Mutation in Intron 6 of the Hamster Mitf Gene Leads to Skipping of the Subsequent Exon and Creates a Novel Animal Model for the Human Waardenburg Syndrome Type II

Corresponding author: Jochen Graw, Institute of Developmental Genetics, D-85764 Neuherberg, Germany., graw{at}gsf.de (E-mail)

Communicating editor: C. KOZAK

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In the course of analysis of ENU-induced mutations in Syrian hamsters, a novel dominant anophthalmic white mutant (Wh^V203) with hearing loss was recovered. Because of this phenotype and a close linkage to the Tpi gene, the Mitf gene was considered as a candidate gene. In the Mitf cDNA, a deletion of 76 bp covering the entire exon 7 was detected. Further molecular analysis revealed a T A exchange 16 bp upstream of the end of intron 6, leading to skipping of exon 7. These 16 bp at the end of intron 6 are identical in hamster, rat, mouse, and humans, indicating high conservation during evolution and a functional importance in splicing. Since the loss of exon 7 changes the open reading frame of the MITF transcript, translation will be stopped after 10 new amino acids. The truncated protein is predicted to contain only a part of the basic region and will miss the two helical domains and the leucine zipper. The Wh^V203 mutation in the Syrian hamster affects the same functional domains of the Mitf transcription factor as the human R124X mutation, causing human Waardenburg syndrome type II. Therefore, the Wh^V203 hamster mutant provides a novel model for this particular syndrome.

SINCE the discovery of the mouse microphthalmia (Mi) mutation more than 50 years ago (HERTWIG 1942 ), numerous mutant alleles have been identified and genetically characterized. The mutations affect particular cell types, which are derived from neural-crest melanocytes. The size of the mutant eyes is reduced because of the affected retinal pigmented epithelium. The mutants frequently develop deafness owing to the lack of inner ear melanocytes. The mutations detected in the mouse are mainly recessive, but semidominant or dominant phenotypes also have been reported. The wild-type allele encodes a basic-helix-loop-helix leucine zipper (bHLHzip) transcription factor and has been referred to as microphthalmia-associated transcription factor (mitf; STEINGRIMSSON et al. 1994 ; YAJIMA et al. 1999 ; HALLSSON et al. 2000 ; THAUNG et al. 2002 ).

In the rat, only one mutation in the Mitf gene has been identified (mib/mib); it leads to depigmentation, microphthalmia, osteopetrosis, and neurological disorders. The mutation is recessive and was characterized as a deletion covering several kilobases of genomic DNA at the Mitf locus. Since no Mitf cDNA was detected, the mutation most likely represents a Mitf-null allele (OPDECAMP et al. 1998 ).

In the zebrafish, a recessive mutation (nacre; nac^w2) also was described recently. The homozygous mutants lack melanophores throughout development, but the retinal pigment epithelium is normal. The mutation was characterized as a C T exchange leading to a premature stop codon. The truncated protein lacks the basic DNA-binding domain and the helix-loop-helix/leucine zipper. It is suggested that the nac^w2 mutation is a loss-of-function mutation in the Mitfa gene. Since the zebrafish genome possesses a second Mitf gene (Mitfb), the loss of Mitfa function can be compensated for at least in some tissues (e.g., the retinal pigmented epithelium; LISTER et al. 1999 , LISTER et al. 2001 ).

Mutations within the human MITF were estimated in 20% of patients suffering from Waardenburg syndrome type II (YAJIMA et al. 1999 ). The mutations lead to dominant phenotypes and affect mostly the basic helix-loop-helix motif and the leucine zipper region (TASSABEHJI et al. 1994 , TASSABEHJI et al. 1995 ; MORELL et al. 1997 ; SMITH et al. 1997 ).

In the Syrian hamster, one dominant mutation in Mitf (W241X) has been reported and designated as anophthalmic white (Wh). It is predicted that this premature stop codon leads to a truncation of the protein in the loop between helix 1 and helix 2 of the bHLHzip region. It prevents the protein from dimerizing or from binding to its DNA target sites (HODGKINSON et al. 1998 ).

