A yeast model reveals biochemical severity associated with each of three variant alleles of galactose‐1P uridylyltransferase segregating in a single family

Abstract

Classic galactosaemia is a potentially lethal inborn error of metabolism that results from profound impairment of galactose-1P uridylyltransferase (GALT). Like many autosomal recessive disorders, classic galactosaemia demonstrates marked allelic heterogeneity; many if not most patients are compound heterozygotes. Owing in part to the fact that most GALT mutations are never observed in patients in the homozygous state, in part to concerns of possible allelic interaction, and in part to the broad range of GALT activity levels associated with the affected, carrier, and control states, definition of the specific functional consequence of individual variant GALT alleles from studies of clinical samples alone can be a challenging task. To overcome this problem we previously developed and applied a null-background yeast system to enable functional analyses of human GALT alleles expressed individually or in defined pairs. We report here the application of this system to characterize three distinct variant alleles of GALT identified within a single family. Of these alleles, one carried a missense mutation (K285N) that has previously been reported and characterized, one carried a nonsense mutation (R204X) that has previously been reported but not characterized, and the third carried a missense substitution (T268N) that was novel. Our studies reported here reconfirm the profound nature of the K285N mutation, demonstrate that the R204X mutation severely compromises both expression and function of human GALT, and finally implicate T268N as one of a very small number of naturally occurring rare but neutral missense polymorphisms in human GALT.

Molecular basis of classic galactosemia from the structure of human galactose 1-phosphate uridylyltransferase

“…4). The p.Lys285Asn variant, commonly detected in Europeans (33), removes three hydrogen bonds involving helix α 6 , likely destabilising the protein. This agrees with our low recombinant yield for this variant (Supplementary Material, Fig.…”

“…It is of note that various cofactors and modifications can alter a metabolic enzyme’s turnover and stability, with functional association to disease (38). As renewed interest in the mechanistic basis of galactosemia has shown that disease-associated mutants exert their effects on misfolding through affecting expression (39), solubility (33,40), stability (31,40,41) and aggregation tendency (31) of hGALT, it is critical to understand the contribution of these factors. The previous lack of consideration of the effects of covalent modification by UMP and divalent metal binding has been in part due to the absence of a crystal structure of the human protein.…”

A Case Study of Monozygotic Twins Apparently Homozygous for a Novel Variant of UDP-Galactose 4′-epimerase (GALE)

“…Yeast growth assays in the presence of galactose: Yeast growth assays were performed as described previously (Chhay et al 2008a).…”

Functional and structural impact of the most prevalent missense mutations in classic galactosemia

“…2001; Chhay et al. 2008), a characterization of these variants focusing on different structural features is still missing. To date, there is no solved three-dimensional structure of the human GALT.…”