expression of the Gollop-Wolfgang complex:
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[18]
[19] [20]
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report of a Brazilian family, American Journal of Medical Genetics 28 (1987) 971e980. C.M. Armour, D.E. Bulman, O. Jarinova, R.C. Rogers, K.B. Clarkson, B.R. DuPont, A. Dwivedi, F.O. Bartel, L. McDonell, C.E. Schwartz, K.M. Boycott, D.B. Everman, G.E. Graham, 17p13.3 microduplications are associated with split-hand/foot malformation and long-bone deficiency (SHFLD), European Journal of Human Genetics 19 (2011) 1144e1151. H. Konishi, M. Sugiyama, K. Mizuno, H. Saito, Y. Yatabe, T. Takahashi, H. Osada, T. Takahashi, Detailed characterization of a homozygously deleted region corresponding to a candidate tumor suppressor locus at distal 17p13.3 in human lung cancer, Oncogene 22 (2003) 1892e1905. N. Heisterkamp, C. Morris, J. Groffen, ABR, an active BCR-related gene, Nucleic Acids Research 17 (1989) 8821e8831. V. Kaartinen, A. Nagy, I. Gonzalez-Gomez, J. Groffen, N. Heisterkamp, Vestibular dysgenesis in mice lacking Abr and Bcr Cdc42/RacGAPs, Developmental Dynamics 223 (2002) 517e525. D.L. Bruno, B.M. Anderlid, A. Lindstrand, C. van Ravenswaaij-Arts, D. Ganesamoorthy, J. Lundin, C.L. Martin, J. Douglas, C. Nowak, M.P. Adam, R.F. Kooy, N. Van der Aa, E. Reyniers, G. Vandeweyer, I. Stolte-Dijkstra, T. Dijkhuizen, A. Yeung, M. Delatycki, B. Borgstrom, L. Thelin, C. Cardoso, B. van Bon, R. Pfundt, B.B. de Vries, A. Wallin, D.J. Amor, P.A. James, H.R. Slater, J. Schoumans, Further molecular and clinical delineation of co-locating 17p13.3 microdeletions and microduplications that show distinctive phenotypes, Journal of Medical Genetics 47 (2010) 299e311.
Contents lists available at SciVerse ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Short clinical report
Split-hand/foot malformation with long-bone deficiency and BHLHA9 duplication: Two cases and expansion of the phenotype to radial agenesis Florence Petit a, b, *, Joris Andrieux c, Bénédicte Demeer d, Louis-Michel Collet e, Henri Copin f, g, i, Elise Boudry-Labis c, Fabienne Escande h, Sylvie Manouvrier-Hanu a, b, Michèle Mathieu-Dramard d, i,1 a
Service de Génétique Clinique, Hôpital Jeanne de Flandre, CHRU, Lille, France Université Lille Nord de France, France Laboratoire de Génétique Médicale, Hôpital Jeanne de Flandre, CHRU, Lille, France d Service de Génétique Clinique, Hôpital Nord, CHU, Amiens, France e Service de Chirurgie orthopédique pédiatrique, Hôpital Nord, CHU, Amiens, France f Service de Médecine et Biologie de la reproduction et de Cytogénétique, Hôpital Nord, CHU, Amiens, France g Université de Picardie Jules Verne, France h Laboratoire de Biochimie et Biologie Moléculaire, Centre de Biologie Pathologie, CHRU, Lille, France i Service de Gynécologie-Obstétrique, Centre multidisciplinaire de diagnostic prénatal, Hôpital Nord, CHU, Amiens, France b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 August 2012 Accepted 19 November 2012 Available online 30 November 2012
Split-hand/foot malformation (SHFM) with long-bone deficiency (SHFLD, MIM#119100) is a rare condition characterised by SHFM associated with long-bone malformation usually involving the tibia. Previous published data reported several unrelated patients with 17p13.3 duplication and SHFLD. Recently, BHLHA9 has been proposed to be the major candidate gene responsible for this limb malformation. Here we report two new patients affected with ectrodactyly harbouring a 17p13.3 duplication detected by array-CGH. Both duplications contain 3 genes including BHLHA9 and are inherited from an unaffected parent. One of the patients presents a complete radial agenesis, expanding the phenotype of SHFLD3. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: BHLHA9 Ectrodactyly SHFLD SHFM 17p13.3 duplication
1. Introduction Split-hand/foot malformation (SHFM) is a congenital limb malformation characterised by a defect of the central rays of hands and/or feet due to the failure of median apical ectodermal ridge activity [1]. This is a genetically heterogeneous group with 6 loci currently known in the non-syndromic forms (SHFM1-6). Among the syndromic SHFM, split-hand/foot malformation with long-bone deficiency (SHFLD, MIM#119100) is a rare condition characterised by the association of SHFM and a long-bone malformation being usually a tibial defect (tibial hemimelia, hypoplasia, aplasia or
