Publications by authors named "Kent Lai"

28 Publications

  • Page 1 of 1

Fragment Screening Reveals Starting Points for Rational Design of Galactokinase 1 Inhibitors to Treat Classic Galactosemia.

ACS Chem Biol 2021 Apr 16;16(4):586-595. Epub 2021 Mar 16.

Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom, OX3 7DQ.

Classic galactosemia is caused by loss-of-function mutations in galactose-1-phosphate uridylyltransferase (GALT) that lead to toxic accumulation of its substrate, galactose-1-phosphate. One proposed therapy is to inhibit the biosynthesis of galactose-1-phosphate, catalyzed by galactokinase 1 (GALK1). Existing inhibitors of human GALK1 (hGALK1) are primarily ATP-competitive with limited clinical utility to date. Here, we determined crystal structures of hGALK1 bound with reported ATP-competitive inhibitors of the spiro-benzoxazole series, to reveal their binding mode in the active site. Spurred by the need for additional chemotypes of hGALK1 inhibitors, desirably targeting a nonorthosteric site, we also performed crystallography-based screening by soaking hundreds of hGALK1 crystals, already containing active site ligands, with fragments from a custom library. Two fragments were found to bind close to the ATP binding site, and a further eight were found in a hotspot distal from the active site, highlighting the strength of this method in identifying previously uncharacterized allosteric sites. To generate inhibitors of improved potency and selectivity targeting the newly identified binding hotspot, new compounds were designed by merging overlapping fragments. This yielded two micromolar inhibitors of hGALK1 that were not competitive with respect to either substrate (ATP or galactose) and demonstrated good selectivity over hGALK1 homologues, galactokinase 2 and mevalonate kinase. Our findings are therefore the first to demonstrate inhibition of hGALK1 from an allosteric site, with potential for further development of potent and selective inhibitors to provide novel therapeutics for classic galactosemia.
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http://dx.doi.org/10.1021/acschembio.0c00498DOI Listing
April 2021

Discovery of Novel Inhibitors Targeting Multi-UDP-hexose Pyrophosphorylases as Anticancer Agents.

Molecules 2020 Feb 3;25(3). Epub 2020 Feb 3.

Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT 84108, USA.

To minimize treatment toxicities, recent anti-cancer research efforts have switched from broad-based chemotherapy to targeted therapy, and emerging data show that altered cellular metabolism in cancerous cells can be exploited as new venues for targeted intervention. In this study, we focused on, among the altered metabolic processes in cancerous cells, altered glycosylation due to its documented roles in cancer tumorigenesis, metastasis and drug resistance. We hypothesize that the enzymes required for the biosynthesis of UDP-hexoses, glycosyl donors for glycan synthesis, could serve as therapeutic targets for cancers. Through structure-based virtual screening and kinetic assay, we identified a drug-like chemical fragment, GAL-012, that inhibit a small family of UDP-hexose pyrophosphorylases-galactose pyro-phosphorylase (GALT), UDP-glucose pyrophosphorylase (UGP2) and UDP--acetylglucosamine pyrophosphorylase (AGX1/UAP1) with an IC of 30 µM. The computational docking studies supported the interaction of GAL-012 to the binding sites of GALT at Trp190 and Ser192, UGP2 at Gly116 and Lys127, and AGX1/UAP1 at Asn327 and Lys407, respectively. One of GAL-012 derivatives GAL-012-2 also demonstrated the inhibitory activity against GALT and UGP2. Moreover, we showed that GAL-012 suppressed the growth of PC3 cells in a dose-dependent manner with an EC of 75 µM with no effects on normal skin fibroblasts at 200 µM. Western blot analysis revealed reduced expression of pAKT (Ser473), pAKT (Thr308) by 77% and 72%, respectively in the treated cells. siRNA experiments against the respective genes encoding the pyrophosphorylases were also performed and the results further validated the proposed roles in cancer growth inhibition. Finally, synergistic relationships between GAL-012 and tunicamycin, as well as bortezomib (BTZ) in killing cultured cancer cells were observed, respectively. With its unique scaffold and relatively small size, GAL-012 serves as a promising early chemotype for optimization to become a safe, effective, multi-target anti-cancer drug candidate which could be used alone or in combination with known therapeutics.
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http://dx.doi.org/10.3390/molecules25030645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038226PMC
February 2020

The Galactose Index measured in fibroblasts of GALT deficient patients distinguishes variant patients detected by newborn screening from patients with classical phenotypes.

Mol Genet Metab 2020 03 9;129(3):171-176. Epub 2020 Jan 9.

Department of Pediatrics, Division of Metabolic Disorders, Emma Children's Hospital, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands. Electronic address:

Background: The high variability in clinical outcome of patients with Classical Galactosemia (CG) is poorly understood and underlines the importance of prognostic biomarkers, which are currently lacking. The aim of this study was to investigate if residual galactose metabolism capacity is associated with clinical and biochemical outcomes in CG patients with varying geno- and phenotypes.

