- Email: [email protected]
split foot malformation with hearing impairment: a case report
International Journal of Pediatric Otorhinolaryngology (2003) 67, 1127 /1132
www.elsevier.com/locate/ijporl
CASE REPORT
Deletion mapping of split hand/split foot malformation with hearing impairment: a case report Kunihiro Fukushimaa,*, Kyoko Nagaib, Haruyo Tsukadab, Akemi Sugataa, Kenichi Sugataa, Norio Kasaia, Namiki Kibayashia, Yukihide Maedaa, Mehmet Gunduza, Kazunori Nishizakia a
Department of Otolaryngology */Head and Neck Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata Cho, Okayama 700-8558, Japan b Department of Otolaryngology */Head and Neck Surgery, Gunnma University School of Medicine, Gunnma, Japan Received 25 March 2003; received in revised form 30 May 2003; accepted 1 June 2003
KEYWORDS Mapping; Split hand/split foot malformation; Hearing impairment
Summary Split hand/split foot malformation (SHFM), which typically appears as lobster-like limb malformation, is a rare clinical condition caused by a partial deletion of chromosome 7q. Hearing impairment sometimes accompanies syndromic SHFM cases; a case of inner and middle ear malformation with SHFM is described in this report. We conducted a genetic evaluation of this patient and found a deleted region that overlaps a previously reported locus of SHFM as well as a DFNB14 locus that can cause nonsyndromic hearing impairment by autosomal recessive inheritance. – 2003 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Split hand/split foot malformation type 1 (SHFM1, OMIM183600) is a congenital malformation syndrome that typically includes an ectrodactyly limb malformation resembling the primary limbs of an arthropod, syndactyly, and median clefts of the hands and feet. The disease itself is considered to be very rare, affecting one out of 90 000 newborn babies [1]. Del Porto et al. first reported a case of a deletion at chromosome 7(q11 /q22) in which the patient
*Corresponding author. Tel.: /81-86-235-7307; fax: /81-86235-7308. E-mail address: [email protected] (K. Fukushima).
had ectrodactyly, microcephalus, and low-set ears [2]. At least nine additional cases have demonstrated the relationship between the partial deletion of 7q and the clinical manifestation of SHFM [3 /8]. In particular, the region of 7q21.2 /q21.3 was frequently shared among cases [3] and found to be the SHFM1 locus. Three other loci for SHFM have also been mapped: SHFM2 on chromosome Xq26, SHFM3 on chromosome 10q24, and SHFM4, the result of a mutation in the TP63 gene, on chromosome 3q27 [3]. Other abnormalities in various degrees may be found in combination with SHFM. Scherer et al. classified syndromic ectrodactyly (SE) into the following nine clinical types [8]. (1) Ectrodactyly, ectodermal dysplasia and cleft lip/palate syndrome (online menderian inheritance in men
0165-5876/03/$ - see front matter – 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0165-5876(03)00193-9
1128
[OMIM: http://www3.ncbi.nlm.nih.gov/Omim/ searchomim.html] #129900), (2) Lacrimoauriculodentodigital syndrome (LADD syndrome: OMIM #149730), (3) Acro /Dermato /Ungual /Lacrimal / Tooth syndrome (ADULT syndrome: OMIM #103285), (4) Ectrodactyly and ectodermal dysplasia without cleft lip/palate (OMIM #129810), (5) Fontaine syndrome (OMIM #183700), (6) Acral / renal /mandibular syndrome (OMIM #200980), (7) Ectrodactyly /Cleft palate syndrome (OMIM #129830), (8) Ectrodactyly and hearing loss (OMIM #220600), and (9) Karsch /Neugebauer syndrome (OMIM #183800). Among these nine subclassifications, hearing loss is a relatively common clinical manifestation. However, the otological or audiological component of SHFM with hearing impairment has not been fully described with further genetic testing, although the presence of hearing impairment can deeply affect the patient’s quality of life. In this study, we present a case of SHFM in which the patient has hearing loss and typical ectrodactyly and in which we found partial deletion of chromosome 7q. Deletion mapping with short nucleotide repeats (SNR) demonstrated the deleted region of 7q that causes the phenotype of hearing loss along with ectrodactyly.
2. Case presentation A full-term baby boy was born with a body weight of 2076 g. His Apgar score was 5 at 10 min and 9 at 30 min. Multiple malformations of his hands and feet (Fig. 1), hypotonic muscles, and difficulty of suckling were noted. Cytological examination revealed a partial deletion of chromosome 7 [del(7)(q11.2q22)], shown in Fig. 2. The baby was referred to the Gunnma Medical School Hospital when he was 4 months old. At the age of 2 years 7 months, he was referred to the otolaryngology clinic there because he was unable to respond to any sound. The boy was 80 cm tall and weighed 9.5 kg, which were each two standard deviations below the age-appropriate Japanese standard. In addition to his small body size, the boy had poor muscle tone. The boy’s right hand appeared normal but his left hand had only four fingers, his right foot had only four toes, and his left foot had only three toes. The boy had auricular malformation including lowset ears, loss of the Crura anthelicis , and stenosis of the external ear canals. Microgenia, hypoplasia of the tongue, and mild laryngomalacia were also observed. Click-evoked ABR testing revealed that the threshold is 80 dB in his right ear and 90 dB in his left ear. Condition orientation reflex testing re-
K. Fukushima et al.
Fig. 1 Radiograph of the patient’s foot, which also shows the absence of a toe.
vealed a sound response at 90 dB. Gross motor movement showed apparent developmental delay; he did not achieve head control until 1 year 7 months of age and he did not master sitting alone until the age of 2 years 1 month. High resolution computed tomography revealed middle ear malformation, including fusion of the malleus and incus, narrowing of the internal ear meatus, hypoplasia of the cochlea, and ballooning of the ampullae of the semicircular canals (Fig. 3). These radiological and clinical manifestations of this case were partly reported elsewhere in Japanese [9].