* Corresponding author. Service de Génétique Clinique, Hôpital Jeanne de Flandre, CHRU, 59037 Lille Cedex, France. Tel.: þ33 3 20 44 49 11; fax: þ33 3 20 44 49 01. E-mail address: fl[email protected] (F. Petit). 1 These authors contributed equally to this work. 1769-7212/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmg.2012.11.002
dysplasia). Different modes of inheritance have been suggested: autosomal dominant with reduced penetrance [2e7], autosomal recessive [8e10] and digenic inheritance [11e13]. Clinical manifestations are highly variable among the same family, and pedigrees often show non-penetrant individuals. Few loci have been associated with SHFLD: loci on chromosomes 1q42.2eq43 and 6q14.1 have been suggested on genome wide linkage analysis of a large SHFLD family [11]; 2q14.2 locus has been reported after breakpoint mapping of a de novo translocation in a single patient [14]. A locus on 17p13.3 (MIM#612576) was first identified by linkage analysis of a large Brazilian family in which 9 individuals were affected by autosomal dominant split-hand/foot malformation and long-bone deficiency of variable expression and incomplete penetrance [15,16]. 17p13.3 duplications were then reported in unrelated families with SHFLD [6,17]. Klopocki et al. (2012) refined the minimal critical region analysing 17 families with such duplications and identified an 11.8 kb region encompassing a single gene, BHLHA9. This gene encodes a putative basic loop helix transcription
F. Petit et al. / European Journal of Medical Genetics 56 (2013) 88e92
factor expressed in the limb bud mesenchyme underlying the apical ectodermal ridge in mouse and zebrafish embryos. Knockdown of BHLHA9 in zebrafish results in shortening of the pectoral fins, confirming its role in limb development [6]. We report two new patients affected with ectrodactyly, both having a 17p13.3 duplication containing BHLHA9 inherited from an unaffected parent. One of them presented a complete radial agenesis, expanding the phenotype of SHFLD3. 2. Clinical report Patient 1 is a 16 months old boy, fourth child of healthy unrelated parents of French origin. Family history was unremarkable, except a maternal aunt with preaxial polydactyly. An anamnios and a single umbilical artery were diagnosed during the second trimester of pregnancy. The boy was born at the gestational age of 36 weeks by iterative caesarean section. His birth weight was
89
2710 g (p10), height 48 cm (p25) and occipito-frontal circumference 34 cm (p50). Examination at birth revealed bilateral congenital malformation of the upper limb (Fig. 1). On the right side, there was a complete radial agenesis with hypoplastic bowed ulna and oligodactyly (3 fingers). On the left side, there was an oligodactyly with 3 fingers and 4 metacarpals, and the radius appeared hypoplastic. He had no lower-limb malformation and no facial dysmorphism. Cardiac ultrasound showed a small atrial septal defect. Abdominoerenal ultrasounds, cervicoethoracoelumbar radiographs and audition tests were normal. Walking was achieved at 13 months. At 16 months old, at last examination, the patient had normal psychomotor development. Patient 2 is a 3 years old girl, second child of healthy unrelated parents of French origin. Family history was unremarkable regarding limb malformation. The mother had surgery for ventricular septal defect during childhood. A bilateral ectrodactyly of the upper limbs was diagnosed by ultrasounds at 20 weeks of
Fig. 1. Clinical and radiological findings in Patient 1. A: Right upper limb: ectrodactyly associated with radial deviation of the wrist and hypoplastic forearm. B: Right upper limb Xray at birth: radial agenesis and hypoplastic bowed ulna. C: Right hand X-ray at 23 months: absence of 1st and 3rd rays. Malposition of the 2nd digit with hypoplastic metacarpal. Fused metacarpals are observed in central ray, most likely corresponding to 3rd and 4th metacarpals. D: Left forearm: ectrodactyly with presence of 3 rays. Middle digit and nail are broad, consistent with fusion of 3rd and 4th rays. E: Left upper limb X-ray at birth: hypoplastic radius. F: Left hand X-ray at 23 months: absence of 1st and 3rd digits. Malposition of the 2nd digit with hypoplastic middle phalange. Broad middle digit consistent with fusion of 3rd and 4th rays. Hypoplasia of 3rd metacarpal. Only one carpal bone visible, consistent with delayed ossification or carpal anomalies.