In this article, we describe a novel dominant allele (Wh^V203) in the Syrian hamster. The phenotype cosegregates with a point mutation in a highly conserved region of intron 6. It leads to skipping of exon 7 of the Mitf gene during the maturation of the transcript.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Animals:
Three-month-old Syrian hamsters (Mesocricetus auratus) were treated with ENU (ethylnitrosourea; 160 mg/kg body weight). Immediately after treatment, the animals were mated with an untreated partner. The eyes of the hamsters were examined with a slit lamp after one drop of 1% atropine without anesthesia (KRATOCHVILOVA 1981 ). Homozygous mutants were obtained by intercrosses of the heterozygotes. In the homozygous mutants, the appearance of the microphthalmic eyes was obvious macroscopically.

General pathology:
A standard pathological procedure was used to determine any morphological abnormalities in the homozygous mutants.

Histology:
Four-day-old animals were killed and the dissected eyes were placed into Carnoy's solution. After 3 hr, the tissues were embedded into JB4 plastic medium (Polysciences, Eppelheim, Germany) according to the manufacturer's suggestion. Serial transversal sections (2–4 µm) were cut with a dry glass knife at an ultramicrotom (OMU4; Reichert, Walldorf, Germany), collected in water drops on glass slides, and stained with methylene blue and basic Fuchsin.

Hearing loss:
Hamsters were exposed to the sound of a "click box" (1000 kHz, 102 dB; MRC Institute of Hearing Research, Nottingham, UK). Usually, the hamsters react immediately to this sound. If no reaction was observed, the hamsters were classified as deaf.

Linkage analysis:
Microphthalmic hamster V203 was mated to the mutant Gapdh/Tpi 4300 (PRETSCH et al. 2000 ). F₁ offspring were backcrossed to wild-type hamsters. F₂ animals were screened for the presence of microphthalmia and the Tpi mutation.

Molecular characterization:
Eyes of wild-type hamster or remnants of the eyes from homozygous V203 hamster mutants were isolated from 1- or 2-day-old hamsters. RNA was isolated according to standard procedures and cDNA was prepared using the Ready-to-Go kit (Amersham-Pharmacia, Freiburg, Germany). The Mitf coding region was amplified using the meso-wh primers L1 and R1 (Table 1) spanning most of the hamster Mitf gene (according to EMBL accession no. AF020900). The PCR amplification product was cloned into pCR-TOPO vector (Fermentas, St. Leon-Rot, Germany) and sequenced commercially (SequiServe, Vaterstetten, Germany). To confirm the 76-bp deletion at the cDNA level, cDNA of five mutants was prepared and sequenced.

Genomic DNA was prepared from spleen of wild-type and homozygous mutant hamster. Based on sequence homologies between the mouse and human Mitf sequences, the primer Ham-Mitf-L3 was designed on the basis of the mouse sequence Z23066 and combined with a hamster-specific primer (Ham-Mitf-R3). To amplify the 3' end of the hamster Mitf intron 6, together with its flanking part of exon 7, a primer based on the (unpublished) intron sequence of the mouse (kindly provided by E. Steingrimsson) was combined with the primer specific for the hamster exon 7 (Table 1).

The computer-aided analysis of deduced amino acid (aa) sequences used the proteomics tools from Expasy (http://www.expasy.ch).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

General characterization:
A novel hamster mutant characterized by red eyes and white belly was recovered in the first generation after paternal treatment with ENU and recorded under the laboratory number V203. Homozygous mutants resulting from intercross matings of heterozygotes are white and exhibit microphthalmia (Fig 1). Since this phenotype is similar to another hamster mutant, Wh (HODGKINSON et al. 1998 ), our new mutation has been referred to as microphthalmic white (Wh^V203).

View larger version (48K): In this window In a new window Download PPT slide   Figure 1. Hamster mutant MhV203: microphthalmic eyes with an albino coat color. The wild-type hamster in the middle is flanked by the homozygous white and microphthalmic WhV203/WhV203 mutant on the right and the heterozygous WhV203 mutant with red eyes and a white belly on the left.