3. Fine deletion mapping The DNA was obtained from peripheral blood by phenol chloroform extraction and amplified by the polymerase chain reaction (PCR) as described previously [10]. Each 10-ml PCR sample contained 10 ng of genomic DNA, 2.0 pmol of each primer (Invitrogen Corp., Carlsbad, CA, USA), 200 mM of each dNTP (Toyobo Inc., Osaka, Japan), 0.25 U of Taq DNA polymerase, and 1 / PCR buffer (Takara Shuzo Inc., Tokyo, Japan). After the initial denaturation step at 94 8C for 2 min, samples were amplified under the following thermal conditions: 95 8C for 30 s, 55 8C for 30 s, and 72 8C for 30 s for
Deletion mapping of split hand/split foot malformation with hearing impairment
1129
Fig. 2 Diagram of chromosome 7q (A), its normal map (B), and a map showing its deletion (C) in the present case.
25 cycles, with a final extension step at 72 8C for 10 min. The chromosomal location was determined from information in the GDB genome database http://gdbwww.gdb.org/. Electrophoresis was performed on a 6% polyacrylamide gel and bands were detected by silver staining. Samples of DNA from three healthy controls were also used to identify the banding pattern of a single allele. The chromosomal locations of the primers used here were (from centromere to telomere): D7S1830, D7S1831, D7S494, D7S669, D7S644, D7S646, D7S651, D7S518,
D7S796, D7S525, D7S1817, D7S486, and D7S480 (Table 1).
4. Sequencing analysis The exons of the PDS gene were amplified by PCR according to previously reported conditions and primers [11]. The products were purified using a commercial kit (QIAquickTM, QIAGEN Inc., Valencia, CA, USA). Half of the PCR products were used
Fig. 3 A computed tomography scan of the patient’s ear, showing fusion of the malleus and incus (A) and malformation of the cochlea (B), resulting in abnormal communication with the internal ear canal.
1130
K. Fukushima et al.
6. Discussion
Table 1 Marker
Map distance (cM)
Result
D7S1830 D7S1831 D7S494 D7S669 D7S644 D7S646 D7S651 D7S518 D7S796 D7S525 D7S1817 D7S486 D7S480
79.38/51.78 49.55 70.60 107.35 119.05 119.78 122.15 122.63 122.83 124.81 126.59 129.26 130.98
NI NI Heterozygous Heterozygous Homozygous Homozygous Homozygous Homozygous Heterozygous NI NI Heterozygous Heterozygous
NI, not informative because of the homozygosity.
for direct sequencing and half were subcloned using a pBluescript subcloning kit (Stratagene, La Jolla, CA, USA). All samples were sequenced using the Big-dyeTM terminator cycle sequencing ready kit with AmplitaqTM DNA polymerase FS (ABI: Applied Biosystems Foster City, CA, USA) and run on an ABI PRISM 373S Genetic Analyzer.
5. Results Each of four markers (D7S644, D7S646, D7S651, and D7S518) demonstrated a single band, while the flanking markers D7S669 and D7S796 demonstrated apparent heterozygosity, the presence of two different alleles (Table 1). In the region between these two markers, a minimum of 4.58 cM and a maximum of 16.48 cM was deleted in this case. This region included the SHFM genetic region and a wider region on the telomeric side. This region apparently excluded the PDS coding region (Fig. 4) and a direct sequence of the PDS coding region demonstrated no apparent mutation.
Fig. 4 Diagrams of the deleted region in the present case, the previously reported SHFM locus, and the DFNB14 locus.
