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Origins, distribution and expression of the Duarte-2 (D2) allele of galactose-1-phosphate uridylyltransferase.

Carney AE, Sanders RD, Garza KR, McGaha LA, Bean LJ, Coffee BW, Thomas JW, Cutler DJ, Kurtkaya NL, Fridovich-Keil JL - Hum. Mol. Genet. (2009)

Bottom Line: Patients with Duarte galactosemia demonstrate reduced GALT activity and carry one profoundly impaired GALT allele (G) along with a second, partially impaired GALT allele (Duarte-2, D2).The p.N314D substitution, however, is not; it is found together with a silent polymorphism, p.L218(TTA), on functionally normal Duarte-1 alleles (D1, also called Los Angeles or LA alleles).Here we present allele-specific qRT-PCR evidence that D2 alleles express less mRNA in vivo than their wild-type counterparts; the difference is small but statistically significant.

View Article: PubMed Central - PubMed

Affiliation: Emory University, Atlanta, USA

ABSTRACT
Duarte galactosemia is a mild to asymptomatic condition that results from partial impairment of galactose-1-phosphate uridylyltransferase (GALT). Patients with Duarte galactosemia demonstrate reduced GALT activity and carry one profoundly impaired GALT allele (G) along with a second, partially impaired GALT allele (Duarte-2, D2). Molecular studies reveal at least five sequence changes on D2 alleles: a p.N314D missense substitution, three intronic base changes and a 4 bp deletion in the 5' proximal sequence. The four non-coding sequence changes are unique to D2. The p.N314D substitution, however, is not; it is found together with a silent polymorphism, p.L218(TTA), on functionally normal Duarte-1 alleles (D1, also called Los Angeles or LA alleles). The HapMap database reveals that p.N314D is a common human variant, and cross-species comparisons implicate D314 as the ancestral allele. The p.N314D substitution is also functionally neutral in mammalian cell and yeast expression studies. In contrast, the 4 bp 5' deletion characteristic of D2 alleles appears to be functionally impaired in reporter gene transfection studies. Here we present allele-specific qRT-PCR evidence that D2 alleles express less mRNA in vivo than their wild-type counterparts; the difference is small but statistically significant. Furthermore, we characterize the prevalence of the 4 bp deletion in GG, NN and DG populations; the deletion appears exclusive to D2 alleles. Combined, these data strongly implicate the 4 bp 5' deletion as a causal mutation in Duarte galactosemia and suggest that direct tests for this deletion, as proposed here, could enhance or supplant current tests, which define D2 alleles on the basis of the presence and absence of linked coding sequence polymorphisms.

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Related in: MedlinePlus

Strategy for allele-specific amplification of (GTCA)2 versus (GTCA)3 5′ GALT sequences. As illustrated, the forward primer specific for the (GTCA)3 allele includes a 91 bp ‘tail’ of S. cerevisiae sequence that increases the size of the corresponding amplicon.
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DDP080F2: Strategy for allele-specific amplification of (GTCA)2 versus (GTCA)3 5′ GALT sequences. As illustrated, the forward primer specific for the (GTCA)3 allele includes a 91 bp ‘tail’ of S. cerevisiae sequence that increases the size of the corresponding amplicon.

Mentions: To facilitate rapid and direct detection of the 4 bp 5′ deletion in human genomic DNA, we designed a multiplex allele-specific PCR-based assay (Fig. 2); allele-specific amplicons are generated using forward primers specific for either the wild-type (GTCA)3 or mutant (GTCA)2 GALT sequences, together with a shared reverse primer. As illustrated in Figure 2, the (GTCA)3-specific primer includes a 91 bp ‘tail’ of non-human (Saccharomyces cerevisiae) sequence that extends the length of the amplicon without compromising annealing specificity. The wild-type amplicon is 403 bp, whereas the corresponding (GTCA)2-specific amplicon, which lacks the 91 bp tail, is 308 bp. This size difference makes for easy visual distinction between the two amplified fragments following agarose gel electrophoresis. Figure 3A illustrates the application of this method to a set of controls, including one sample homozygous for the (GTCA)3 wild-type allele, one homozygous for the (GTCA)2 D2 allele and one heterozygous for the (GTCA)2 and (GTCA)3 alleles.


