Apparent digenic inheritance of Waardenburg syndrome type 2 (WS2) and autosomal recessive ocular albinism (AROA)
Apparent digenic inheritance of Waardenburg syndrome type 2 (WS2) and autosomal recessive ocular albinism (AROA)R. Morell1,*,+, R. A. Spritz2, L. Ho2, J. Pierpont3, W. Guo5, T. B. Friedman1,4,5,+ and J. H. Asher Jr 1,5
1Department of Zoology, 4Department of Pediatrics and Human Development and 5Genetics Program, Michigan State University, East Lansing, MI, USA, 2Department of Medical Genetics, University of Wisconsin, Madison, WI, USA and 3Department of Pediatrics, University of Arizona Health Sciences Center, Tucson, AZ, USA
Received September 30, 1996;Revised and Accepted February 21, 1997
Waardenburg syndrome (WS) is a clinically and genetically heterogeneous disease accounting for >2% of the congenitally deaf population. It is characterized by deafness in association with pigmentary anomalies and various defects of neural crest-derived tissues. At least four types are recognized (WS1, WS2, WS3 and WS4) on the basis of clinical and genetic criteria. Two previously described families seemed to delineate a new subtype characterized by WS2 in conjunction with ocular albinism (OA). Since mutations in the MITF gene are responsible for some instances of WS2, we screened for mutations in one of the WS2-OA families and discovered a 1 bp deletion in exon 8 of MITF. OA previously has been associated with compound heterozygosity for a mutant TYR allele and the TYRR402Q allele, a functionally significant polymorphism that is associated with moderately reduced tyrosinase catalytic activity. In this family, all of the individuals with the OA phenotype are either homozygous or heterozygous for TYRR402Q, and heterozyous for the 1 bp deletion in MITF. This suggests that the WS2-OA phenotype may result from digenic interaction between a gene for a transcription factor (MITF) and a gene that it regulates (TYR).
Waardenburg syndrome (WS) is characterized by pigmentary anomalies, deafness and various defects of neural crest-derived tissues (1 ), and accounts for >2% of the congenitally deaf population (2 ). Ever since the first description of WS, the full scope of its clinical features has been controversial as all features show considerable variation in expressivity and reduced penetrance, even within families. Dystopia canthorum, the lateral displacement of the inner canthi relative to the outer canthi, allows for the distinction between type 1 and type 2 Waardenburg syndrome (WS1, MIM 193500 and WS2, MIM 193510) (3 -6 ). Dystopia canthorum is 97% penetrant in families with WS1, and its uniform absence defines a family as WS2. Additional specific clinical features define other types of WS, such as upper limb abnormalities in WS3 (MIM 148820) and aganglionic megacolon in WS4 (MIM 277580). Each of the clinical features of WS has been reported to segregate as a distinct entity in other families.
Several families have been reported in whom WS and ocular albinism (OA) appeared to co-segregate as a distinct genetic syndrome. Bard (7 ) described a family with features typical of WS2 as well as ocular albinism (OA). Because OA, which is usually either an autosomal recessive or an X-linked recessive disorder, was the most prominent presenting feature in this family showing apparent dominant inheritance, and because WS2 and OA occurred together in most affected family members, the author proposed that a single novel dominant disorder was segregating. A second WS family with apparent OA was described by Goldberg (8 ). Lewis (9 ) reported a family with an apparent dominant form of OA associated with sensorineural deafness (MIM 103470), and suggested that Bard's family (7 ) had the same disorder.
So-called `autosomal recessive ocular albinism' (AROA) is now known to represent clinically mild instances of oculocutaneous albinism type I (OCA1, MIM 203100) or oculocutaneous albinism type II (OCA2, MIM 203200), autosomal recessive disorders that result from mutations in the tyrosinase (TYR) or P genes, respectively (10 , reviewed in ref. 11 ). In the case of tyrosinase-related AROA, Fukai et al. (12 ) showed that AROA can result from compound heterozygosity for a mutant allele of TYR and a polymorphic allele (TYRR402Q) encoding a form of tyrosinase with reduced catalytic activity. Subsequent studies have shown that this is a very frequent cause of AROA (R.A. Spritz, unpublished data).