Hearing impairment is one of clinical symptoms that are found in combination with ectrodactyly [12 /14]. The association of particular chromosomal region and ectrodactyly has already reported. Sharland et al. [15] reported a case of tetramelic ectrodactyly that demonstrated complex chromosomal rearrangement (5q11.2, 5q34, 7q21.2, 7q31.3 and 9q22.1) without any apparent chromosomal deletions. In that case, no additional deficit other than a minor facial anomaly was observed. Genuardi et al. [16] also reported a case of bilateral SHFM with balanced translocation of t(2;7)(q21.1;q22.1) that showed autosomal dominant inheritance. Our result clearly demonstrated that the ectrodactyly with hearing loss can be caused by the same chromosomal abnormality, a deletion in 7q21.2 /q21.3, as reported in various types of non-SE. The locus of ectrodactyly can be genetically mapped from somatic cell hybrid lines [17] and the minimum chromosomal region of SHFDassociated deletions is PON (168820) /D7S812 / SHFD1 /D7S811 /ASNS (108370). Marinoni et al. [6] reported a case of SHFM with developmental delay that showed the deletion of 7q21.2 /q22.1 and the absence of D7S 527, D7S 479, and D7S 554. With the result from deletion mapping, the deleted chromosomal region in the present case included the previously reported SHFD-associated region as indicated in Fig. 4. Thus, the same factor found in the same genomic region may play the central role in the cause of non-SE and ectrodactyly with hearing loss. Recently, Tackels-Horne et al. [18] described two families with a form of SHFM in which deficiency of the central rays in the appendicular skeleton was associated with sensorineural hearing loss. Variably expressed SHFM was found in nine of 15 presumed gene carriers, and mild to moderate sensorineural hearing loss in seven. In these two families, Tackels-Horne et al. [18] found that the disorder was linked to markers on 7q21, the region of the SHFM1 locus, with a combined maximum load score of 4.37 at theta /0.0 for D7S527 at 80% penetration. This region again overlapped with the deleted region in the present case. Among the families that were reported by Tackels-Horne et al. [18], one presumed gene carrier showed hearing impairment without ectrodactyly, and it may be possible that nonsyndromic hearing loss is caused by an allelic variance of SHFM with hearing loss. Three different nonsyndromic deafness genes have been mapped near the SHFM locus: DFNB14 [19], DFNB17 [20], and PDS (DFNB4) [11,21]. The critical region of DFNB14 was reported
Deletion mapping of split hand/split foot malformation with hearing impairment
between D7S527 and D7S3047, spanning a 15-cM region [19], and the PDS gene was considered to be a telomeric border of this region [19]. The critical region of the DFNB17 was D7S2487, D7S655, and D7S480, and the PDS gene was located on its centromeric border [20]. Among these three adjacent genes, one gene that overlaps with the deleted region in the present case is DFNB14 (Fig. 4). Interestingly, Fraser et al. [13] and Wildervank et al. [14] have reported cases of SHFM with hearing loss that involve multiple affected siblings born to parents with normal hearing. These facts suggest that some cases of SHFM with deafness may be inherited by an autosomal recessive mode; for example, DFNB14 produces an autosomal recessive phenotype of deafness in Lebanon. So far, no responsible genes were found either for SHFM or DFNB14, although many genes including DLX5 and DLX6 , two homeobox genes that are homologous to the Drosophila distalless gene 7q22 [22], and DSS1 (D eleted in the S plit hand/S plit foot SHFM1 region, MIM #601285) have unique expression patterns in the developing mouse limb bud [23]. However, these three specific genes were not broken by chromosomal abnormalities [23] or damaged by point mutations [24,25]. The present case contained interesting middle and inner ear anomalies and this is the first report that revealed the radiological findings of the temporal bone of SHFM with hearing loss. However, detailed pathological findings of the temporal bone that should be compared to the radiological finding are not available. The presence of a middle ear anomaly implied the possibility of mixed hearing loss, previous reports of SHFM with hearing loss only mentioned ‘‘deafness’’ or ‘‘sensorineural hearing loss’’ with no description of any middle or inner ear malformation. Needless to say, bone conductive audiometry to determine the reliable hearing threshold of sensorineural componet of hearing was required to confirm the conductive hearing loss, it was currently unable because of the patient’s mental retardation. Among the possible candidate genes, abnormality in the PDS gene can cause inner ear anomalies, including an enlarged vestibular aquaduct with or without Mondini malformation [26]; the middle ear malformation found in the present case is not a common phenotype of PDS mutation. Unfortunately, the present case did not received any diagnostic or screening testing for hearing impairment until the boy grew up to 2 years and 7 months in his age. The anomalies in his fingers and toes were the typical risk factor for hearing impairment and the hearing test should have been considered. Not only the establishment of newborn
1131
hearing screening, but the further understanding of such a typical syndromic deafness can accelerate the achievement of early identification and intervention of the hearing impaired children.
Acknowledgements This project is partly supported by grants from the Ministry of Health, Labor, and Welfare and a grant for young scientists from the Ministry of Education, Culture, Sports, Science and Technology.
