- Email: [email protected]
Two different PAX3 gene mutations causing Waardenburg syndrome type I
Molecular and Cellular Probes (1996) 10, 229–231
Short Communication
Two different PAX3 gene mutations causing Waardenburg syndrome type I G. Wildhardt,1,2∗ A. Winterpacht,2 K. Hilbert,2 H. Menger2 and B. Zabel2 1
Laboratory Dres. Kapp, Breuer et al., Bahnhofplatz 2, D-55116 Mainz and 2Children’s Hospital, University of Mainz, Langenbeckstr. 1, D-55101 Mainz, Germany (Received 20 December 1995, Accepted 22 December 1995)
Waardenburg syndrome (WS) is a form of autosomal dominant inherited deafness combined with specific congenital anomalies. WS types I and III are correlated with mutations in the PAX3 gene on chromosome 2q37. In this report we describe two mutations in the human PAX3 gene causing WS type I in two families. One mutation is an insertion in the paired box domain resulting in a protein termination within the paired box. The second mutation is a base pair substitution producing an arginine to cysteine amino acid change in the homeobox region. 1996 Academic Press Limited KEYWORDS: Waardenburg syndrome, deafness, PAX3, paired box, homeobox.
Waardenburg syndrome (WS) is the most frequent form of congenital deafness in humans (1 in 42 000). The inheritance is autosomal dominant with variable expression and incomplete penetrance. Three clinical types are distinguished. Type I (WS1, MIM 193500) is the most common form and is characterized by sensorineural deafness, heterochromia iridis, dystopia canthorum, hypopigmentation of the skin and scalp hair (white forelock), broad nasal root and synophrys. Type II (WS2, MIM 193510) presents as type I, but without dystopia canthorum. Type III (WS3, 148820) or Klein-Waardenburg syndrome is similar to WS1 but includes upper limb abnormalities.1,2 Linkage analysis led to the identification of PAX3 as a WS candidate gene, located at chromosome 2q35–37. The gene was found to be mutated in some cases of WS1 and WS3. It is highly homologous to the murine pax3 locus,3 a developmental control gene responsible for the splotch phenotype in mice. It contains highly conserved sequence motifs typical of
DNA binding proteins (paired domain, octapeptide and homeobox), identifying PAX3 as transcription factor.4 None of the WS2 families were linked to the PAX3 gene locus.5 However, some WS2 cases were shown to be due to mutations of the human microphthalmia (MITF) gene localized at 3p12.3–p14.6 Several different PAX3 gene mutations have been reported in families with Waardenburg syndrome.7 Most occur in the highly conserved paired domain and in the homeobox of PAX3. The way in which they affect the normal functioning of PAX3 is a matter of speculation, as the target genes of this transcription factor are yet to be identified. Here we report mutations in two families with WS1. DNA was isolated from peripheral lymphocytes using standard protocols. Amplification was carried out with Taq polymerase (Boehringer) as described by the manufacturer, under the following conditions: 10 m Tris-HCl (pH 8·3), 1·5 m MgCl2, 50 m KCl and 0·1% gelatin, 500 ng DNA and 10 pmole of each
∗ Please address all correspondence to: Dr G. Wildhardt, Children’s Hospital, University of Mainz, Langenbeckstr. 1, D-55101 Mainz, Germany.
0890–8508/96/030229+03 $18.00/0
229
1996 Academic Press Limited
230
G. Wildhardt et al. Paired box
Octapeptide
Homeobox
Wildtype
Mutant 1
Mutant 2
Fig. 1. Nucleotide sequence showing normal and mutant exon 2 and 6 of the PAX3 gene. The primers for amplification are GW 33a 5′-CAGTGACTTTTCCCTTGCTTC-3′, GW 34a 5′-CCGGTCTTCCCCAACAG-3′ (exon 2) and GW C 5′-TTCATCAGTGAAATCCTTAAA-3′, GW D 5-′CTAATCTCCTTGACTCTTCC-3′ (exon 6).