Some cases of WS2 are caused by mutations in the MITF gene (13 -15 ). The MITF protein is thought to be a transcription factor that regulates the TYR gene (16 -18 ), suggesting a possible link between WS2, resulting from a MITF mutation, and AROA, resulting from reduced tyrosinase function. Accordingly, we re-ascertained the family originally described by Bard (7 ) and screened for mutations in both the MITF and TYR genes. We find that both a mutant allele of MITF and a functionally significant polymorphism of TYR are segregating in the MSU11 family, probably accounting for the juxtaposition of WS2 and AROA.
In the MSU11 family, generation III has been described previously (7 ) (Fig. 1 A). All of these individuals were contacted again in 1993 and 1994, and new ophthalmalogical and audiological evaluations made of III-3, who had exhibited the mildest pigmentary defects and no visual acuity defects. In addition, evaluations were made of individuals II-3 and II-4, who had not been described fully (7 ), and individuals in generation IV, all of whom were born after 1978. A summary of findings is presented in Table 1 .
WS is a clinically and genetically heterogeneous group of pigmentation-deafness disorders caused, in part, by mutations in the transcription factor genes PAX3 (WS1 and WS3) and MITF (WS2) (13 -15 ,21 -31 ). All of the clinical features of WS show reduced penetrance and variable expressivity, even within families segregating a single mutation. This observation suggests that genetic background may play a major role in the etiology of some of the clinical attributes of these syndromes in both human (32 -34 ) and murine (35 ) models of WS.
Pandya et al. (33 ) recently posited an oligogenic epistasis model of WS in which clinical features involve genes in a `melanocyte cluster'. Under this model, inter- and intra-family variability in WS could result from different combinations of mutations in genes for receptors and their ligands, or for transcription factors and their target genes, within the `melanocyte cluster'. Thus, the most important background genes that modulate the clinical features of WS may be those whose expression is regulated directly by the PAX3 or MITF transcription factors. The MITF protein is thought to regulate the expression of various pigmentation genes by binding at a promoter element termed the M-box (17 ). In particular, MITF has been shown to transactivate the promoter of the tyrosinase gene TYR (16 ,36 ), which encodes the rate-limiting enzyme of melanin biosynthesis.
The MITF mutation segregating in MSU11 is a one base deletion in exon 8, which shifts the reading frame and results in a premature translational termination codon in exon 9. Transcripts with premature translational termination are often degraded by mechanisms not yet fully understood (37 ). However, transcripts containing nonsense or frameshift mutations in their final exon may escape this nonsense RNA decay mechanism. One example of this is a frameshift in the final exon of the [beta]-globin gene that results in a mild, dominant-negative form of [beta]-thalassemia (38 ). The MITF frameshift described in this study is unlike other MITF mutations in that translational termination is predicted to occur within exon 9, the last translated exon. It is possible that the corresponding truncated protein product might have novel transactivation capabilities, perhaps accounting for the OA phenotype in association with WS2. However, truncated MITF proteins expressed from mutant cDNA constructs fail to transactivate the TYR promoter, and fail to dimerize with normal MITF protein (15 ), suggesting that the hypothetical truncated MITF protein polypeptide resulting from the exon 8 frameshift described here is very unlikely to act in a dominant-negative manner, at least with regard to regulation of the TYR gene. It seems more likely that the MITF exon 8 frameshift results in haploinsufficiency of MITF function, with consequent down-regulation of TYR gene expression.
Homozygosity for mutations in the TYR gene results in greatly reduced tyrosinase catalytic activity and OCA1. Most individuals with OCA1 are compound heterozygotes for two different mutant alleles of TYR (10 ,11 ). OCA1 shows considerable phenotypic overlap with AROA. Individuals with tyrosinase-related AROA typically are compound heterozygotes for a mutant allele of TYR and a polymorphic allele (TYRR402Q) encoding a form of tyrosinase with reduced catalytic activity-although still within the normal range (12 ). Heterozygotes and even homozygotes for TYRR402Q are phenotypically normal (39 ), whereas compound heterozygotes for OCA1 mutant alleles and TYRR402Q may manifest AROA (12 ). Nevertheless, most OCA1/ TYRR402Q compound heterozygotes are also phenotypically normal, underscoring the importance of epistatic phenomena that modulate the pigmentary phenotype.
