Human Molecular Genetics, 2002, Vol. 11, No. 25 3231-3236
© 2002 Oxford University Press
SLUG (SNAI2) deletions in patients with Waardenburg disease
1Instituto de Biología Molecular y Celular del Cáncer (IBMCC), Centro de Investigación del Cáncer, CSIC/Universidad de Salamanca, Campus Unamuno, 37007 Salamanca, Spain and 2Academic Unit of Medical Genetics, St Mary's Hospital, Manchester M13 0JH, UK
Received August 21, 2002; Accepted October 4, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Waardenburg syndrome (WS; deafness with pigmentary abnormalities) is a congenital disorder caused by defective function of the embryonic neural crest. Depending on additional symptoms, WS is classified into four types: WS1, WS2, WS3 and WS4. WS1 and WS3 are caused by mutations in PAX3, whereas WS2 is heterogenous, being caused by mutations in the microphthalmia (MITF) gene in some but not all affected families. The identification of Slugh, a zinc-finger transcription factor expressed in migratory neural crest cells, as the gene responsible for pigmentary disturbances in mice prompted us to analyse the role of its human homologue SLUG in neural crest defects. Here we show that two unrelated patients with WS2 have homozygous deletions in SLUG which result in absence of the SLUG product. We further show that Mitf is present in Slug-deficient cells and transactivates the SLUG promoter, and that Slugh and Kit genetically interact in vivo. Our findings further define the locus heterogeneity of WS2 and point to an essential role of SLUG in the development of neural crest-derived human cell lineages: its absence causes the auditory–pigmentary symptoms in at least some individuals with WS2.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Waardenburg syndrome is a rare (1/40 000) disorder with pigmentary abnormalities and sensorineural deafness, usually inherited in an autosomal dominant manner (1). The main subtypes of WS are type 1 (WS1), associated with dystopia canthorum and type 2 (WS2), without dystopia. WS is a disorder of the neural crest or its derivatives and molecular dissection of WS provides an avenue for investigating neural crest differentiation. WS1 (and the rare type 3 and type 4 variants WS3 and WS4) represent failures of several neural crest derivatives, whereas WS2 appears to be a specific failure of melanocytes. WS1 and WS3 are caused by mutations in PAX3 (2–4), whereas WS2 is heterogenous (5). Some WS2 families have mutations in the microphthalmia (MITF) gene and occasional cases are caused by mutations in endothelin 3 or its receptor EDNRB (these are more usually associated with Hirschsprung disease in the rare WS4 variant), but for many cases the cause is unidentified. SLUG is one possible candidate (6). This zinc finger transcription factor is a marker of neural crest cells in Xenopus, zebrafish and chick embryos and probably has a functional role in formation of premigratory neural crest (reviewed in 7). In the mouse, the corresponding gene, Slugh, is expressed in migratory but not premigratory neural crest cells and is not essential for neural crest development (7). We recently showed that mice homozygous for loss-of-function mutations of Slugh often have a white forehead blaze and areas of depigmentation on the ventral body, tail and feet (6), indicating that the function of Slug is necessary for normal development of melanocytes. Because of the phenotypic similarity of these mice to patients with WS2, we set out to investigate a possible role of the human SLUG (SNAI2) gene in Waardenburg syndrome.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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An initial series of DNAs from 38 unrelated patients, all with features of Waardenburg syndrome [but not necessarily fulfilling the formal diagnostic criteria of Liu et al. (8)] and negative for MITF mutations, were screened by Southern blotting for SLUG gene rearrangements or deletions using a full-length SLUG cDNA probe (6). As shown in Figure 1, two subjects showed deletions of SLUG spanning the entire SLUG coding region. As a control for differences in DNA loading, the blot was stripped and rehybridized with a probe for the KIT gene (6), which maps to chromosome segment 4q11–q12. No significant differences in DNA loading were observed with this control probe (Fig. 1C). We tested the extent of the SLUG deletions by analysis of two closely flanking markers, STSG29942 and STSG54511. Both of these markers, which flank the SLUG locus, were not homozygously deleted in the two subjects, indicating that the deletions in these patients are relatively short. To confirm these data and accurately determine SLUG copy number, we developed a real-time quantitative PCR assay for part of exon 1 of the gene. As shown in Table 1, the target sequence was not detected in subjects B and D, or in Slugh-/- mice. These results thus confirm SLUG deletions in subjects B and D.