Methods: Galactose Metabolite Profiling (GMP) was used to determine residual galactose metabolism in fibroblasts of CG patients. The association between the galactose index (GI) defined as the ratio of the measured metabolites [UC]Gal-1-P/ [C]UDP-galactose, and both intellectual and neurological outcome and galactose-1-phosphate (Gal-1-P) levels was investigated.

Results: GMP was performed in fibroblasts of 28 patients and 3 control subjects. The GI of the classical phenotype patients (n = 22) was significantly higher than the GI of four variant patients detected by newborn screening (NBS) (p = .002), two homozygous p.Ser135Leu patients (p = .022) and three controls (p = .006). In the classical phenotype patients, 13/18 (72%) had a poor intellectual outcome (IQ < 85) and 6/12 (50%) had a movement disorder. All the NBS detected variant patients (n = 4) had a normal intellectual outcome (IQ ≥ 85) and none of them has a movement disorder. In the classical phenotype patients, there was no significant difference in GI between patients with a poor and normal clinical outcome. The NBS detected variant patients had significantly lower GI levels and thus higher residual galactose metabolism than patients with classical phenotypes. There was a clear correlation between Gal-1-P levels in erythrocytes and the GI (p = .001).

Conclusions: The GI was able to distinguish CG patients with varying geno- and phenotypes and correlated with Gal-1-P. The data of the NBS detected variant patients demonstrated that a higher residual galactose metabolism may result in a more favourable clinical outcome. Further research is needed to enable individual prognostication and treatment in all CG patients.
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http://dx.doi.org/10.1016/j.ymgme.2020.01.002DOI Listing
March 2020

Novel mRNA-Based Therapy Reduces Toxic Galactose Metabolites and Overcomes Galactose Sensitivity in a Mouse Model of Classic Galactosemia.

Mol Ther 2020 01 19;28(1):304-312. Epub 2019 Sep 19.

Division of Medical Genetics, Department of Pediatrics, University of Utah, Salt Lake City, UT 84108, USA. Electronic address:

Classic galactosemia (CG) is a potentially lethal inborn error of galactose metabolism that results from deleterious mutations in the human galactose-1 phosphate uridylyltransferase (GALT) gene. Previously, we constructed a GalT (GalT-deficient) mouse model that exhibits galactose sensitivity in the newborn mutant pups, reduced fertility in adult females, impaired motor functions, and growth restriction in both sexes. In this study, we tested whether restoration of hepatic GALT activity alone could decrease galactose-1 phosphate (gal-1P) and plasma galactose in the mouse model. The administration of different doses of mouse GalT (mGalT) mRNA resulted in a dose-dependent increase in mGalT protein expression and enzyme activity in the liver of GalT-deficient mice. Single intravenous (i.v.) dose of human GALT (hGALT) mRNA decreased gal-1P in mutant mouse liver and red blood cells (RBCs) within 24 h with low levels maintained for over a week. Repeated i.v. injections increased hepatic GalT expression, nearly normalized gal-1P levels in liver, and decreased gal-1P levels in RBCs and peripheral tissues throughout all doses. Moreover, repeated dosing reduced plasma galactose by 60% or more throughout all four doses. Additionally, a single intraperitoneal dose of hGALT mRNA overcame the galactose sensitivity and promoted the growth in a GalT newborn pup.
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http://dx.doi.org/10.1016/j.ymthe.2019.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952165PMC
January 2020

A novel phosphoglucomutase-deficient mouse model reveals aberrant glycosylation and early embryonic lethality.

J Inherit Metab Dis 2019 09 21;42(5):998-1007. Epub 2019 Jun 21.

Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah.

Patients with phosphoglucomutase (PGM1) deficiency, a congenital disorder of glycosylation (CDG) suffer from multiple disease phenotypes. Midline cleft defects are present at birth. Overtime, additional clinical phenotypes, which include severe hypoglycemia, hepatopathy, growth retardation, hormonal deficiencies, hemostatic anomalies, frequently lethal, early-onset of dilated cardiomyopathy and myopathy emerge, reflecting the central roles of the enzyme in (glycogen) metabolism and glycosylation. To delineate the pathophysiology of the tissue-specific disease phenotypes, we constructed a constitutive Pgm2 (mouse ortholog of human PGM1)-knockout (KO) mouse model using CRISPR-Cas9 technology. After multiple crosses between heterozygous parents, we were unable to identify homozygous life births in 78 newborn pups (P = 1.59897E-06), suggesting an embryonic lethality phenotype in the homozygotes. Ultrasound studies of the course of pregnancy confirmed Pgm2-deficient pups succumb before E9.5. Oral galactose supplementation (9 mg/mL drinking water) did not rescue the lethality. Biochemical studies of tissues and skin fibroblasts harvested from heterozygous animals confirmed reduced Pgm2 enzyme activity and abundance, but no change in glycogen content. However, glycomics analyses in serum revealed an abnormal glycosylation pattern in the Pgm2 animals, similar to that seen in PGM1-CDG.
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http://dx.doi.org/10.1002/jimd.12110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739163PMC
September 2019

Discovery of novel inhibitors of human galactokinase by virtual screening.

J Comput Aided Mol Des 2019 04 26;33(4):405-417. Epub 2019 Feb 26.

NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD, 20850, USA.