primer in a total volume of 100 ll. After initial heating to 95°C for 8 min the enzyme was added (5 U ll−1) and the PCR was preformed; 15 s 96°C, 1 min 55°C for exon 2 and 1 min 50°C for exon 6, 2 min 72°C for 30 cycles in a Perkin Elmer (system 9600) thermocycler. The PCR products were either cloned into pUC 19 (exon 2) or sequenced directly (exon 6). Sequencing was carried out on an ABI 373A automatic sequencing apparatus, using the dye deoxy terminator cycle sequencing kit and the protocols from ABI. In the first family a 7-year-old boy with sensorineural deafness, heterochromia iridis, dystopia canthorum and broad nasal root was the index case. His mother, who carried the same mutation, had the following symptoms: dystopia canthorum, broad nasal root, hypoplasic alae and grey hair since the age of 16. We identified a single base pair insertion (T) in position 292 of the coding sequence (exon 2) resulting in a stop codon in exon 3 within the paired domain. The truncated protein has a shorter paired domain and lacks the octapeptide and the homeobox. In the second family the index case was a 4-yearold girl with deafness, dystopia canthorum, broad nasal root and synophrys. We identified a heterozygote C to T substitution in position 811 (exon 6) which causes an amino acid change (arg to cys) in
the homeobox. The mutated protein could have an altered affinity for DNA sequences compared to the wild type protein. Arginine in position 271 is highly conserved in homeobox proteins from yeast to man among eukaryotes.8 This amino acid is involved in binding to the phosphate backbone in the major groove of DNA9 and is just three amino acids away from the residue responsible for sequence specific binding. This cysteine residue in the altered protein may form a disulfide bridge with another cysteine which could have a dramatic influence on the protein structure. The molecular basis of the intra- and interfamilial variability of the WS1 phenotype is still unclear. The phenotype may be due to loss-of-function mutations and/or influenced by additional loci that modify or regulate the WS phenotype.10 References 1. McKusick, V. M. (1994). Mendelian Inheritance in Man, 11th ed. Baltimore: Johns Hopkins University Press. 2. Waardenburg, P. J. (1951). A new syndrome combining developmental anomalies of the eyelids, eyebrows and nose root with pigmentary defects of the iris and head hair and with congenital deafness. American Journal of Human Genetics 3, 195–253.
PAX3 gene mutations 3. Tassabehji, M., Newton, V. E., Leverton, K., Turnbull, K., Seemanova, E., Kunze, J., Sperling, K., Strachen, T. & Read, A. P. (1994). PAX3 gene structure and mutations: close analogies between Waardenburg syndrome and Splotch mouse. Human Molecular Genetics 3, 1069–74. 4. Goulding, M. D., Chaepakis, G., Deutsch, U., Erselius, J. & Gruss, P. (1991). Pax3, a novel murine DNA binding protein expressed during early neurogenesis. European Journal of Molecular Biology 10, 1135–47. 5. Farrer, L. A., Arnos, K. S., Asher, J. H. Jr., Baldwin, C. T., Diehl, S. R., Friedman, T. B., Greenberg, J. et al. (1994). Locus heterogeneity for Waardenburg syndrome is predictive of clinical subtypes. American Journal of Human Genetics 55, 728–37. 6. Hughes, A. E., Newton, V. E., Liu, X. Z. & Read, A. P. (1994). A gene for Waardenburg Syndrome type 2 maps close to the human homologue of the microphthalmia gene at chromosome 3p12–p14.1. Nature Genetics 7, 509–12.
231
7. Tassabehji, M., Newton, V., Lui, X. Z., Brady, A., Donnai, D., Krajewska-Walasek, M., Murday, V., Norman, A., Obersztyn, E., Reardon, W., Rice, J. C., Trembath, R., Wieacker, P., Whiteford, M., Winter, R. & Read, A. (1995). The mutational Spectrum in Waardenburg syndrome. Human Molecular Genetics 4, 2131–7. 8. Lalwani, A. K., Brister, R., Fex, J., Grundfast, K. M., Ploplis, B., San Agustin, T. B. & Wilcox, E. R. (1995). Further elucidation of the genomic structure of PAX3, and identification of two different point mutations within the PAX3 homeobox that cause Waardenburg syndrom type I in two families. American Journal of Human Genetics 56, 75– 83. 9. Scott, M. P., Tamkun, J. W. & Hartzell, G. W. (1989). The structure and function of the homeodomain. Biochemical Biophysical Acta 989, 25–48. 10. Strachan, T. & Read, A. P. (1994). PAX genes. Current Opinion in Genetics and Development 4, 427–38.