References [1] V.A. McKusick, W. Lurie, M. Smith, Split hand/foot malformation, type 1: SHFM1, http://www.ncbi.nlm.nih.gov/ htbin-post/Omim/dispmim?183600. [2] G. Del Porto, E. D’Alessandro, C. De Matteis, M.L. Lo Re, M. Ivaldi, C. Di Fusco, Delezione interstiziale del braccio lungo del cromosoma 7 e sue correlazioni cliniche, Pathologica 75 (Suppl) (1983) 268 /271. [3] E.H. Tajara, M. Varella-Garcia, A.C.T. Gusson, Interstitial long-arm deletion of chromosome 7 and ectrodactyly, Am. J. Med. Genet. 32 (1989) 192 /194. [4] M.A. Morey, R.R. Higgins, Ectro-amelia syndrome associated with an interstitial deletion of 7q, Am. J. Med. Genet. 35 (1990) 95 /99. [5] S.H. Roberts, H.E. Hughes, S.J. Davies, A.L. Meredith, Bilateral split hand and split foot malformation in a boy with a de novo interstitial deletion of 7q21.3, J. Med. Genet. 28 (1991) 479 /481. [6] J.C. Marinoni, R.E. Stevenson, J.P. Evans, D. Geshuri, M.C. Phelan, C.E. Schwartz, Split foot and developmental retardation associated with a deletion of three microsatellite markers in 7q21.2 /q22.1, Clin. Genet. 47 (1995) 90 /95. [7] C. McElveen, M.V. Carvajal, D. Moscatello, J. Towner, Y. Lacassie, Ectrodactyly and proximal/intermediate interstitial deletion 7q, Am. J. Med. Genet. 56 (1995) 1 /5. [8] S.W. Scherer, P. Poorkaj, H. Massa, S. Soder, T. Allen, M. Nunes, et al., Physical mapping of the split hand/split foot locus on chromosome 7 and implication in syndromic ectrodactyly, Hum. Mol. Genet. 3 (1994) 1345 /1354. [9] H. Tsukada, K. Nagai, Y. Ooshima, K. Kigata, T. Kamei, A case report with chromosomal abnormality in 7q deletion syndrome combined with congenital malformation of middle and inner ear, Otolaryngol. Head Neck Surg. [Tokyo] 69 (1997) 889 /893. [10] K. Fukushima, N. Kasai, Y. Ueki, K. Nishizaki, K. Sugata, S. Hirakawa, et al., A gene for fluctuating, progressive autosomal dominant nonsyndromic hearing loss, DFNA16, maps to chromosome 2q23 /24.3, Am. J. Hum. Genet. 65 (1999) 141 /150. [11] L.A. Everett, B. Glaser, J.C. Beck, J.R. Idol, A. Buchs, M. Heyman, et al., Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS), Nat. Genet. 17 (1997) 411 /422. [12] A. Birch-Jensen, Congenital deformities of the upper extremities, Op Ex Domo Biol Hered Hum U Hafniensis 19 (1949). [13] G.R. Fraser, The Causes of Profound Deafness in Childhood, Johns Hopkins University Press, Baltimore, 1976, p. 70.
1132 [14] L.S. Wildervanck, Deafness associated with split hands and feet in two siblings. A new syndrome. (Abstract), in: Proceedings of the Eleventh International Congress of Genetics, Hague 1963, pp. 286 /287. [15] M. Sharland, M.A. Patton, L. Hill, Ectrodactyly of hands and feet in a child with a complex translocation including 7q21.2, Am. J. Med. Genet. 39 (1991) 413 /414. [16] M. Genuardi, M.G. Pomponi, V. Sammito, A. Bellussi, M. Zollino, G. Neri, Split hand/split foot anomaly in a family segregating a balanced translocation with breakpoint on 7q22.1, Am. J. Med. Genet. 47 (1993) 823 /831. [17] S.W. Scherer, P. Poorkaj, T. Allen, J. Kim, D. Geshuri, M. Nunes, et al., Fine mapping of the autosomal dominant split hand/split foot locus on chromosome 7, band q21.3 / q22.1, Am. J. Hum. Genet. 55 (1994) 12 /20. [18] D. Tackels-Horne, A. Toburen, E. Sangiorgi, F. Gurrieri, X. de Mollerat, R. Fischetto, et al., Split hand/split foot malformation with hearing loss: first report of families linked to the SHFM1 locus in 7q21, Clin. Genet. 59 (2001) 28 /36. [19] M. Mustapha, N. Salem, D. Weil, E. el-Zir, J. Loiselet, C. Petit, Identification of a locus on chromosome 7q31, DFNB14, responsible for prelingual sensorineural non-syndromic deafness, Eur. J. Hum. Genet. 6 (1998) 548 /551. [20] J.H. Greinwald, Jr, S. Wayne, A.H. Chen, D.A. Scott, R.I. Zbar, M.I. Kraft, et al., Localization of a novel gene for nonsyndromic hearing loss (DFNB17) to chromosome region 7q31, Am. J. Med. Genet. 30 (1998) 107 /113.
K. Fukushima et al. [21] X.C. Li, L.A. Everett, A.K. Lalwani, D. Desmukh, T.B. Friedman, E.D. Green, E.R. Wilcox, A mutation in PDS causes non-syndromic recessive deafness, Nat. Genet. 18 (1998) 215 /217. [22] A. Simeone, D. Acampora, M. Pannese, M. D’Esposito, A. Stornaiuolo, M. Gulisano, et al., Cloning and characterization of two members of the vertebrate Dlx gene family, Proc. Natl. Acad. Sci. USA 91 (1994) 2250 /2254. [23] M.A. Crackower, S.W. Scherer, J.M. Rommens, C.C. Hui, P. Poorkaj, S. Soder, et al., Characterization of the split hand/split foot malformation locus SHFM1 at 7q21.3 / q22.1 and analysis of a candidate gene for its expression during limb development, Hum. Mol. Genet. 5 (1996) 571 / 579. [24] S.W. Scherer, P. Poorkaj, T. Allen, J. Kim, D. Geshuri, M. Nunes, et al., Fine mapping of the autosomal dominant split hand/split foot locus on chromosome 7, band q21.3 / q22.1, Am. J. Hum. Genet. 55 (1994) 12 /20. [25] J. Ignatius, S. Knuutila, S.W. Scherer, B. Trask, J. Kere, et al., Split hand/split foot malformation, deafness, and mental retardation with a complex cytogenetic rearrangement involving 7q21.3, Am. J. Med. Genet. 33 (1996) 507 / 510. [26] S. Usami, S. Abe, M.D. Weston, H. Shinkawa, G. Van Camp, W.J. Kimberling, Non-syndromic hearing loss associated with enlarged vestibular aqueduct is caused by PDS mutations, Hum. Genet. 104 (1999) 188 /192.