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We next searched for point mutations in the SLUG gene in the remaining 36 WS2 patients where we did not detect homozygous deletion of the SLUG gene. Primers were designed to amplify each coding exon with flanking intron sequence from genomic DNA, and the PCR products were sequenced. We failed to detect any SLUG mutations in DNA from the 36 non-deleted patients. Because we have not examined the non-coding part of the gene, it is possible that a more extensive analysis of the entire gene might detect additional mutations in our panel of patients. The possibility of a population polymorphism was excluded as we detect no SLUG deletions in 150 controls studied.
Patients were not selected for any particular mode of inheritance. Of the 38, 13 had a clearly dominant family history, 12 (including the two deletion cases) had pedigrees fully compatible with recessive inheritance and 13 were classified as possible recessive. The ‘possibly recessive’ category included patients where a parent or more distant relative was reported to have some minor pigmentary change such as early graying or a white forelock. In some cases patients with similar histories have turned out to be homozygous for connexin 26 mutations (A.P.R., unpublished), showing that such pedigrees can be compatible with recessive inheritance. Connexin 26 mutations were excluded in our patients.
Subject B was a 15-year-old girl of Bangladeshi origin. She had profound bilateral sensorineural hearing loss, reported to be congenital and heterochromia irides, but no other dysmorphic features or pigmentary changes. Her non-consanguinous parents were normal, as were four siblings. As a child she had suffered fits of unknown origin; CT scan revealed no abnormality. In this regard, Wv homozygotes can show audiogenic seizures, which may be related to the fits described.
Subject D was a 3-year-old Dutch boy who had a 60 dB hearing loss and unilateral heterochromia. All other investigations (ophthalmology, thyroid function, ECG) were normal. He was the only child of normal unrelated parents, born after an uneventful pregnancy and delivery. There was no recorded family history of hearing loss or pigmentary abnormalities.
Mice (Fig. 2) and humans lacking Slugh/SLUG show neural crest dysfunction. In the human cases this manifests as heterochromia irides and hearing loss, both attributable to patchy absence of melanocytes (1). Hearing loss has not been assessed in the Slugh-/- mice, but we have observed hyperactivity and circling in some mutant animals, a behaviour suggestive of hearing impairment. In the Slugh-/- mice, the phenotypes are influenced by genetic background (6, unpublished observations). Mapping and identifying modifiers of the phenotype may help identify phenotypic determinants in human disease.
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The molecular mechanism by which SLUG regulates embryonic development is not known. Thus, we next examined the expression of Pax3, mi (the mouse homologue of human MITF) and Sox10 in the Slugh-/- mouse model. As shown in Figure 3, Pax3, mi and Sox10 mRNA were present in Slug-deficient cells coming from skin. These results are congruent with the observations that, in mouse embryos, mi is expressed earlier than Slugh in the neural crest where melanocytes originate (7,9). This led us to hypothesize that mi/MITF directly regulates SLUG. To test this hypothesis,
1777 bp of the promoter region of the human SLUG gene were cloned upstream of a luciferase reporter gene (pGL3-basic). To directly assess the ability of MITF to activate transcription from DNA sequences present in the SLUG promoter, an expression vector containing an MITF cDNA was co-transfected into NIH-3T3 cells along with the reporter vector containing the SLUG promoter. Co-expression of MITF resulted in an approximately three-fold increased in luciferase activity compared with the activity with the empty vector (Fig. 4). These results strongly suggest that MITF interacts with a sequence in the SLUG promoter. Indeed, there is a potential MITF binding site, CATGTG, that fits the E box consensus sequence CA[C/T]GTG 1396 bp upstream of the transciptional start. When this E box was mutated, the promoter no longer responded to MITF activation (Fig. 4). It seems likely that failure to activate SLUG explains why MITF mutations cause Waardenburg syndrome.