Short Communication
Two different PAX3 gene mutations causing Waardenburg syndrome type I G. Wildhardt,1,2∗ A. Winterpacht,2 K. Hilbert,2 H. Menger2 and B. Zabel2 1
Laboratory Dres. Kapp, Breuer et al., Bahnhofplatz 2, D-55116 Mainz and 2Children’s Hospital, University of Mainz, Langenbeckstr. 1, D-55101 Mainz, Germany (Received 20 December 1995, Accepted 22 December 1995)
Waardenburg syndrome (WS) is a form of autosomal dominant inherited deafness combined with specific congenital anomalies. WS types I and III are correlated with mutations in the PAX3 gene on chromosome 2q37. In this report we describe two mutations in the human PAX3 gene causing WS type I in two families. One mutation is an insertion in the paired box domain resulting in a protein termination within the paired box. The second mutation is a base pair substitution producing an arginine to cysteine amino acid change in the homeobox region. 1996 Academic Press Limited KEYWORDS: Waardenburg syndrome, deafness, PAX3, paired box, homeobox.
Waardenburg syndrome (WS) is the most frequent form of congenital deafness in humans (1 in 42 000). The inheritance is autosomal dominant with variable expression and incomplete penetrance. Three clinical types are distinguished. Type I (WS1, MIM 193500) is the most common form and is characterized by sensorineural deafness, heterochromia iridis, dystopia canthorum, hypopigmentation of the skin and scalp hair (white forelock), broad nasal root and synophrys. Type II (WS2, MIM 193510) presents as type I, but without dystopia canthorum. Type III (WS3, 148820) or Klein-Waardenburg syndrome is similar to WS1 but includes upper limb abnormalities.1,2 Linkage analysis led to the identification of PAX3 as a WS candidate gene, located at chromosome 2q35–37. The gene was found to be mutated in some cases of WS1 and WS3. It is highly homologous to the murine pax3 locus,3 a developmental control gene responsible for the splotch phenotype in mice. It contains highly conserved sequence motifs typical of
DNA binding proteins (paired domain, octapeptide and homeobox), identifying PAX3 as transcription factor.4 None of the WS2 families were linked to the PAX3 gene locus.5 However, some WS2 cases were shown to be due to mutations of the human microphthalmia (MITF) gene localized at 3p12.3–p14.6 Several different PAX3 gene mutations have been reported in families with Waardenburg syndrome.7 Most occur in the highly conserved paired domain and in the homeobox of PAX3. The way in which they affect the normal functioning of PAX3 is a matter of speculation, as the target genes of this transcription factor are yet to be identified. Here we report mutations in two families with WS1. DNA was isolated from peripheral lymphocytes using standard protocols. Amplification was carried out with Taq polymerase (Boehringer) as described by the manufacturer, under the following conditions: 10 m Tris-HCl (pH 8·3), 1·5 m MgCl2, 50 m KCl and 0·1% gelatin, 500 ng DNA and 10 pmole of each
∗ Please address all correspondence to: Dr G. Wildhardt, Children’s Hospital, University of Mainz, Langenbeckstr. 1, D-55101 Mainz, Germany.
0890–8508/96/030229+03 $18.00/0
229
1996 Academic Press Limited
230
G. Wildhardt et al. Paired box
Octapeptide
Homeobox
Wildtype
Mutant 1
Mutant 2
Fig. 1. Nucleotide sequence showing normal and mutant exon 2 and 6 of the PAX3 gene. The primers for amplification are GW 33a 5′-CAGTGACTTTTCCCTTGCTTC-3′, GW 34a 5′-CCGGTCTTCCCCAACAG-3′ (exon 2) and GW C 5′-TTCATCAGTGAAATCCTTAAA-3′, GW D 5-′CTAATCTCCTTGACTCTTCC-3′ (exon 6).