www.elsevier.com/locate/ijporl
CASE REPORT
Deletion mapping of split hand/split foot malformation with hearing impairment: a case report Kunihiro Fukushimaa,*, Kyoko Nagaib, Haruyo Tsukadab, Akemi Sugataa, Kenichi Sugataa, Norio Kasaia, Namiki Kibayashia, Yukihide Maedaa, Mehmet Gunduza, Kazunori Nishizakia a
Department of Otolaryngology */Head and Neck Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata Cho, Okayama 700-8558, Japan b Department of Otolaryngology */Head and Neck Surgery, Gunnma University School of Medicine, Gunnma, Japan Received 25 March 2003; received in revised form 30 May 2003; accepted 1 June 2003
KEYWORDS Mapping; Split hand/split foot malformation; Hearing impairment
Summary Split hand/split foot malformation (SHFM), which typically appears as lobster-like limb malformation, is a rare clinical condition caused by a partial deletion of chromosome 7q. Hearing impairment sometimes accompanies syndromic SHFM cases; a case of inner and middle ear malformation with SHFM is described in this report. We conducted a genetic evaluation of this patient and found a deleted region that overlaps a previously reported locus of SHFM as well as a DFNB14 locus that can cause nonsyndromic hearing impairment by autosomal recessive inheritance. – 2003 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Split hand/split foot malformation type 1 (SHFM1, OMIM183600) is a congenital malformation syndrome that typically includes an ectrodactyly limb malformation resembling the primary limbs of an arthropod, syndactyly, and median clefts of the hands and feet. The disease itself is considered to be very rare, affecting one out of 90 000 newborn babies [1]. Del Porto et al. first reported a case of a deletion at chromosome 7(q11 /q22) in which the patient
*Corresponding author. Tel.: /81-86-235-7307; fax: /81-86235-7308. E-mail address: [email protected] (K. Fukushima).
had ectrodactyly, microcephalus, and low-set ears [2]. At least nine additional cases have demonstrated the relationship between the partial deletion of 7q and the clinical manifestation of SHFM [3 /8]. In particular, the region of 7q21.2 /q21.3 was frequently shared among cases [3] and found to be the SHFM1 locus. Three other loci for SHFM have also been mapped: SHFM2 on chromosome Xq26, SHFM3 on chromosome 10q24, and SHFM4, the result of a mutation in the TP63 gene, on chromosome 3q27 [3]. Other abnormalities in various degrees may be found in combination with SHFM. Scherer et al. classified syndromic ectrodactyly (SE) into the following nine clinical types [8]. (1) Ectrodactyly, ectodermal dysplasia and cleft lip/palate syndrome (online menderian inheritance in men
0165-5876/03/$ - see front matter – 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0165-5876(03)00193-9
1128
[OMIM: http://www3.ncbi.nlm.nih.gov/Omim/ searchomim.html] #129900), (2) Lacrimoauriculodentodigital syndrome (LADD syndrome: OMIM #149730), (3) Acro /Dermato /Ungual /Lacrimal / Tooth syndrome (ADULT syndrome: OMIM #103285), (4) Ectrodactyly and ectodermal dysplasia without cleft lip/palate (OMIM #129810), (5) Fontaine syndrome (OMIM #183700), (6) Acral / renal /mandibular syndrome (OMIM #200980), (7) Ectrodactyly /Cleft palate syndrome (OMIM #129830), (8) Ectrodactyly and hearing loss (OMIM #220600), and (9) Karsch /Neugebauer syndrome (OMIM #183800). Among these nine subclassifications, hearing loss is a relatively common clinical manifestation. However, the otological or audiological component of SHFM with hearing impairment has not been fully described with further genetic testing, although the presence of hearing impairment can deeply affect the patient’s quality of life. In this study, we present a case of SHFM in which the patient has hearing loss and typical ectrodactyly and in which we found partial deletion of chromosome 7q. Deletion mapping with short nucleotide repeats (SNR) demonstrated the deleted region of 7q that causes the phenotype of hearing loss along with ectrodactyly.
2. Case presentation A full-term baby boy was born with a body weight of 2076 g. His Apgar score was 5 at 10 min and 9 at 30 min. Multiple malformations of his hands and feet (Fig. 1), hypotonic muscles, and difficulty of suckling were noted. Cytological examination revealed a partial deletion of chromosome 7 [del(7)(q11.2q22)], shown in Fig. 2. The baby was referred to the Gunnma Medical School Hospital when he was 4 months old. At the age of 2 years 7 months, he was referred to the otolaryngology clinic there because he was unable to respond to any sound. The boy was 80 cm tall and weighed 9.5 kg, which were each two standard deviations below the age-appropriate Japanese standard. In addition to his small body size, the boy had poor muscle tone. The boy’s right hand appeared normal but his left hand had only four fingers, his right foot had only four toes, and his left foot had only three toes. The boy had auricular malformation including lowset ears, loss of the Crura anthelicis , and stenosis of the external ear canals. Microgenia, hypoplasia of the tongue, and mild laryngomalacia were also observed. Click-evoked ABR testing revealed that the threshold is 80 dB in his right ear and 90 dB in his left ear. Condition orientation reflex testing re-
K. Fukushima et al.
Fig. 1 Radiograph of the patient’s foot, which also shows the absence of a toe.
vealed a sound response at 90 dB. Gross motor movement showed apparent developmental delay; he did not achieve head control until 1 year 7 months of age and he did not master sitting alone until the age of 2 years 1 month. High resolution computed tomography revealed middle ear malformation, including fusion of the malleus and incus, narrowing of the internal ear meatus, hypoplasia of the cochlea, and ballooning of the ampullae of the semicircular canals (Fig. 3). These radiological and clinical manifestations of this case were partly reported elsewhere in Japanese [9].