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gestation. Foetal karyotype on amniotic fluid was normal at standard resolution 46,XX. She was born at term with weight 2760 g (p9e25), height 49 cm (p25e50) and occipito-frontal circumference 33 cm (p9). Examination at birth confirmed a bilateral asymmetric ectrodactyly, with absence of the third digit on the left hand and two digits on the right hand. These two digits comprised three phalanges. Considering the large metacarpals, we imply that these rays may correspond to a fusion of rays IeII and IVeV, but the interpretation is difficult (Fig. 2). There were neither forearm nor lower-limb malformation, and no facial dysmorphism. She also presented a small umbilical hernia and an atrial septal defect which closed spontaneously. At 3 years old, at the last examination, the patient had normal psychomotor and growth development. 2.1. Genomic rearrangement In both patients, array-CGH analysis using the 60K array AgilentÒ revealed 17p13.3 duplication (Fig. 3), confirmed by qPCR using primers in the BHLHA9 and ABR genes. No additional aberration has been identified by array-CGH. For Patient 1 a standard metaphase karyotype performed on leukocytes was normal 46,XY. TBX5 sequencing performed in the
hypothesis of HolteOram syndrome revealed no mutation for the proband. Array-CGH revealed a 17p13.3 duplication arr[hg18] 17p13.3(908,011 2,933,334e1,131,284 31,195,508 2) spanning 198 kbe287 kb (flanking non duplicated probes located at 908,011 bp and 1,195,508 bp from 17p telomere). This imbalance was confirmed by quantitative PCR performed for the proband and his parents, showing that the duplication was inherited from the unaffected father. Patient 2 has a slightly larger duplication arr[hg18] 17p13.3(849,124 2,879,373e1,131,284 31,195,508 2) spanning 252 kbe346 kb (flanking non duplicated probes located at 849,124 bp and 1,195,508 bp from 17p telomere). This imbalance was confirmed by qPCR performed for the proband and his parents, showing that the duplication was inherited from the unaffected father. Overlapping duplications identified in both patients involved 3 genes: the entire BHLHA9, part of ABR and TUSC5 genes (Fig. 3). 3. Discussion 17p13.3 duplications encompassing BHLHA9 gene have been recently identified as the most common aetiology of SHFLD [6]. We
Fig. 2. Clinical and radiological findings in Patient 2. A,E: Right upper limb: ectrodactyly with only two rays present. B,F: Left upper limb: ectrodactyly with absence of 3rd ray and hypoplastic 1st and 5th digits. C,D: Right and left upper limbs X-rays at birth: no anomaly of forearm bones. G: Right hand X-ray at 7 months: presence of only two rays with short metacarpals and three phalanges. H: Left hand X-ray at 7 months: absence of 3rd ray. Hypoplasia of 2nd and 5th middle phalanges. Two carpal bones visible, which is consistent with the age of the patient.
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Fig. 3. Schematic overview of the 17p13.3 duplications identified in Patients 1 and 2 showing annotated genes. The minimal and maximal duplicated regions are indicated in blue by large and narrow bars respectively (hg18). The minimal critical region (MCR) described by Klopocki et al., 2012 is indicated in light blue.
report herein two new patients presenting with bilateral asymmetric ectrodactyly and a 17p13.3 duplication containing ABR, BHLHA9 and TUSC5. High phenotypic variability is observed in patients with SHFLD harbouring a 17p13.3 duplication, and tibial involvement has been described in approximately 60% of affected 17p duplication carriers [6]. For the 2 patients reported here, only the upper limbs were involved, with asymmetrical ectrodactyly. Patient 1 has a complete unilateral radial agenesis and a hypoplastic bowed ulna. In the hypothesis of a HolteOram syndrome, we sequenced TBX5 and did not find a mutation in Patient 1. This is the first description of forearm malformation associated with a 17p13.3 duplication, expanding the phenotype of SHFLD3. Refining the minimal 17p13.3 critical region in 17 SHFLD families with 17p13.3 duplication Klopocki et al. (2012) identified BHLHA9 as the major candidate gene. It encodes a putative basic helixeloopehelix transcription factor expressed in the limb bud mesenchyme underlying the apical ectodermal ridge in mouse and zebrafish embryos. Knock-down of BHLHA9 in zebrafish by antisense morpholino oligonucleotides results in shortening of the pectoral fins, confirming its role in limb development in a dosage dependant manner. Whole mount in situ hybridisation in mice embryos revealed BHLHA9 expression in both forelimb and hindlimb, with no other major expression domain [6]. To our knowledge, Bhlha9 