tional mutations in the PAX3 gene. The first one concerns a missense mutation within the paired do- main region of exon 2. The same mutation was re-.
HUMAN MUTATION Supplement 1:S145-S147 (1998)
MUTATION IN BRIEF
tification of Two
s causing
the Paired nstrearn of the
Horneodomain Frans A. Hal,'* Moni ue P.A. Geurds,' Cor W.R.J. Cremers,' Ben C.J. Hamel,' and Edwin C.M. Mariman
P
'Department of H u m n Genetics, University Hospital Nijmegen, 6500 HB Nijmegen, The Netherlands Department of Oto-Rhino-Laryngobg-y,University Hospital Nijmegen, 6500 HB Nijmegen, The Netherlands
2
Communicated by David C. Page
INTRODUCTION
Waaidenburg syndrome (WS) is an autosomal dominant disorder characterized by varying combinations of congenital deafness, dystopia canthorum (outward displacement of the inner canthi of the eyes), and abnormal pigmentation of the eyes, hair, and skin. WS can be clinically divided in two main subtypes on the basis of the presence (WS1, MIM 193500) or absence (WS2, MIM 193510) of dystopia canthorum. Individuals having features of WS1 plus limb abnormalities are classified as WS type 3 (WS3, MIM 148820).Additional features occasionally seen in WS include spina bifida and Hirschsprung's disease. Mutations in the MITF gene on chromosome 3~12.3-p14.1 have been reported in patients with WS2 (Tassabehjiet al., 1994), whereas both WS1 and WS3 are caused by mutations in the gene for PAX3 on chromosome 2q35. A wide range of different PAX3 mutations has been described in the literature, including missense mutations, small deletions, frame shift mutations, and mutations that preclude proper splicing of the gene (Baldwin et al., 1995; Lalwani et al., 1995; Hol et al., 1995; Zlotogora et al., 1995). The majority of these mutations affect at least one of the highly conserved domains, i.e., the paired domain, the octapeptide sequence, or the homeodomain, respectively. Recently, investigators identified PAX3 mutations in the 3 ' part of the gene (Baldwinet al., 1995).In those cases the corresponding putative truncated protein would still contain all the conserved domains. Here we describe two additional mutations in the PAX3 gene. The first one concerns a missense mutation within the paired domain region of exon 2. The same mutation was reported previously in an unrelated patient by other 0 1998 WILEY-LISS, INC.
investigators (Pierpont et al., 1994).The second one concerns a nonsense mutation in exon 6 downstream of the homeodomain, which co-segregateswith WSl in a four-generationfamily. MATERIALS AND METHODS Patients
We report here on two unrelated four-generation families with classical signs of WS 1. For family 1, only the index patient #3911 was available. Clinical examination of this patient revealed dystopia canthorum and confluent eyebrows as the only visible WS features. Information provided by the patient suggests that at least 10 other family members show signs of WS, including two cases of deafness. Family 2 was referred to us by the department of otolaryngology. The index patient #6260 displayed dystopia canthorum, deafness, confluent eyebrows, and heterochromia irides; however, clinical features were observed to vary within the family. One member was born with spina bifida, which is known to be associated with WS1 (Hol et al., 1995). Unfortunately, she died 2 1 days after birth, and no material was available for genetic analysis. It is not known whether this patient showed any specificsign of WS1. Another family member displayed premature greying as the sole phenotypic feature and was designated as unaffected.
Received 12 October 1995; accepted 4 August 1996. *Correspondenceto: Dr. Frans A. Hols, Department of Human Genetics, University Hospital Nijmegen, 6500 HB Nijmegen, The Netherlands. Contract grant sponsor: Dutch Rinses Beatrii Fonds; Contract grant numbers: 93-005 and 95-0521.
S146
HOL ET AL.
SSCP Analysis Genomic DNA was extracted from whole blood of normal subjects and patients according to the procedure described by Miller et al. (1988).SSCP analysis of the exons of the PAX3 gene was performed as described previously (Hol et al., 1995) using primers located in the flankingintronic sequences. Mutations, as revealed by shifted bands, were characterized by direct sequencing of purified PCR products and confirmed by cloning of the PCR products followed by sequencing of at least three independent mutant clones. RESULTS AND DISCUSSION In this report, we discuss two different point mutations in the human PAX3 gene that are associated with WSl. Both mutations could not be detected in 50 healthy control individuals. In patient #3911, a missense mutation (A-G) was found within the paired domain region of exon 2, causing a methionine to valine substitution at codon 62 (M62V) (Fig. la). The same mutation has been detected previously in an unrelated patient (Pierpont et al., 1994). Interestingly, the deletion identified by Tassabehji et al. (1992) in a WS1 family results in the loss of six amino acids including the methionine residue mutated in our patient. These findings indicate that the methionine at position 62 A codon #
59
60
63
62
61
64
I l e V a l G l u Met A l a H i s ATC GTG GAG ATG GCC CAC
Normal
5'
-
Mutant
5'
-
ATC GTG GAG GTG GCC CAC I l e V a l Glu V a l Ala His
codon #
278 279 280 281 282 283
A l a G 1 A l a Asn G l n L e u ' Z I G C C AAT CAA CTG
B
Normal
5'
-
Mutant
5'
-
v
-
v
GCT GGG GCC AAT TAA CTG Asn ***
-
3'
-
3'
-
3'
-
3'
Partial cDNA and protein sequence of exon 2 (a) and exon 6 (b) of the PAX3 gene showing the missense mutation (M62V) detected in patient #3911 (family 1) and the nonsense mutation (Q282X) detected in patient #6260 (family 2), respectively. Arrowheads mark the altered nucleotides. The boxed sequence represents the 3' end of the homeodomain in exon 6. FIGURE 1.