3. Fine deletion mapping The DNA was obtained from peripheral blood by phenol chloroform extraction and amplified by the polymerase chain reaction (PCR) as described previously [10]. Each 10-ml PCR sample contained 10 ng of genomic DNA, 2.0 pmol of each primer (Invitrogen Corp., Carlsbad, CA, USA), 200 mM of each dNTP (Toyobo Inc., Osaka, Japan), 0.25 U of Taq DNA polymerase, and 1 / PCR buffer (Takara Shuzo Inc., Tokyo, Japan). After the initial denaturation step at 94 8C for 2 min, samples were amplified under the following thermal conditions: 95 8C for 30 s, 55 8C for 30 s, and 72 8C for 30 s for
Deletion mapping of split hand/split foot malformation with hearing impairment
1129
Fig. 2 Diagram of chromosome 7q (A), its normal map (B), and a map showing its deletion (C) in the present case.
25 cycles, with a final extension step at 72 8C for 10 min. The chromosomal location was determined from information in the GDB genome database http://gdbwww.gdb.org/. Electrophoresis was performed on a 6% polyacrylamide gel and bands were detected by silver staining. Samples of DNA from three healthy controls were also used to identify the banding pattern of a single allele. The chromosomal locations of the primers used here were (from centromere to telomere): D7S1830, D7S1831, D7S494, D7S669, D7S644, D7S646, D7S651, D7S518,
D7S796, D7S525, D7S1817, D7S486, and D7S480 (Table 1).
4. Sequencing analysis The exons of the PDS gene were amplified by PCR according to previously reported conditions and primers [11]. The products were purified using a commercial kit (QIAquickTM, QIAGEN Inc., Valencia, CA, USA). Half of the PCR products were used
Fig. 3 A computed tomography scan of the patient’s ear, showing fusion of the malleus and incus (A) and malformation of the cochlea (B), resulting in abnormal communication with the internal ear canal.
1130
K. Fukushima et al.
6. Discussion
Table 1 Marker
Map distance (cM)
Result
D7S1830 D7S1831 D7S494 D7S669 D7S644 D7S646 D7S651 D7S518 D7S796 D7S525 D7S1817 D7S486 D7S480
79.38/51.78 49.55 70.60 107.35 119.05 119.78 122.15 122.63 122.83 124.81 126.59 129.26 130.98
NI NI Heterozygous Heterozygous Homozygous Homozygous Homozygous Homozygous Heterozygous NI NI Heterozygous Heterozygous
NI, not informative because of the homozygosity.
for direct sequencing and half were subcloned using a pBluescript subcloning kit (Stratagene, La Jolla, CA, USA). All samples were sequenced using the Big-dyeTM terminator cycle sequencing ready kit with AmplitaqTM DNA polymerase FS (ABI: Applied Biosystems Foster City, CA, USA) and run on an ABI PRISM 373S Genetic Analyzer.
5. Results Each of four markers (D7S644, D7S646, D7S651, and D7S518) demonstrated a single band, while the flanking markers D7S669 and D7S796 demonstrated apparent heterozygosity, the presence of two different alleles (Table 1). In the region between these two markers, a minimum of 4.58 cM and a maximum of 16.48 cM was deleted in this case. This region included the SHFM genetic region and a wider region on the telomeric side. This region apparently excluded the PDS coding region (Fig. 4) and a direct sequence of the PDS coding region demonstrated no apparent mutation.
Fig. 4 Diagrams of the deleted region in the present case, the previously reported SHFM locus, and the DFNB14 locus.
