Mutations In The Ube3a Gene

The gene E6AP ubiquitin protein ligase (UBE3A), although mapping to the critical AS region, was considered an unlikely candidate for the syndrome, in view of its expression from both parental alleles in lymphocytes and fibroblasts of AS and PWS patients (Matsuura et al., 1997). However, mutation analysis of this gene in AS patients without large 15q deletions, paternal UPD15, or imprinting mutations has led to the identification of loss of function mutations in several cases (Kishino et al., 1997; Matsuura et al., 1997).

The first UBE3A mutations identified in AS patients were a de novo duplication of five nucleotides, a maternally inherited splice site mutation, a de novo two nucleotide deletion, and a de novo amino acid substitution (Kishino et al., 1997; Matsuura et al., 1997). The first three mutations result in frameshifts and premature translation termination. Subsequently, many more UBE3A mutations have been identified in patients with AS (Table 1) (Kishino et al., 1997; Matsuura et al., 1997; Fang et al., 1999; Malzac et al., 1998; Baumer et al., 1999; Tsai et al., 1998; Russo et al., 2000).

The UBE3A gene had been known to encode the E6-associated protein, a protein interacting with the human papilloma virus, to promote the nonlysosomal degradation of the p53 protein (Huibregtse et al., 1991; Scheffner et al., 1990). The gene encodes an E3 ubiquitin protein ligase that is probably involved in the ubiquitination of a diverse range of proteins (Scheffner et al., 1993; Huibregtse et al., 1993). The UBE3A gene encodes a member of a family of functionally related proteins defined by a conserved C-terminal 350- amino acid ''hect'' domain. Hect E3 proteins appear to be important in substrate recognition and in ubiquitin transfer. RT-PCR experiments provided evidence for several isoforms differing at their N termini. Each of the mRNAs was expressed in all cell lines tested. Additional 5' untranslated exons were also identified (Kishino and Wagstaff, 1998). In fact, at least 16 exons were identified, including six exons that encode the 5' UTR. The gene spans approximately 120 kb, with transcription oriented from telomere to centromere. Two processed UBE3A pseudogenes to chromosomes 2 and 21 have also been identified (Kishino and Wagstaff, 1998).

TABLE 1 Mutations in UBE3A in AS

Nucleotide Substitutions

TABLE 1 Mutations in UBE3A in AS

Nucleotide Substitutions

TGT -

TAT

Cys21Tyr

(Matsuura et al., 1997)

GCT -

ACT

Ala178Tur

(Matsuura et al., 1997)

TGG -

TAG

Trp305Ter

(Fang et al., 1999)

TCG -

CCG

Ser34Pro

(Malzac et al., 1998)

CGA -

TGA

Arg417Ter

(Matsuura et al., 1997)

CGA -

TGA

Arg482Ter

(Malzac et al., 1998)

CGT -

TGT

Arg506Cys

(Baumer et al., 1999)

TAT -

TAG

Tyr533Ter

(Fang et al., 1999)

TGG -

TGA

Trp768Ter

(Tsai et al., 1998)

ATA -

AAA

Ile804Lys

(Fang et al., 1999)

GAA -

TAA

Glu167Ter

(Russo et al., 2000)

Splicing Mutations

Splicing Mutations

Small Deletions

1 nt del codon 89 5 nt del codon 107

2 nt del codon 131

14 nt del codon 291 1 nt del codon 311

1 nt del codon 321 7 nt del codon 324 4 nt del codon 369

2 nt del codon 447 10 nt del codon 483 1 nt del codon 596

3 nt del codon 781

4 nt del codon 835

15 nt del codon 851

(Fang et al., 1999) (Fang et al., 1999) (Fang et al., 1999) (Malzac et al., 1998) (Malzac et al., 1998) (Fang et al., 1999) (Fang et al., 1999) (Fang et al., 1999) (Matsuura et al., 1997) (Malzac et al., 1998) (Baumer et al., 1999) (Fang et al., 1999) (Fang et al., 1999) (Fang et al., 1999)

Small Insertions

1

nt

ins

codon

20

(Malzac et al.