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Like mi mutants, Kit and Scf (stem cell factor) mutant mice exhibit striking coat colour phenotypes due to loss of viable melanocytes (10–12). These genes co-ordinately modulate the development and survival of the melanocyte lineage. Kit functions upstream of mi (13) and it has been demonstrated that Kit and mi interact in vivo, resulting in profound melanocyte loss in compound heterozygous mice (14). Thus if Slug mediates the effect of mi on melanocyte survival, Kit and Slugh should genetically interact in vivo similarly to Kit and mi. To examine genetic interactions between Kit and Slugh in vivo, we generated double heterozygotes for a Slugh null allele and the Wv mutation of Kit. Complete absence of functional Kit produces mice devoid of melanocytes from birth (Fig. 5D) but heterozygous Wv/+ mice are born black with occasional white spots (Fig. 5A). Similarly, homozygous mice with the targeted mutation of the Slugh gene are born black with occasional white spots (Fig. 2). In the context of Wv/+ heterozygosity, additional loss of a single Slugh allele produced extensive white spotting from birth (Fig. 5B and C). Similar to the genetic cooperativity between Kit and mi, these observations demonstrate genetic cooperativity between Kit and Slugh for melanocytes.
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In summary, we have shown that mice lacking Slugh have patchy deficiency of melanocytes, a phenotype similar to human Waardenburg syndrome. We showed that some human patients with Waardenburg syndrome carry homozygous deletions of SLUG as their only detected genetic abnormality, thus defining a recessive form of type 2 WS. Preliminary investigations of the role of SLUG in melanocyte development show that it is a downstream target of mi/MITF, which acts on an E-box sequence in the SLUG promoter and that Slugh Kit double heterozygous mice show similar melanocyte defects to mi Kit double heterozygotes, suggesting that SLUG may be the main effector of mi/MITF action in melanoblasts. The frequency and phenotypic range of humans deficient for SLUG remain to be determined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Subjects
DNAs from 38 unrelated individuals with WS2 were studied. All patients lack detectable MITF gene mutations. The two affected have unaffected parents and are sporadic cases. None of these patients' parents are consanguinous.
Southern blot analysis
Blood was obtained with informed consent and genomic DNAs were prepared from peripheral blood lymphocytes. DNAs were digested with restriction endonucleases as described (15), separated by electrophoresis in 0.8% agarose, transferred to Hybond-N (Amersham). Filters were UV cross-linked and hybridized to a 32P-radiolabelled full-length SLUG cDNA probe. DNA loading was monitored by reprobing the filters with a full-length mouse Kit cDNA.
Real-time PCR quantification
Real-time quantitative PCR (16) was developed and carried out for the detection and quantitation of both mouse and human SLUG genes. The exon 1 primers SLUGF (5'-ATGCCGCGCTCCTTCCT-3') and SLUGB (5'-TGTGTCCAGTTCGCT-3') were used in conjunction with a fluorogenic probe SLUGP [5'-(FAM)-CATTTCAACGCCTCCAARAAG-CC-(TAMRA)-3']. The fluorogenic probe contained a 3' blocking phosphate group to prevent probe extension during PCR. Fluorogenic PCRs were set up in a reaction volume of 50 µl using the TaqMan PCR Core Reagent kit (PE Biosystems). Fluorogenic probes were custom-synthesized by PE Biosystems. PCR primers were synthesized by Isogen. Each reaction contained 5 µl of 10x buffer; 300 nM of each amplification primer; 25 nM fluorogenic probe; 200 µM of each dNTP; 1.25 U AmpliTaq Gold, 2 mM MgCl2 and 10 ng DNA. DNA amplifications were carried out in a 96-well reaction plate format in a PE Applied Biosystems 5700 Sequence Detector.
Thermal cycling was initiated with a denaturation step of 10 min at 95°C followed by 40 cycles of 95°C for 15 s, 55°C for 30 s and 72°C for 1 min. Multiple negative water blanks were tested and a calibration curve determined in parallel with each analysis. DNAs (10 ng each) from Slugh+/+, Slugh+/-, and Slugh-/- mice were used for constructing the calibration curve. The ß-actin endogenous control (PE Biosystem) was included to relate both mouse and human SLUG samples to total genomic DNA in each sample.