primer in a total volume of 100 ll. After initial heating to 95°C for 8 min the enzyme was added (5 U ll−1) and the PCR was preformed; 15 s 96°C, 1 min 55°C for exon 2 and 1 min 50°C for exon 6, 2 min 72°C for 30 cycles in a Perkin Elmer (system 9600) thermocycler. The PCR products were either cloned into pUC 19 (exon 2) or sequenced directly (exon 6). Sequencing was carried out on an ABI 373A automatic sequencing apparatus, using the dye deoxy terminator cycle sequencing kit and the protocols from ABI. In the first family a 7-year-old boy with sensorineural deafness, heterochromia iridis, dystopia canthorum and broad nasal root was the index case. His mother, who carried the same mutation, had the following symptoms: dystopia canthorum, broad nasal root, hypoplasic alae and grey hair since the age of 16. We identified a single base pair insertion (T) in position 292 of the coding sequence (exon 2) resulting in a stop codon in exon 3 within the paired domain. The truncated protein has a shorter paired domain and lacks the octapeptide and the homeobox. In the second family the index case was a 4-yearold girl with deafness, dystopia canthorum, broad nasal root and synophrys. We identified a heterozygote C to T substitution in position 811 (exon 6) which causes an amino acid change (arg to cys) in
the homeobox. The mutated protein could have an altered affinity for DNA sequences compared to the wild type protein. Arginine in position 271 is highly conserved in homeobox proteins from yeast to man among eukaryotes.8 This amino acid is involved in binding to the phosphate backbone in the major groove of DNA9 and is just three amino acids away from the residue responsible for sequence specific binding. This cysteine residue in the altered protein may form a disulfide bridge with another cysteine which could have a dramatic influence on the protein structure. The molecular basis of the intra- and interfamilial variability of the WS1 phenotype is still unclear. The phenotype may be due to loss-of-function mutations and/or influenced by additional loci that modify or regulate the WS phenotype.10 References 1. McKusick, V. M. (1994). Mendelian Inheritance in Man, 11th ed. Baltimore: Johns Hopkins University Press. 2. Waardenburg, P. J. (1951). A new syndrome combining developmental anomalies of the eyelids, eyebrows and nose root with pigmentary defects of the iris and head hair and with congenital deafness. American Journal of Human Genetics 3, 195–253.
PAX3 gene mutations 3. Tassabehji, M., Newton, V. E., Leverton, K., Turnbull, K., Seemanova, E., Kunze, J., Sperling, K., Strachen, T. & Read, A. P. (1994). PAX3 gene structure and mutations: close analogies between Waardenburg syndrome and Splotch mouse. Human Molecular Genetics 3, 1069–74. 4. Goulding, M. D., Chaepakis, G., Deutsch, U., Erselius, J. & Gruss, P. (1991). Pax3, a novel murine DNA binding protein expressed during early neurogenesis. European Journal of Molecular Biology 10, 1135–47. 5. Farrer, L. A., Arnos, K. S., Asher, J. H. Jr., Baldwin, C. T., Diehl, S. R., Friedman, T. B., Greenberg, J. et al. (1994). Locus heterogeneity for Waardenburg syndrome is predictive of clinical subtypes. American Journal of Human Genetics 55, 728–37. 6. Hughes, A. E., Newton, V. E., Liu, X. Z. & Read, A. P. (1994). A gene for Waardenburg Syndrome type 2 maps close to the human homologue of the microphthalmia gene at chromosome 3p12–p14.1. Nature Genetics 7, 509–12.
231
7. Tassabehji, M., Newton, V., Lui, X. Z., Brady, A., Donnai, D., Krajewska-Walasek, M., Murday, V., Norman, A., Obersztyn, E., Reardon, W., Rice, J. C., Trembath, R., Wieacker, P., Whiteford, M., Winter, R. & Read, A. (1995). The mutational Spectrum in Waardenburg syndrome. Human Molecular Genetics 4, 2131–7. 8. Lalwani, A. K., Brister, R., Fex, J., Grundfast, K. M., Ploplis, B., San Agustin, T. B. & Wilcox, E. R. (1995). Further elucidation of the genomic structure of PAX3, and identification of two different point mutations within the PAX3 homeobox that cause Waardenburg syndrom type I in two families. American Journal of Human Genetics 56, 75– 83. 9. Scott, M. P., Tamkun, J. W. & Hartzell, G. W. (1989). The structure and function of the homeodomain. Biochemical Biophysical Acta 989, 25–48. 10. Strachan, T. & Read, A. P. (1994). PAX genes. Current Opinion in Genetics and Development 4, 427–38.