Three different frameshift mutations of the tyrosinase gene in type IA oculocutaneous albinism.

Mutations in the gene for the pigment-producing enzyme tyrosinase are responsible for type IA (tyrosinase-negative) oculocutaneous albinism (OCA). Most reported mutations have been single base substitutions. We now report three different frameshift mutations in three unrelated individuals with type IA OCA. The first individual has a single base deletion within a series of five guanidines, resulting in a premature stop codon in exon I on one allele and a missense mutation at codon 382 in exon III on the homologous allele. The second individual is a genetic compound of two separate frameshift mutations, including both the same exon I single base deletion found in the first individual and a deletion of a thymidine-guanidine pair, within the sequence GTGTG, forming a termination codon (TAG) in exon I on the homologous allele. The third individual has a single base insertion in exon I on one allele and a missense mutation at codon 373 in exon III on the homologous allele. The two missense mutations occur within the copper Bbinding region and may interfere with either copper binding to the enzyme or oxygen binding to the copper. These five different mutations disrupt tyrosinase function and are associated with a total lack of melanin biosynthesis.     

A frequent tyrosinase gene mutation associated with type I-A (tyrosinase-negative) oculocutaneous albinism in Puerto Rico.

We have determined the mutations in the tyrosinase gene from 12 unrelated Puerto Rican individuals who have type I-A (tyrosinase-negative) oculocutaneous albinism (OCA). All but one individual are of Hispanic descent. Nine individuals were homozygous for a missense mutation (G47D) in exon I at codon 47. Two individuals were heterozygous for the G47D mutation, with one having a missense mutation at codon 373 (T373K) in the homologous allele and the other having an undetermined mutation in the homologous allele. One individual with negroid features was homozygous for a nonsense mutation (W236X). The population migration between Puerto Rico and the Canary Islands is well recognized. Analysis of three individuals with OCA from the Canary Islands showed that one was a compound heterozygote for the G47D mutation and for a novel missense mutation (L216M), one was homozygous for a missense mutation (P81L), and one was heterozygous for the missense mutation P81L. The G47D and P81L missense mutations have been previously described in extended families in the United States. Haplotypes were determined using four polymorphisms linked to the tyrosinase locus. Haplotype analysis showed that the G47D mutation occurred on a single haplotype, consistent with a common founder for all individuals having this mutation. Two different haplotypes were found associated with the P81L mutation, suggesting that this may be either a recurring mutation for the tyrosinase gene or a recombination between haplotypes.     

Mutations of the tyrosinase gene in patients with oculocutaneous albinism from various ethnic groups in Israel.

We have analyzed the tyrosinase (TYR) gene in 38 unrelated patients with oculocutaneous albinism (OCA), derived from several different ethnic groups of the diverse population of Israel. We detected TYR gene mutations in 23 of the 34 patients with apparent type I (i.e., tyrosinase-deficient) OCA and in none of the patients with other clinical forms of albinism. Among Moroccan Jews with type IA (i.e., tyrosinase-negative) OCA, we detected a highly predominant mutant allele containing a missense substitution, Gly47Asp (G47D). This mutation occurs on the same haplotype as in patients from the Canary Islands and Puerto Rico, suggesting that the G47D mutation in these ethnically distinct populations may stem from a common origin.     

Variation in the tyrosinase gene associated with a white humpback whale (Megaptera novaeangliae)

Tyrosinase-negative oculocutaneous albinism (OCA1A) is characterized by lifelong white hair and skin, a phenotype that has been described in most mammalian species worldwide. Tyrosinase is the key enzyme in melanin biosynthesis, and mutations in the tyrosinase gene result in OCA1A. We examined sequence variation at exon 1 of the tyrosinase gene in 66 humpback whale samples collected from the east coast of Australia, including an anomalously white humpback whale known as "Migaloo." We identified 3 novel variants, including a cytosine deletion that results in a premature stop codon in exon 1. The deletion truncates the tyrosinase protein including the putative catalytic domains that are essential for tyrosinase enzymatic activity. Migaloo was homozygous for this deletion, suggesting that the albino phenotype is a consequence of inactive tyrosinase caused by the frameshift in the tyrosinase gene.     