Because of the phenotypic similarity to several Mitf mutants in the mouse and the known linkage between the Tpi and Mitf genes (http://www.informatics.jax.org), Wh^V203 was tested for linkage with a recently detected Tpi mutation in hamster (PRETSCH et al. 2000 ). We observed only three recombinants in 48 F₂ hamsters tested. This is statistically significantly different (P 0.001, ² test) from a random distribution of 1:1, which would be expected if two loci were at different chromosomes. The genetic distance calculated between Tpi and Wh^V203 is 6.3 ± 3.6 cM.

Microphthalmia:
In the wild-type eye of a 4-day-old hamster (Fig 2A), the cornea, iris, lens, and retina are well developed and regularly arranged. In contrast, severe defects in the eye of homozygous Wh^V203 mutants (Fig 2B) are recovered. The eye globe is distorted and the cornea is malformed; the iris cannot be recognized. The residual lens shows degenerated fiber cells, which become liquefied in the part closed to the cornea. Except in agglomerated pigmented cells, no differentation of the retinal cell layers was observed.

View larger version (102K): In this window In a new window Download PPT slide   Figure 2. Histology of hamster eyes at postnatal day 4. (A) A section of a wild-type hamster eye. The cornea (c) and the lens are clearly separated; the lens epithelium (le) consists of one cell layer; the lens fibers (f), the iris (i), and the retina (r) are normally developed. (B) The eye globe of a homozygous WhV203 mutant at postnatal day 4 is smaller than that of the wild type. The lens epithelium (le) is multilayered; the lens contains mainly liquefacted mass, and in the posterior part shortened lens fibers are present. The development of the retinal layers (r) is disturbed; they exhibit excessive growth and folding.

Hearing loss:
Hearing loss was tested using a click box. Wild-type animals demonstrated in all cases an immediate adverse reflex (n = 15); among the six homozygous mutants tested, none was able to hear the ultrasound. The response from the heterozygotes was intermediate; 12 out of 14 were positive, whereas 2 showed only a very weak reaction.

Viability and fertility:
In the intercrosses of heterozygotes, normal numbers of offspring with the expected 1:2:1 ratio of homozygous and heterozygous mutants and wild types were found. There was a slight reduction in the number of homozygous females (² = 5.4). The outcross of the homozygous mutants revealed a low fertility of the males. However, the analysis of their sperm cells revealed a normal number of spermatozoa in the epididymis and normal population of developing germ cells in the testes. Homozygous female mutants become pregnant very rarely and never bred any offspring. The pathological examination did not reveal any abnormalities except in the eye. In particular, no indications for osteopetrosis were found by X-ray examination (A. LUZ, personal communication).

Molecular analysis:
For a molecular characterization of the mutation, cDNA was prepared from the eye or its remnants within the first two days after birth. Previous Mitf sequence information in hamsters (HODGKINSON et al. 1998 ) covered only a part of the Mitf gene; this fragment was amplified using the primer pair L1/R1 (Table 1). On the basis of the sequence homology between mouse and human (accession nos. Z23066 and Z29678; primer pair L3/R3), we could amplify a fragment of an additional 400 bp containing exon 1m (exon nomenclature according to HALLSSON et al. 2000 ), which overlaps with the main downstream fragment. Both fragments resulted in a complete cDNA sequence of the hamster Mitf gene starting at position 91; the regular stop codon is at position 1347 (Fig 3A). The newly identified 5' end of the hamster Mitf mRNA corresponds to the mouse Mitfa mRNA, which is enriched in the retinal pigmented epithelium of the mouse embryo (AMAE et al. 1998 ).

View larger version (133K): In this window In a new window Download PPT slide   Figure 3. Comparison of wild-type and V203 cDNA sequences. (a) The entire Mitf cDNA sequence of the hamster is given. Start codon ATG in exon 1m (according to Z23066; position 91–93) is underlined and in boldface type. Moreover, differences from the already published sequence (AF020900) at the 3' end are also underlined and in boldface type. The deduced amino acid composition is given above the cDNA sequence. The skipped exon 7 in the WhV203 mutant is indicated by a dashed line between positions 707 and 782; the changed open reading frame leads to 10 new amino acids, which are underlined and in boldface type. (b) Amplification of partial Mitf cDNA from wild-type and homozygous WhV203 mutants. The PCR-amplified cDNA fragment from the wild type is obviously larger than the corresponding fragment from the homozygous V203 hamster. The size difference is due to the skipping of exon 7.