Hearing impairment is one of clinical symptoms that are found in combination with ectrodactyly [12 /14]. The association of particular chromosomal region and ectrodactyly has already reported. Sharland et al. [15] reported a case of tetramelic ectrodactyly that demonstrated complex chromosomal rearrangement (5q11.2, 5q34, 7q21.2, 7q31.3 and 9q22.1) without any apparent chromosomal deletions. In that case, no additional deficit other than a minor facial anomaly was observed. Genuardi et al. [16] also reported a case of bilateral SHFM with balanced translocation of t(2;7)(q21.1;q22.1) that showed autosomal dominant inheritance. Our result clearly demonstrated that the ectrodactyly with hearing loss can be caused by the same chromosomal abnormality, a deletion in 7q21.2 /q21.3, as reported in various types of non-SE. The locus of ectrodactyly can be genetically mapped from somatic cell hybrid lines [17] and the minimum chromosomal region of SHFDassociated deletions is PON (168820) /D7S812 / SHFD1 /D7S811 /ASNS (108370). Marinoni et al. [6] reported a case of SHFM with developmental delay that showed the deletion of 7q21.2 /q22.1 and the absence of D7S 527, D7S 479, and D7S 554. With the result from deletion mapping, the deleted chromosomal region in the present case included the previously reported SHFD-associated region as indicated in Fig. 4. Thus, the same factor found in the same genomic region may play the central role in the cause of non-SE and ectrodactyly with hearing loss. Recently, Tackels-Horne et al. [18] described two families with a form of SHFM in which deficiency of the central rays in the appendicular skeleton was associated with sensorineural hearing loss. Variably expressed SHFM was found in nine of 15 presumed gene carriers, and mild to moderate sensorineural hearing loss in seven. In these two families, Tackels-Horne et al. [18] found that the disorder was linked to markers on 7q21, the region of the SHFM1 locus, with a combined maximum load score of 4.37 at theta /0.0 for D7S527 at 80% penetration. This region again overlapped with the deleted region in the present case. Among the families that were reported by Tackels-Horne et al. [18], one presumed gene carrier showed hearing impairment without ectrodactyly, and it may be possible that nonsyndromic hearing loss is caused by an allelic variance of SHFM with hearing loss. Three different nonsyndromic deafness genes have been mapped near the SHFM locus: DFNB14 [19], DFNB17 [20], and PDS (DFNB4) [11,21]. The critical region of DFNB14 was reported
Deletion mapping of split hand/split foot malformation with hearing impairment
between D7S527 and D7S3047, spanning a 15-cM region [19], and the PDS gene was considered to be a telomeric border of this region [19]. The critical region of the DFNB17 was D7S2487, D7S655, and D7S480, and the PDS gene was located on its centromeric border [20]. Among these three adjacent genes, one gene that overlaps with the deleted region in the present case is DFNB14 (Fig. 4). Interestingly, Fraser et al. [13] and Wildervank et al. [14] have reported cases of SHFM with hearing loss that involve multiple affected siblings born to parents with normal hearing. These facts suggest that some cases of SHFM with deafness may be inherited by an autosomal recessive mode; for example, DFNB14 produces an autosomal recessive phenotype of deafness in Lebanon. So far, no responsible genes were found either for SHFM or DFNB14, although many genes including DLX5 and DLX6 , two homeobox genes that are homologous to the Drosophila distalless gene 7q22 [22], and DSS1 (D eleted in the S plit hand/S plit foot SHFM1 region, MIM #601285) have unique expression patterns in the developing mouse limb bud [23]. However, these three specific genes were not broken by chromosomal abnormalities [23] or damaged by point mutations [24,25]. The present case contained interesting middle and inner ear anomalies and this is the first report that revealed the radiological findings of the temporal bone of SHFM with hearing loss. However, detailed pathological findings of the temporal bone that should be compared to the radiological finding are not available. The presence of a middle ear anomaly implied the possibility of mixed hearing loss, previous reports of SHFM with hearing loss only mentioned ‘‘deafness’’ or ‘‘sensorineural hearing loss’’ with no description of any middle or inner ear malformation. Needless to say, bone conductive audiometry to determine the reliable hearing threshold of sensorineural componet of hearing was required to confirm the conductive hearing loss, it was currently unable because of the patient’s mental retardation. Among the possible candidate genes, abnormality in the PDS gene can cause inner ear anomalies, including an enlarged vestibular aquaduct with or without Mondini malformation [26]; the middle ear malformation found in the present case is not a common phenotype of PDS mutation. Unfortunately, the present case did not received any diagnostic or screening testing for hearing impairment until the boy grew up to 2 years and 7 months in his age. The anomalies in his fingers and toes were the typical risk factor for hearing impairment and the hearing test should have been considered. Not only the establishment of newborn
1131
hearing screening, but the further understanding of such a typical syndromic deafness can accelerate the achievement of early identification and intervention of the hearing impaired children.
Acknowledgements This project is partly supported by grants from the Ministry of Health, Labor, and Welfare and a grant for young scientists from the Ministry of Education, Culture, Sports, Science and Technology.