Mutations of the tyrosinase gene in Indo-Pakistani patients with type I (tyrosinase-deficient) oculocutaneous albinism (OCA).

Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders characterized by deficient synthesis of melanin pigment. Type I (tyrosinase-deficient) OCA results from mutations of the tyrosinase gene (TYR gene) encoding tyrosinase, the enzyme that catalyzes the first two steps of melanin biosynthesis. Mutations of the TYR gene have been identified in a large number of patients, most of Caucasian ethnic origin, with various forms of type I OCA. Here, we present an analysis of the TYR gene in eight Indo-Pakistani patients with type I OCA. We describe four novel TYR gene mutations and a fifth mutation previously observed in a Caucasian patient.     

A Tyrosinase missense mutation causes albinism in the Wistar rat

Tyrosinase serves as a key enzyme in the synthesis of melanin. In humans mutations in the TYR gene are associated with type 1 oculocutaneous albinism (OCA1) that leads to reduced or absent pigmentation of skin, hair and eye. Various mutations causing OCA in man, mouse, rabbit and cattle have been identified throughout the Tyrosinase gene including nonsense, missense, frameshift and splice site alterations. Here we report a missense substitution at codon R299H in exon 2 of the Tyr gene in the albino Wistar rat. As this very exchange has already been described in OCA patients, our findings reinforce the significance of this region for normal catalytic activity of tyrosinase protein.     

Tyrosinase gene mutations in oculocutaneous albinism 1 (OCA1): definition of the phenotype

Oculocutaneous albinism (OCA) is a common human genetic condition resulting from mutations in at least twelve different genes. OCA1 results from mutations of the tyrosinase gene and presents with the life-long absence of melanin pigment after birth (OCA1A) or with the development of minimal-to-moderate amounts of cutaneous and ocular pigment (OCA1B). Other types of OCA have variable amounts of cutaneous and ocular pigment. We hypothesized that white hair at birth indicates OCA1 and tested this in a sample of 120 probands with OCA and white hair at birth. We found that 102 (85%) of the probands had OCA1 with one or two identifiable tyrosinase gene mutations, with 169 (83%) of the 204 OCA1 tyrosinase gene alleles having identifiable mutations and 35 (17%) having no identifiable change in the coding, splice junction, or proximal promoter regions of the gene. The inability to identify the mutation was more common with OCA1B (24/35, 69%) than with OCA1A (11/35, 31%) alleles. Seven probands with no tyrosinase gene mutations were found to have OCA2 with one or two P gene mutations, and in eleven, no mutations were detected in either gene. We conclude that (1) the presence of white hair at birth is a useful clinical tool suggesting OCA1 in a child or adult with OCA, although OCA2 may also have this presentation; (2) the molecular analysis of the tyrosinase and P genes are necessary for precise diagnosis; and (3) the presence of alleles without identifiable mutations of the tyrosinase gene, particularly in OCA1B, suggests that more complex mutation mechanisms of this gene are common in OCA.Electronic database Information: accession numbers and URLs for data presented in this article are as follows:Albinism Database, , for a list of published mutations of the tyrosinase geneOnline Mendelian Inheritance in Man (OMIM), , for OCA1 (MIM 203100), OCA2 (MIM 203200)     

Human oculocutaneous albinism caused by single base insertion in the tyrosinase gene

Tyrosinase-negative oculocutaneous albinism (OCA) is an inborn error of metabolism, characterized by a complete lack of melanin pigments in the eyes and skin. We have isolated and characterized the tyrosinase gene of one affected child (S.S.) with tyrosinase-negative OCA. Sequence analysis reveals a single-base insertion in the exon 2 that shifts the reading frame and introduces a premature termination signal (TGA codon) after the amino acid residue 298. Functional analysis of the mutated gene indicates that such a truncated tyrosinase lacking one potential copper-binding region is catalytically inactive. We therefore conclude that the albino phenotype of the patient S.S. is a consequence of the inactive tyrosinase caused by the nonsense mutation in the tyrosinase gene.     

ApoB gene nonsense and splicing mutations in a compound heterozygote for familial hypobetalipoproteinemia.