In the PCR products, we confirmed the alternative splicing at the beginning of exon 6 as observed in humans (HODGKINSON et al. 1993 ) or in mouse (STEINGRIMSSON et al. 1994 ). According to the peak areas in the sequencing profiles, it is estimated that the additional 18-bp fragment in exon 6 is present in about half of the cDNA.

In the 3' part of the Mitf gene we observed four polymorphic sites in our hamsters as compared to the database (AF020900). Two polymorphisms are silent (position 870 GAC GAT encoding Asp; position 1179 AAA AAG encoding Lys). In contrast, the change from GAC GGC at position 1106 will lead to an exchange from Asp to Gly, and the AGC GGC exchange at position 1165 is considered to switch Ser to Gly.

In general, the Mitf amino acid sequence is highly conserved between mouse and hamster. The first 302 amino acids are even identical, and among the next 67 amino acids only four substitutions were observed. All these alterations are downstream of the helix-loop-helix motif (aa 236–251) or the leucine zipper (aa 261–282); a PROSITE scan suggests that the putative phosphorylation sites are not affected.

The obvious difference between the wild-type hamster and the Wh^V203 mutant is the reduced size of the L1/R1 PCR product in the mutant (Fig 3B). Sequence analysis revealed a deletion of 76 bp between positions 725 and 800 (Fig 3A). On the basis of the human exon boundaries (TASSABEHJI et al. 1994 ), it is concluded that the missing 76 bp reflects the loss of exon 7 in the Wh^V203 mutants.

The cause for skipping exon 7 was found in intron 6; from genomic DNA, we amplified a region covering the 3' end of intron 6 and the 5' region of exon 7 (primer pair intron6-L2/exon7-R1; Table 1). An exchange of T A was observed in intron 6, 16 bp upstream of its boundary to exon 7 (Fig 4). The substitution was confirmed in several independent sequences from wild-type and homozygous mutant mice. Moreover, sequence comparison of this particular region showed that it is highly conserved among human, mouse, rat, and hamster; in particular, the last 16 bp are identical in these species, indicating a functional importance of this element in splicing. Therefore, this mutation is strongly suggested to be causative for the skipping of exon 7 and for the resulting phenotype.

View larger version (36K): In this window In a new window Download PPT slide   Figure 4. Point mutation in a conserved region at the end of intron 6. A fragment covering the boundary between intron 6 and exon 7 was amplified from genomic DNA. A base-pair exchange from A T was observed (in boldface type and underlined) 16 bp upstream of the 3' end of intron 6; the beginning of exon 7 is in green. The comparison between the genomic sequence of hamster, mouse, rat, and humans demonstrates the identity of the last 16 bp in intron 6; the mutation in the hamster V203 affects the first of them, indicating a functional importance of this stretch during splicing. (*) Identical bases in all species.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Mitf gene belongs to a group of genes, which are expressed during development of neural-crest-derived melanocytes. Mitf is activated by Pax3 (WATANABE et al. 1998 ; VACHTENHEIM and NOVOTNA 1999 ), Sox10 (BONDURAND et al. 2000 ), Wnt3a (DORSKY et al. 2000 ), and onecut-2 (JACQUEMIN et al. 2001 ). As a basic helix-loop-helix/leucine-zipper transcription factor, Mitf protein itself regulates other genes like MyoD, Myf5, c-Met, c-Kit, tyrosinase, Trp-1, Qnr-71, Ednrb, and Edn3 (TACHIBANA 1999 ). Therefore, mutations in these genes may cause similar syndromes. To understand the molecular mechanisms underlying these syndromes, a detailed analysis of a variety of homologous mutations in different species is important. Moreover, the comparison of conserved regions vs. polymorphic sites will allow also more detailed knowledge of which part of a gene might be important for its functions.