Cycle threshold (Ct) readings from Slug+/+, Slug+/-, and Slug-/- mice were utilized to establish standard curves for each reaction series. The Ct was computed for each sample using Sequence Detection Software (Applied Biosystems) and these settings were automatically used for further computations. Standard curves were generated by plotting the mean of triplicate Ct values versus the log of the Slug copy number. The copy numbers for unknown samples were determined by applying the mean Ct value of triplicates to the standard curve. Although equal amounts of DNA were used (10 ng each), a ß-actin endogenous control was included to relate both mouse and human Slug/SLUG samples to total genomic DNA in each sample.
Analysis of genomic DNA
The three exons of the human SLUG gene plus adjacent non-coding and flanking sequences were amplified in duplicate from DNA of patients without SLUG deletions by PCR. The nucleotide sequences of at least six independent clones of each were determined.
Mouse crosses
Animals were housed under non-sterile conditions in a conventional animal facility. Mice heterozygous and homozygotes for the Slugh
1 mutation generated by removing the genomic sequences of the entire Slugh protein-coding region (Slugh1 mutant mice) have been described previously (7). W/Wv and breeding pairs were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Heterozygous Slug+/- mice were bred to Wv mice to generate compound heterozygotes. F1 animals were crossed to obtain null Slug-/- mice heterozygous for Wv deficiency.
Plamid constructs and luciferase assays
The 1777 bp upstream promoter sequence of SLUG was isolated from a P1 clone containing the SLUG gene (Genome Systems) and cloned into the luciferase reporter plasmid pGL3-basic (Promega) and termed pSLUG-1777. The pSLUG-1777mut is identical to pSLUG-1777 except that the E-box sequence (CATGTG) in position -1396 is substituted with the mutant sequence gAgGTG (mutated nucleotides in lower case). Human MITF cDNA was generated by RT–PCR of total RNA from human melanocytes. Nucleotide sequence was verified by sequencing and the cDNA was cloned into the expression plasmid pEF-BOS (17). For reporter assays, NIH-3T3 cells were transfected using Dual-Luciferase (Promega) with normalization to Renilla luciferase, and mean±SE was determined from at least three data points. NIH-3T3 cells were maintained in DMEM supplemented with 10% FBS.
Reverse transcription–PCR
To analyse expression of Slug, Pax3, mi and Sox10 in Slugh+/+, Slugh+/- and Slugh-/- mice, RT was performed according to the manufacturer's protocol in a 20 µl reaction containing 50 ng of random hexamers, 3 µg of total RNA and 200 units of Superscript II RNase H- reverse transcriptase (GIBCO/BRL). The sequences of the specific primers were as follows: Slugh, sense primer 5'-GCCTCCAAAAAGCCAAACTA-3' and antisense primer 5'-CACAGTGATGGGGCTGTATG-3'; Sox10, sense primer 5'-CATGAACGCCTTCATGGTGT-3' and antisense primer 5'-AGCTCAGTCACATCAAAGGT-3'; Pax3, sense primer 5'-CTAAGATCCTGTGCAGGTAC-3' and antisense primer 5'-GGTTGCTAAACCAGACCTGC-3'; Mitf, sense primer 5'-GTATGAACACGCACTCTCGA-3' and antisense primer 5'-CTTCTGCGCTCATACTGCTC-3'. Amplification of ß-actin RNA served as a control to assess the quality of each RNA sample. The PCR products were confirmed by hybridization with specific internal probes.
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ACKNOWLEDGEMENTS |
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We are indebted to members of lab 13 for helpful discussions and specific thanks to Professor R. González-Sarmiento for his unconditional help and support. We are grateful to Dr T. Gridley for the Slug mutant mice. This work was supported by DGCYT (SAF2000-0148, and BIO2000-0453-P4-02), Junta de Castilla y León (CSI3/01, and CSI1/02), FIS (01/0114 and PI020138), and NIH grant (1 R01 CA79955-01). A.R.G. is a scholarship holder from CSIC-GLAXO. Work in Manchester was supported by EC grant QLRT-1999-00988 to A.P.R.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +34 923238403; Fax: +34 923294813; Email: isg{at}usal.es
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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