Two novel apoB gene mutations were identified in a patient (CM) with phenotypic homozygous hypobetalipoproteinemia. Haplotype analysis of the apoB alleles from this patient and his family members revealed him to be a genetic compound for the disease. In contrast to previous studies of other hypobetalipoproteinemic patients, no clues existed as to where in the apoB gene the molecular defects resided. Therefore, it was necessary to characterize the apoB genes of the patient by sequence analysis. The apoB gene contains 29 exons and is 43 kb in length. The gene encodes a 14.1 kb mRNA and a 4563 amino acid protein. Both apoB alleles from the patient were cloned via 26 sets of polymerase chain reactions (PCR). These clones contained a total of approximately 24 kb of apoB gene sequence, including regions 5' and 3' to the coding region, 29 exons, and the intron/exon junctions. Complete DNA sequence analysis of these clones showed that each apoB allele had a mutation. In the paternal apoB allele, there was a splicing mutation. The first base of the dinucleotide consensus sequence (GT) in the 5' splice donor site in intron 5 was replaced by a T. It is likely that this base substitution interferes with proper splicing and results in the observed absence of plasma apoB. In the maternal apoB allele, there was a nonsense mutation. The first base of the Arg codon (CGA) at residue 412 in exon 10 was replaced by a T, resulting in a termination codon (TGA). The nonsense mutation is likely to terminate translation after residue 411 resulting in a severely truncated protein only 9% of the length of B-100.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oculocutaneous albinism type 1: link between mutations,tyrosinase conformational stability,and enzymatic activity

Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. Two subtypes of OCA1 have been described: severe OCA1A with complete absence of tyrosinase activity and less severe OCA1B with residual tyrosinase activity. Here, we characterize the recombinant human tyrosinase intramelanosomal domain and mutant variants, which mimic genetic changes in both subtypes of OCA1 patients. Proteins were prepared using site‐directed mutagenesis, expressed in insect larvae, purified by chromatography, and characterized by enzymatic activities, tryptophan fluorescence, and Gibbs free energy changes. The OCA1A mutants showed very low protein expression and protein yield and are enzymatically inactive. Mutants mimicking OCA1B were biochemically similar to the wild type, but exhibited lower specific activities and protein stabilities. The results are consistent with clinical data, which indicates that OCA1A mutations inactivate tyrosinase and result in severe phenotype, while OCA1B mutations partially inactivate tyrosinase and result in OCA1B albinism.     

High residual arylsulfatase A (ARSA) activity in a patient with late-infantile metachromatic leukodystrophy.

We identified a patient suffering from late-infantile metachromatic leukodystrophy (MLD) who has a residual arylsulfatase A (ARSA) activity of about 10%. Fibroblasts of the patient show significant sulfatide degradation activity exceeding that of adult MLD patients. Analysis of the ARSA gene in this patient revealed heterozygosity for two new mutant alleles: in one allele, deletion of C 447 in exon 2 leads to a frameshift and to a premature stop codon at amino acid position 105; in the second allele, a G-->A transition in exon 5 causes a Gly309-->Ser substitution. Transient expression of the mutant Ser309-ARSA resulted in only 13% enzyme activity of that observed in cells expressing normal ARSA. The mutant ARSA is correctly targeted to the lysosomes but is unstable. These findings are in contrast to previous results showing that the late-infantile type of MLD is always associated with the complete absence of ARSA activity. The expression of the mutant ARSA protein may be influenced by particular features of oligodendrocytes, such that the level of mutant enzyme is lower in these cells than in others.     

The etiology of oculocutaneous albinism (OCA) type II: the pink protein modulates the processing and transport of tyrosinase