plays an essential role in the correct functioning of the PAX3 transcription factor and that its removal or exchange leads to the WS1 phenotype. A novel nonsense mutation was identified in exon 6 of the PAX3 gene in patient #6260 of a second family with classical signs of WS1. The mutation (C+T) changes the codon for glutamine at position 282 into a premature stop codon (Q282)0, which is positioned shortly after the homeodomain (Fig. lb). The mutation creates an Msel site (5 '-AATT-3 ') allowing the detection of the mutation by restriction digestion of the exon 6 PCR fragment. In this way we were able to show that the mutation co-segregated with the WS1 phenotype in this family (Z = +3.6 at 8 = 0.0). Although premature greying is recognized as a sign of WS, the individual displaying this feature appeared not to carry the mutation. This illustrates that premature greying as the sole WS characteristic in relatives of WS patients is not sufficient to make a reliable diagnosis. Since the mutation is located shortly after the homeodomain,the predicted truncated peptide retains all conserved domains that are important for its DNA binding capacity. It is interesting to note that the clinical characteristics are the same as in patients with mutations affecting one or more of the conserved domains. Assuming that neither the mutated RNA nor the protein is subject to rapid degradation, the results suggest that the 3 ' part of the protein is of considerable functional importance. In addition, one could argue that the truncated product does not exert a dominant negative effect, in which case a more severe phenotype, toward the condition observed in homozygotes,would be expected (Zlotogora et al., 1995). Baldwin et al. (1995) reported on four similar nonsense mutations observed in typical WS1 patients, all of them leading to truncated proteins with intact conserved domains. Together, these data support the concept that the Cterminal part of the human PAX3 is essential for the proper functioning of the protein. Interestingly, Chalepakis et al. (1994) presented evidence that the C-terminal portion of the murine Pax-3 gene harbours a transcription-activating domain. In conclusion, the mutations presented here contribute to the identification of functionally important regions of the PAX3 gene, i.e., the methionine codon at position 62 and the downstream segment of the gene encoding the C-terminal part of the PAX3 protein. ACKNOWLEDGMENTS We thank prof. dr. H.H. Ropers for helpful discussions. We also greatly acknowledge S. van der VeldeVisser and E. van Rossum-Boenders for cell culture
IDENTIFICATION OF TWO PAX3 MUTATIONS
and EBV transformations. This work was supported by the Dutch Prinses Beatrix Fonds grant nr 93-005 and 95-0521. REFERENCES Baldwin CT, Hoth CF, Macina RA, Milunsky A (1995) Mutations in PAX3 that cause Waardenburg syndrome type I: Ten new mutations and review of the literature. Am J Med Genet 58:115-122. Chalepakis G, Jones FS, Edelman GM, Gruss P (1994) Pax-3 contains domains for transcription activation and transcription inhibition. Proc Natl Acad Sci USA 91:12745-12749. Hol FA, Hamel BCJ, Geurds MPA, Mullaart RA, Barr FG, Macina RA, Mariman ECM (1995) A frameshift mutation in the gene for PAX3 in a girl with spina bifida and mild signs of Waardenburg syndrome. J Med Genet 32:52-56. Hoth CF, Milunski A, Lipsky N, Sheffer R, Clarren SK, Baldwin C T (1993) Mutations in the paired domain of the human PAX3 gene cause Klein-Waardenburg syndrome (WS-111) as well as Waardenburg syndrome type I (WS-I). Am J Hum Genet 52:455-462. Lalwani AK, Brister R, Fex J, Grundfast KM, Ploplis B, San Agustin
5147
TB, Wilcox ER (1995) Further elucidation of the genomic structure of PAX3, and identification of two different point mutations within the PAX3 homeobox that cause WS type I in two families. Am J Hum Genet 56:75-83. Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucl Acids Res 16:1215. Pierpont JW, Doolan LD, Amann K, Snead GR, Erickson RP (1994) A single base pair substitution within the paired box of PAX3 in an individual with Waardenburg syndrome type 1 (WS1). Human Mutat 4:227-228. Tassabehji M, Read AI: Newton VE, Harris R, Balling R, Gruss I: Strachan T (1992) Waardenburg’s syndrome patients have mutations in the human homologue of the Pax-3 paired box gene. Nature 355635-636. Tassabehji M, Newton VE, Read A P (1994) Waardenburg syndrome type 2 caused by mutations in the human microphthalmia (MITF) gene. Nature Genet 8:251-255. Zlotogora J, Lerer I, Bar-David S, Ergaz Z, Abeliovich D (1995) Homozygosity for Waardenburg syndrome. Am J Hum Genet 56:1173-1178.