The TYRR402Q polymorphism is segregating in the MSU11 family, as is a mutant allele of the MITF gene. Several members of the MSU11 family are affected not only with WS2, but with OA-a feature not usually associated with WS2. Moreover, OA usually segregates as an autosomal recessive condition, as stated above. However, the individuals in MSU11 with OA are actually heterozygous for the mutant MITF allele and either heterozygous or homozygous for the TYRR402Q polymorphism. By contrast, individuals who have two normal MITF alleles are phenotypically normal regardless of the TYR genotype. Even if the mutant alleles of MITF were expressed, the resulting proteins would be unlikely to transactivate the TYR promoter (15 ); thus, the occurrence of OA in the TYRR402Q/---individuals in MSU11 seems likely to be the result of down-regulation of the TYR locus through haploinsufficiency of MITF. If so, this would appear to be the first example of oligogenic, or in this case digenic, inheritance in WS.
The family reported by Bard (7 ) (generations II and III in Fig. 3 1) were re-ascertained, and pedigree and clinical information collected regarding generation IV. We refer to this family as MSU11 (19 ). Two 20 ml blood samples were collected from all available members of MSU11, and DNA was prepared as described (20 ).
Each of the nine exons of MITF were PCR amplified from the genomic DNA as described by Tassabehji et al. (14 ). The PCR products were radiolabeled with [[alpha]-33P]CTP incorporated during the amplification reaction (40 ), diluted with 2 vols of sequencing loading buffer, denatured at 95oC for 5 min, and then electrophoresed through 0.5* MDE gels (AT Biochem) for SSCP analysis as described (21 ,22 ). A variant band was observed in exon 8 amplicons from individual MSU11-IV-4. These PCR products were cloned in pGEM-T (Promega) and sequenced using an ABI 373A DNA sequencer (Applied Biosystems).
To confirm the presence of the MITF mutation in genomic DNA, mutation-specific PCR was performed by substituting the primer (5'-AACCGACAGGAGAAACTGGG-3') for the forward exon 8 PCR primer, and using the normal reverse primer (5'-CTGTTTCTACTGTCTTGAAGTCGG-3'). As a control, primers for tetranucleotide repeat marker D6S1040 were included in the reaction tube. The reaction conditions were: each primer 0.25 [mu]M, each dNTP 100 [mu]M, 1.5 mM MgCl2, 50 mM KCl, 1.5 U of thermostable DNA polymerase and 50 ng of genomic DNA in a total of 50 [mu]l; cycled 30 times at 94oC (30 s)-55.5oC (45 s)-72oC (45 s) with a 5 min final extension at 72oC. The PCR products were resolved in 3% NuSeive 3:1 gels (FMC) and visualized by ethidium bromide staining. Under these conditions, constant bands of ~250 bp, corresponding to the alleles of D6S1040, were observed in all individuals, while the mutant allele-specific band migrated at 77 bp. As an independent confirmation of genotype, the exon 8 PCR products with a 1 bp deletion were resolved from the normal PCR products on a sequencing gel for all individuals in MSU11 (data not shown).
Affected family members were screened for pathologic mutations of the TYR gene and were genotyped for the TYRR402Q allele by simultaneous SSCP/heteroduplex analysis using 0.5* MDE gels as described (41 ). This method detects ~95% of known TYR mutations. Furthermore, all members of MSU11 were genotyped directly for the TYRR402Q allele. PCR amplifications of TYR exon 4 were carried out as above using the primers 5'-ACATCTTTCCATGTCTCCAG- 3' and 5'-ATTCAGCAATTCCTCTGAAA-3'. The 350 bp PCR products were gel purified and then cycle sequenced in both directions using the n-Taq Cycle Sequencing kit (USB), [[alpha]-33P]CTP and one or the other of the PCR primers.
We would like to thank members of the `MSU11' family for their help and enthusiasm. We would also like to thank Dr Martin Pearlman (Lansing Ophthalmology, Lansing, MI), Dr David Kaufman (College of Osteopathic Medicine, Michigan State University) and Dr Robert Erickson (Department of Pediatrics, University of Arizona Health Sciences Center, Tucson, AZ) for their help in the clinical evaluations. The authors wish to acknowledge the instructive and valuable suggestions made by an anonymous reviewer of this manuscript. This research was supported by grants: DC01160 (to J.H.A. and T.B.F.) from the National Institutes on Deafness and other Communication Disorders, AR39892 (to R.A.S.) from the National Institutes of Health, and fellowship grant for medical students #91005090 (to J.P.) from the American Heart Association.
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*To whom correspondence should be addressed. Tel: +1 301 402 4249; Fax: +1 301 402 5354; Email: morellr@helix.nih.gov
+Present address: Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders (NIDCD), NIH, 5 Research Court, Rockville, MD 20850, USA
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