1998)

2

nt

ins

codon

59

(Baumer et al

., 1999)

1

nt

ins

codon

105

(Russo et al.,

2000)

1

nt

ins

codon

237

(Baumer et al

., 1999)

1

nt

ins

codon

648

(Malzac et al.

1998)

1

nt

ins

codon

654

(Russo et al.,

2000)

4

nt

ins

codon

662

(Fang et al., 1999)

3

nt

ins

codon

803

(Malzac et al.

1998)

1

nt

ins

codon

816

(Malzac et al.

1998)

5

nt

ins

codon

836

(Kishino et al.

, 1997)

16 nt ins codon 845 (Baumer et al., 1999)

16 nt ins codon 845 (Baumer et al., 1999)

Complex Rearrangement 26 nt del and 1 nt ins (nt 2230) (Malzac et al., 1998)

The public databases contain the following UBE3A mutations In AS (http://archive.uwcm.ac.uk/uwcm/mg/search/228487.html).

The early studies on the expression of UBE3A concluded that there was no evidence of imprinting (of any alternatively spliced form) in the tissues studied (Kishino et al., 1997; Matsuura et al., 1997; Nakao et al., 1994). Subsequent studies, however, clearly demonstrated that in mice and humans there is imprinted expression of UBE3A and that there is maternal-only expression in specific regions of the brain, particularly in the hippocampus, cerebellum, Purkinje cells, and cells of the olfactory bulb in mice (Albrecht et al., 1997; Vu and Hoffman, 1997; Rougeulle et al., 1997). Animals exhibiting partial paternal UPD for the syntenic AS region had striking reduction of Ube3a expression in the above cells, with variably decreased levels in other brain parts, as compared to controls. If extrapolated to the same human tissues, it was thought that such differences might explain retardation, seizures, and ataxia in AS patients (Albrecht et al., 1997). Indeed, when UBE3A expression in various tissues including brain samples of normal, PWS, and AS subjects was compared, the results confirmed major reductions of all UBE3A alternatively spliced mRNA in AS brain samples (Rougeulle et al., 1997).

Transgenic mice with the maternal or paternal UBE3A genes knocked out have been generated (Jiang et al., 1998). These mice were compared with their wild- type (m+/p+) littermates. Mice with paternal deficiency (m+/p—) were essentially similar to wild-type mice. The phenotype of mice with a maternal deficiency (m—/p+) resembles that of human AS with motor dysfunction, inducible seizures, and a context-dependent learning deficit. The absence of detectable expression of UBE3A in hippocampal neurons and Purkinje cells in m—/p+ mice, indicating imprinting with silencing of the paternal allele, correlated well with the neurologic and cognitive impairments. Long-term potentiation in the hippocampus was severely impaired. The cytoplasmic abundance of p53 was found to be greatly increased in Purkinje cells and in a subset of hippocampal neurons in m—/p+ mice, as well as in a deceased AS patient. The authors suggested that failure of UBE3A to ubiquitinate target proteins and promote their degradation could be a key aspect of the pathogenesis of AS (Jiang et al., 1998).

Not all ''3 Nos'' biparental AS cases (no deletion, no paternal UPD15, no IC mutations) have proven to have a mutation in the exons of UBE3A (Figure 1). Even after the identification of all the exons of UBE3A, the determination of the several alternatively spliced isoforms, and extensive mutation analyses, only about 5% of AS patients have mutations in the UBE3A gene (or about 20% of the AS patients with no deletion, no UPD, no IC mutations). It is therefore possible that mutations in other genes within the critical region could cause AS, or that these patients have a phenotype similar to AS but due to genes or genomic regions outside of chromosome 15q11- q13. For example, AS and Rett syndrome may have similar phenotypes and it is possible that some cases diagnosed as AS may have mutations in the recently identified Rett syndrome gene MECP2 (Amir et al., 1999).

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