Oculocutaneous albinism (OCA) is caused by reduced or deficient melanin pigmentation in the skin, hair, and eyes. OCA has different phenotypes resulting from mutations in distinct pigmentation genes involved in melanogenesis. OCA type 2 (OCA2), the most common form of OCA, is an autosomal recessive disorder caused by mutations in the P gene, the function(s) of which is controversial. In order to elucidate the mechanism(s) involved in OCA2, our group used several antibodies specific for various melanosomal proteins (tyrosinase, Tyrp1, Dct, Pmel17 and HMB45), including a specific set of polyclonal antibodies against the p protein. We used confocal immunohistochemistry to compare the processing and distribution of those melanosomal proteins in wild type (melan-a) and in p mutant (melan-p1) melanocytes. Our results indicate that the melanin content of melan-p1 melanocytes was less than 50% that of wild type melan-a melanocytes. In contrast, the tyrosinase activities were similar in extracts of wild type and p mutant melanocytes. Confocal microscopy studies and pulse-chase analyses showed altered processing and sorting of tyrosinase, which is released from melan-p1 cells to the medium. Processing and sorting of Tyrp1 was also altered to some extent. However, Dct and Pmel17 expression and subcellular localization were similar in melan-a and in melan-p1 melanocytes. In melan-a cells, the p protein showed mainly a perinuclear pattern with some staining in the cytoplasm where some co-localization with HMB45 antibody was observed. These findings suggest that the p protein plays a major role in modulating the intracellular transport of tyrosinase and a minor role for Tyrp1, but is not critically involved in the transport of Dct and Pmel17. This study provides a basis to understand the relationship of the p protein with tyrosinase function and melanin synthesis, and also provides a rational approach to unveil the consequences of P gene mutations in the pathogenesis of OCA2.     

A third mutation at the CpG dinucleotide of codon 504 and a silent mutation at codon 506 of the HEX A gene.

Two CpG mutations at codon 504 of the gene encoding the alpha-subunit of beta-hexosaminidase (the HEX A gene) have been identified previously: (1) a C deletion resulting in premature termination of the alpha-subunit and (2) a G----A transition resulting in 504Arg----His substitution, in patients with infantile Tay-Sachs disease and juvenile GM2 gangliosidosis, respectively. This prompted a search for a C----T transition in the same dinucleotide, as would be expected from the mechanism of CpG mutagenesis. Such a mutation, which results in a substitution of cysteine for arginine, was found in a patient with chronic GM2 gangliosidosis, in compound heterozygosity with the known 269Gly----Ser allele. The biochemical phenotype of the 504Arg----Cys mutation was examined by site-directed mutagenesis of the alpha-subunit cDNA and transfection of Cos-1 cells. The expression of the mutagenized cDNA with the cysteine substitution gave rise to an alpha-subunit with the same defects as those resulting from expression of mutagenized cDNA with the histidine substitution--i.e., secretion primarily as the alpha-monomer rather than as the alpha alpha dimer, along with absence of enzymatic activity. The 504Arg----Cys/269Gly----Ser genotype of the chronic GM2 gangliosidosis patient is shared by her sibling, who has mild adult-onset GM2 gangliosidosis, implying that the clinical differences between them must be attributed to other factors. The family is unique in yet another respect--namely, that the normal allele of the mother and of a 504Arg----Cys heterozygous sibling has a silent mutation, a G----A transition in the wobble position of the glutamic acid codon at position 506.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 9-bp deletion (2320del9) on the background of the arylsulfatase A pseudodeficiency allele in a metachromatic leukodystrophy patient and in a patient with nonprogressive neurological symptoms

A 9-bp deletion (2320del9) was detected in the arylsulfatase A genes of a patient with late infantile metachromatic leukodystrophy and of a patient with nonprogressive neurological symptoms and very low arylsulfatase A activity. Both patients are heterozygous for the deletion, which involves codons 406–408 and causes loss of a Ser-Asp-Thr tract in the predicted protein. In both patients the 9-bp deletion lies in a pseudodeficiency allele. The patient with metachromatic leukodystrophy carries the common 459 + 1G > A mutation in the other allele. The other patient is homozygous for the pseudodeficiency allele, and consequently is a compound heterozygote for a metachromatic leukodystrophy allele and a pseudodeficiency allele. We hypothesize that the compound heterozygosity predisposes to the development of nonprogressive neurological symptoms in the presence of additional, still unknown, genetic or nongenetic factors. Received: 18 April 1997 / Accepted: 16 August 1997     

Homozygous tyrosinase gene mutation in an American black with tyrosinase-negative (type IA) oculocutaneous albinism

We have identified a tyrosinase gene mutation in an American black with classic, tyrosinase-negative oculocutaneous albinism. This mutation results in an amino acid substitution (Cys----Arg) at codon 89 of the tyrosinase polypeptide. The proband is homozygous for the substitution, suggesting that this mutation may be frequently associated with tyrosinase-negative oculocutaneous albinism in blacks.