References [1] V.A. McKusick, W. Lurie, M. Smith, Split hand/foot malformation, type 1: SHFM1, http://www.ncbi.nlm.nih.gov/ htbin-post/Omim/dispmim?183600. [2] G. Del Porto, E. D’Alessandro, C. De Matteis, M.L. Lo Re, M. Ivaldi, C. Di Fusco, Delezione interstiziale del braccio lungo del cromosoma 7 e sue correlazioni cliniche, Pathologica 75 (Suppl) (1983) 268 /271. [3] E.H. Tajara, M. Varella-Garcia, A.C.T. Gusson, Interstitial long-arm deletion of chromosome 7 and ectrodactyly, Am. J. Med. Genet. 32 (1989) 192 /194. [4] M.A. Morey, R.R. Higgins, Ectro-amelia syndrome associated with an interstitial deletion of 7q, Am. J. Med. Genet. 35 (1990) 95 /99. [5] S.H. Roberts, H.E. Hughes, S.J. Davies, A.L. Meredith, Bilateral split hand and split foot malformation in a boy with a de novo interstitial deletion of 7q21.3, J. Med. Genet. 28 (1991) 479 /481. [6] J.C. Marinoni, R.E. Stevenson, J.P. Evans, D. Geshuri, M.C. Phelan, C.E. Schwartz, Split foot and developmental retardation associated with a deletion of three microsatellite markers in 7q21.2 /q22.1, Clin. Genet. 47 (1995) 90 /95. [7] C. McElveen, M.V. Carvajal, D. Moscatello, J. Towner, Y. Lacassie, Ectrodactyly and proximal/intermediate interstitial deletion 7q, Am. J. Med. Genet. 56 (1995) 1 /5. [8] S.W. Scherer, P. Poorkaj, H. Massa, S. Soder, T. Allen, M. Nunes, et al., Physical mapping of the split hand/split foot locus on chromosome 7 and implication in syndromic ectrodactyly, Hum. Mol. Genet. 3 (1994) 1345 /1354. [9] H. Tsukada, K. Nagai, Y. Ooshima, K. Kigata, T. Kamei, A case report with chromosomal abnormality in 7q deletion syndrome combined with congenital malformation of middle and inner ear, Otolaryngol. Head Neck Surg. [Tokyo] 69 (1997) 889 /893. [10] K. Fukushima, N. Kasai, Y. Ueki, K. Nishizaki, K. Sugata, S. Hirakawa, et al., A gene for fluctuating, progressive autosomal dominant nonsyndromic hearing loss, DFNA16, maps to chromosome 2q23 /24.3, Am. J. Hum. Genet. 65 (1999) 141 /150. [11] L.A. Everett, B. Glaser, J.C. Beck, J.R. Idol, A. Buchs, M. Heyman, et al., Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS), Nat. Genet. 17 (1997) 411 /422. [12] A. Birch-Jensen, Congenital deformities of the upper extremities, Op Ex Domo Biol Hered Hum U Hafniensis 19 (1949). [13] G.R. Fraser, The Causes of Profound Deafness in Childhood, Johns Hopkins University Press, Baltimore, 1976, p. 70.
1132 [14] L.S. Wildervanck, Deafness associated with split hands and feet in two siblings. A new syndrome. (Abstract), in: Proceedings of the Eleventh International Congress of Genetics, Hague 1963, pp. 286 /287. [15] M. Sharland, M.A. Patton, L. Hill, Ectrodactyly of hands and feet in a child with a complex translocation including 7q21.2, Am. J. Med. Genet. 39 (1991) 413 /414. [16] M. Genuardi, M.G. Pomponi, V. Sammito, A. Bellussi, M. Zollino, G. Neri, Split hand/split foot anomaly in a family segregating a balanced translocation with breakpoint on 7q22.1, Am. J. Med. Genet. 47 (1993) 823 /831. [17] S.W. Scherer, P. Poorkaj, T. Allen, J. Kim, D. Geshuri, M. Nunes, et al., Fine mapping of the autosomal dominant split hand/split foot locus on chromosome 7, band q21.3 / q22.1, Am. J. Hum. Genet. 55 (1994) 12 /20. [18] D. Tackels-Horne, A. Toburen, E. Sangiorgi, F. Gurrieri, X. de Mollerat, R. Fischetto, et al., Split hand/split foot malformation with hearing loss: first report of families linked to the SHFM1 locus in 7q21, Clin. Genet. 59 (2001) 28 /36. [19] M. Mustapha, N. Salem, D. Weil, E. el-Zir, J. Loiselet, C. Petit, Identification of a locus on chromosome 7q31, DFNB14, responsible for prelingual sensorineural non-syndromic deafness, Eur. J. Hum. Genet. 6 (1998) 548 /551. [20] J.H. Greinwald, Jr, S. Wayne, A.H. Chen, D.A. Scott, R.I. Zbar, M.I. Kraft, et al., Localization of a novel gene for nonsyndromic hearing loss (DFNB17) to chromosome region 7q31, Am. J. Med. Genet. 30 (1998) 107 /113.
K. Fukushima et al. [21] X.C. Li, L.A. Everett, A.K. Lalwani, D. Desmukh, T.B. Friedman, E.D. Green, E.R. Wilcox, A mutation in PDS causes non-syndromic recessive deafness, Nat. Genet. 18 (1998) 215 /217. [22] A. Simeone, D. Acampora, M. Pannese, M. D’Esposito, A. Stornaiuolo, M. Gulisano, et al., Cloning and characterization of two members of the vertebrate Dlx gene family, Proc. Natl. Acad. Sci. USA 91 (1994) 2250 /2254. [23] M.A. Crackower, S.W. Scherer, J.M. Rommens, C.C. Hui, P. Poorkaj, S. Soder, et al., Characterization of the split hand/split foot malformation locus SHFM1 at 7q21.3 / q22.1 and analysis of a candidate gene for its expression during limb development, Hum. Mol. Genet. 5 (1996) 571 / 579. [24] S.W. Scherer, P. Poorkaj, T. Allen, J. Kim, D. Geshuri, M. Nunes, et al., Fine mapping of the autosomal dominant split hand/split foot locus on chromosome 7, band q21.3 / q22.1, Am. J. Hum. Genet. 55 (1994) 12 /20. [25] J. Ignatius, S. Knuutila, S.W. Scherer, B. Trask, J. Kere, et al., Split hand/split foot malformation, deafness, and mental retardation with a complex cytogenetic rearrangement involving 7q21.3, Am. J. Med. Genet. 33 (1996) 507 / 510. [26] S. Usami, S. Abe, M.D. Weston, H. Shinkawa, G. Van Camp, W.J. Kimberling, Non-syndromic hearing loss associated with enlarged vestibular aqueduct is caused by PDS mutations, Hum. Genet. 104 (1999) 188 /192.