Origins, distribution and expression of the Duarte-2 (D2) allele of galactose-1-phosphate uridylyltransferase.

Carney AE, Sanders RD, Garza KR, McGaha LA, Bean LJ, Coffee BW, Thomas JW, Cutler DJ, Kurtkaya NL, Fridovich-Keil JL - Hum. Mol. Genet. (2009)

Strategy for allele-specific amplification of (GTCA)2 versus (GTCA)3 5′ GALT sequences. As illustrated, the forward primer specific for the (GTCA)3 allele includes a 91 bp ‘tail’ of S. cerevisiae sequence that increases the size of the corresponding amplicon.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667289&req=5

DDP080F2: Strategy for allele-specific amplification of (GTCA)2 versus (GTCA)3 5′ GALT sequences. As illustrated, the forward primer specific for the (GTCA)3 allele includes a 91 bp ‘tail’ of S. cerevisiae sequence that increases the size of the corresponding amplicon.
Mentions: To facilitate rapid and direct detection of the 4 bp 5′ deletion in human genomic DNA, we designed a multiplex allele-specific PCR-based assay (Fig. 2); allele-specific amplicons are generated using forward primers specific for either the wild-type (GTCA)3 or mutant (GTCA)2 GALT sequences, together with a shared reverse primer. As illustrated in Figure 2, the (GTCA)3-specific primer includes a 91 bp ‘tail’ of non-human (Saccharomyces cerevisiae) sequence that extends the length of the amplicon without compromising annealing specificity. The wild-type amplicon is 403 bp, whereas the corresponding (GTCA)2-specific amplicon, which lacks the 91 bp tail, is 308 bp. This size difference makes for easy visual distinction between the two amplified fragments following agarose gel electrophoresis. Figure 3A illustrates the application of this method to a set of controls, including one sample homozygous for the (GTCA)3 wild-type allele, one homozygous for the (GTCA)2 D2 allele and one heterozygous for the (GTCA)2 and (GTCA)3 alleles.

Bottom Line: Patients with Duarte galactosemia demonstrate reduced GALT activity and carry one profoundly impaired GALT allele (G) along with a second, partially impaired GALT allele (Duarte-2, D2).The p.N314D substitution, however, is not; it is found together with a silent polymorphism, p.L218(TTA), on functionally normal Duarte-1 alleles (D1, also called Los Angeles or LA alleles).Here we present allele-specific qRT-PCR evidence that D2 alleles express less mRNA in vivo than their wild-type counterparts; the difference is small but statistically significant.

View Article: PubMed Central - PubMed

Affiliation: Emory University, Atlanta, USA

ABSTRACT
Duarte galactosemia is a mild to asymptomatic condition that results from partial impairment of galactose-1-phosphate uridylyltransferase (GALT). Patients with Duarte galactosemia demonstrate reduced GALT activity and carry one profoundly impaired GALT allele (G) along with a second, partially impaired GALT allele (Duarte-2, D2). Molecular studies reveal at least five sequence changes on D2 alleles: a p.N314D missense substitution, three intronic base changes and a 4 bp deletion in the 5' proximal sequence. The four non-coding sequence changes are unique to D2. The p.N314D substitution, however, is not; it is found together with a silent polymorphism, p.L218(TTA), on functionally normal Duarte-1 alleles (D1, also called Los Angeles or LA alleles). The HapMap database reveals that p.N314D is a common human variant, and cross-species comparisons implicate D314 as the ancestral allele. The p.N314D substitution is also functionally neutral in mammalian cell and yeast expression studies. In contrast, the 4 bp 5' deletion characteristic of D2 alleles appears to be functionally impaired in reporter gene transfection studies. Here we present allele-specific qRT-PCR evidence that D2 alleles express less mRNA in vivo than their wild-type counterparts; the difference is small but statistically significant. Furthermore, we characterize the prevalence of the 4 bp deletion in GG, NN and DG populations; the deletion appears exclusive to D2 alleles. Combined, these data strongly implicate the 4 bp 5' deletion as a causal mutation in Duarte galactosemia and suggest that direct tests for this deletion, as proposed here, could enhance or supplant current tests, which define D2 alleles on the basis of the presence and absence of linked coding sequence polymorphisms.

Show MeSH
Related in: MedlinePlus