knock-down in mice has not been performed yet. At present, little is known about this gene function. A role in the regulation of survival and/or proliferation of the cells in the progress zone of the developing bud is suggested by Klopocki et al. (2012), but the molecular pathway has not been studied. About the two other genes concerned by the duplication in our patients, no involvement in limb development is currently known. TUSC5 expression might play a role in the pathogenesis of lung cancer, acting as a tumour suppressor [18]. ABR (active BCR-related) gene encodes a protein that is similar to the protein encoded by the BCR (breakpoint cluster region) gene located on chromosome 22 and involved in reciprocal translocations in Philadelphia chromosome-positive chronic myelogenous leukaemia [19]. It contains a GTPase-activating protein domain, a domain found in members of the Rho family of GTP-binding proteins. Functional studies in mice determined that this protein plays a role in vestibular morphogenesis [20]. Adult double null mutant for ABR and BCR genes displays abnormal saccule and utricule, responsible for impaired balance and motor coordination. Cochlea and semicircular canals are not affected and therefore no deafness is observed. No limb malformations were reported in these knock-out mice. In our two patients presenting ABR duplication, no vestibular impairment is reported. None of our patients had neurodevelopmental problems at the age of last examination, consistent with the fact that YWHAE gene
was not concerned by the duplication. This gene has been associated with autistic features and other behavioural symptoms, speech and motor delay, in patients sharing more proximal or larger 17p13.3 duplications [21]. The reason why the reported larger duplications involving BHLHA9 are not associated with limb malformation is unknown. This could be explained either by nonpenetrance or by the disruption of BHLHA9 regulatory elements in smaller duplications [17]. Incomplete penetrance has already been described in families reported by Armour et al. (2011) and Klopocki et al. (2012), only 30e50% of duplication carriers being affected with SHFLD [6,17]. The two patients reported herein inherited the duplication from their unaffected father. Thus, a second molecular event is likely to explain this observation and further studies are necessary to identify these modifiers in order to decode the pathogenesis of SHFLD3. More striking is the asymmetry observed in some patients having only one limb affected, suggesting the involvement of stochastic factors as well. Acknowledgements The authors thank Guillaume Jedraszak and Richard Gouron for their contribution to improve the manuscript. This work was partially supported by the French national clinical research program (Programme Hospitalier de Recherche Clinique). References [1] P.H. Duijf, H. van Bokhoven, H.G. Brunner, Pathogenesis of split-hand/splitfoot malformation, Human Molecular Genetics 12 (Spec No 1) (2003) R51e R60. [2] H.E. Hoyme, K.L. Jones, W.L. Nyhan, R.M. Pauli, M. Robinow, Autosomal dominant ectrodactyly and absence of long bones of upper or lower limbs: further clinical delineation, The Journal of Pediatrics 111 (1987) 538e543. [3] R.N. Sener, E. Isikan, H.B. Diren, B.S. Sayli, F. Sener, Bilateral split-hand with bilateral tibial aplasia, Pediatric Radiology 19 (1989) 258e260. [4] F. Majewski, W. Kuster, B. ter Haar, T. Goecke, Aplasia of tibia with split-hand/ split-foot deformity. Report of six families with 35 cases and considerations about variability and penetrance, Human Genetics 70 (1985) 136e147. [5] E. Majewski, T. Goecke, P. Meinecke, Ectrodactyly and absence (hypoplasia) of the tibia: are there dominant and recessive types? American Journal of Medical Genetics 63 (1996) 185e189. [6] E. Klopocki, S. Lohan, S.C. Doelken, S. Stricker, C.W. Ockeloen, R. Soares Thiele de Aguiar, K. Lezirovitz, R.C. Mingroni Netto, A. Jamsheer, H. Shah, I. Kurth, R. Habenicht, M. Warman, K. Devriendt, U. Kordass, M. Hempel, A. Rajab, O. Makitie, M. Naveed, U. Radhakrishna, S.E. Antonarakis, D. Horn, S. Mundlos, Duplications of BHLHA9 are associated with ectrodactyly and tibia hemimelia inherited in non-Mendelian fashion, Journal of Medical Genetics 49 (2012) 119e125. [7] V.M.D. Kaloustian, W.A. Mnaymneh, Bilateral tibial aplasia with lobster-claw hands. A rare genetic entity, Acta Paediatrica Scandinavica 62 (1973) 77e78. [8] I. Witters, K. Devriendt, P. Moerman, J. Caudron, C. Van Hole, J.P. Fryns, Bilateral tibial agenesis with ectrodactyly (OMIM 119100): further evidence for autosomal recessive inheritance, American Journal of Medical Genetics 104 (2001) 209e213.
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