In this article, we describe the entire Mitf gene in the Syrian hamster. The Mitf gene, in both mouse and humans, has a very complex structure in its 5' region. The first four possible exons (1a, 1h, 1b, and 1m) in front of exon 2 lead to tissue-specific alternatively spliced transcripts (AMAE et al. 1998 ; HALLSSON et al. 2000 ). On the basis of the corresponding sequence information in the mouse, we amplified the 5' part of the Mitf cDNA from the hamster retinal pigmented epithelium. It corresponds to mouse Mitf-1m and indicates a strong conservation of the tissue-specific alternative splice products at least for rodents. Additionally, a few polymorphic sites in the 3' part of the coding region were found.

During this study, we characterized a novel dominant mutation in the hamster Mitf gene. An exchange of T A 16 bp upstream of the splice donor site of exon 7 leads to a loss of this exon during splicing. Since exon 7 consists of 76 bp, its loss changes the open reading frame and after 10 novel amino acids (after position 211) a stop codon occurs, truncating the protein just in front of the helix-loop-helix and leucine zipper motif.

The skipping of exon 7 as a consequence of the T A substitution 16 bp upstream of the intron 6/exon 7 border demonstrates the importance of this particular base for the splicing mechanism even if the sequence at this position does not fit the conserved sequence in mammalian nuclear pre-mRNA introns, which is recognized by the U2 snRNA (STALEY and GUTHRIE 1998 ). However, the most downstream 16 bp of intron 6 are identical in human, mouse, rat, and hamster, indicating their functional importance. Farther upstream in the intron, the degree of homology is lower.

Heterozygous Mitf^V203 mutants are normal agouti, but with a white belly and red eyes, whereas the homozygous mutants are white with closed eyes and suffer also from sterility and hearing loss. Two dominant mutations in humans [one of Indian origin (LALWANI et al. 1998 ) and the other from northern Europe (NOBUKUNI et al. 1996 )] leading to a similar phenotype have been characterized recently. They affect the same functional region of the Mitf transcription factor by a stop codon at amino acid position 214 (R214X), which is also in front of the helix-loop-helix and leucine zipper motif. In both families, hearing loss was the most common finding, followed by ocular pigmentary disturbances. However, heterochromia iridis occurs more frequently in the Indian family than in the affected members of the European family, indicating additional effects of different genetic backgrounds. Since the new hamster mutation Mitf^V203 affects the same region, it might be considered as an excellent model for this mutation in humans.

The other already described dominant hamster mutation, Wh, has been found downstream and leads to a stop codon between helix 1 and helix 2 (W241X). This nonsense mutation destabilizes the Mh-Mitf mRNA and prevents the encoded basic helix-loop-helix leucine zipper protein from dimerizing or binding to its DNA target sites (HODGKINSON et al. 1998 ). The mutation leads to a highly pleiotropic phenotype exhibiting dominant spotting coat colors and causing homozygotes to be deaf, blind, white, and sterile (ASHER and JAMES 1982 ).

In addition to the mutations mentioned above, several other mutations are described in mice and humans that affect the basic region and the first helix of the helix-loop-helix motif of the Mitf protein. In the mouse, the alleles Mi^wh (I212N), mi (R215), Mi^or (R216L), and mi^vt (D222N) are reported to touch this particular region also (for a detailed overview see STEINGRIMSSON et al. 1994 ; MOORE 1995 ). Recently, Mitf^Mi-H and Mitf^Rorp were detected; in the Mitf^Mr-H mutants a splicing defect also was shown to be causative for the phenotype (THAUNG et al. 2002 ).

The increasing list of mutations in the Mitf gene is a hint of its functional importance. However, more detailed comparative studies are necessary to deduce genotype-phenotype correlations, considering the mutations available even in different species.

	FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AJ458438 and AJ458439.
² Present address: GSF-National Research Center for Environment and Health, Institute of Human Genetics, D-85764 Neuherberg, Germany.

	ACKNOWLEDGMENTS

The authors thank Erika Bürkle, Dagmar Reinl, and Monika Stadler for expert technical assistance. Eirikur Steingrimsson (Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, Reykjavik, Iceland) supported us with unpublished sequence information on mouse Mitf intron sequences. Oligonucleotides were obtained from Utz Linzner, GSF-Institute of Experimental Genetics.

Manuscript received December 16, 2002; Accepted for publication March 7, 2003.

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