Classic Galactosemia is a potentially lethal autosomal recessive metabolic disorder caused by deficient galactose-1-phosphate uridyltransferase (GALT) that results in the buildup of galactose-1-phosphate (gal-1-p) in cells. Galactokinase (GALK1) is the enzyme responsible for converting galactose into gal-1-p. A pharmacological inhibitor of GALK1 is hypothesized to be therapeutic strategy for treating galactosemia by reducing production of gal-1-p. In this study, we report the discovery of novel series of GALK1 inhibitors by structure-based virtual screening (VS). Followed by an extensive structural modeling and binding mode analysis of the active compounds identified from quantitative high-throughput screen (qHTS), we developed an efficient pharmacophore-based VS approach and applied for a large-scale in silico database screening. Out of 230,000 compounds virtually screened, 350 compounds were cherry-picked based on multi-factor prioritization procedure, and 75 representing a diversity of chemotypes exhibited inhibitory activity in GALK1 biochemical assay. Furthermore, a phenylsulfonamide series with excellent in vitro ADME properties was selected for downstream characterization and demonstrated its ability to lower gal-1-p in primary patient fibroblasts. The compounds described herein should provide a starting point for further development of drug candidates for the GALK1 modulation in the Classic Galactosemia.
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http://dx.doi.org/10.1007/s10822-019-00190-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459016PMC
April 2019

Prevalence of epithelial abnormalities and high-risk human papilloma virus in cervicovaginal Pap smears of population subgroups as a guide toward evidence-based best practice.

Diagn Cytopathol 2019 Jul 23;47(7):648-652. Epub 2019 Feb 23.

Department of Pediatrics, University of Utah, Salt Lake City, Utah, USA.

Background: The 2012 American Society for Colposcopy and Cervical Pathology Consensus Guidelines provide information for managing abnormal cervical cancer screening tests and cancer precursors. According to these guidelines for Pap smear diagnosis of Atypical squamous cells of undetermined significance, reflex high risk (HR) human papilloma virus (HPV) genotyping is required among women 21 years of age or older. Whereas, in women of 30 to 65 years of age, HR-HPV can be ordered by the clinicians as part of co-testing with any diagnosis and every 5 years with a negative Cervico-Vaginal Pap test (CVPT).

Methods: A retrospective review of the CoPath database of the Pathology Department at the University of Florida, College of Medicine Jacksonville, FL, was performed to identify North Florida (NF) women who underwent CVPT and HR-HPV testing between 2006 and 2014. The women were stratified by race and age, respectively.

Results: The study included 19,933 CVPTs. Significant differences in the outcomes' distributions were found among age and race groups, respectively. Highest prevalence of HPV positivity was found in African American women, and in 14- to 20-year-old women, respectively. Twenty- to 30-year-old women had the highest percentage (59%) of epithelial abnormality. The most common HR-HPV genotypic distribution was other HR-HPV.

Conclusions: This study underscores the importance of using both HR-HPV and CVPT for screening for cervical cancer, and confirms the need for special focus on managing high-risk populations subgroups, such as African American women, and women of ages 14 to 20 years especially in high-risk populations.
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http://dx.doi.org/10.1002/dc.24158DOI Listing
July 2019

Effect of genotype on galactose-1-phosphate in classic galactosemia patients.

Mol Genet Metab 2018 11 23;125(3):258-265. Epub 2018 Aug 23.

Department of Pathology, University of Utah, Salt Lake City, UT, USA; ARUP Laboratories, Salt Lake City, UT, USA.

Impaired activity of galactose-1-phosphate uridyltransferase (GALT) causes classic galactosemia (OMIM 230400), characterized by the accumulation of galactose-1-phosphate (GAL1P) in patients' red blood cells (RBCs). Our recent study demonstrated a correlation between RBC GAL1P and long-term outcomes in galactosemia patients. Here, we analyze biochemical and molecular results in 77 classic galactosemia patients to evaluate the association between GALT genotypes and GAL1P concentration in RBCs. Experimental data from model organisms were also included to assess the correlation between GAL1P and predicted residual activity of each genotype. Although all individuals in this study showed markedly reduced RBC GALT activity, we observed significant differences in RBC GAL1P concentrations among galactosemia genotypes. While levels of GAL1P on treatment did not correlate with RBC GALT activities (p = 0.166), there was a negative nonlinear correlation between mean GAL1P concentrations and predicted residual enzyme activity of genotype (p = 0.004). These studies suggest that GAL1P levels in RBCs on treatment likely reflect the overall functional impairment of GALT in patients with galactosemia.
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http://dx.doi.org/10.1016/j.ymgme.2018.08.012DOI Listing
November 2018

The Leloir Pathway of Galactose Metabolism - A Novel Therapeutic Target for Hepatocellular Carcinoma.

Anticancer Res 2016 12;36(12):6265-6271

Division of Medical Genetics, Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT, U.S.A.

Hepatocellular carcinoma (HCC) is one of the most lethal types of cancer worldwide, with poor prognosis and limited treatments. In order to identify novel therapeutic targets that will lead to development of effective therapies with manageable side effects, we tested the hypothesis that knocking-down galactokinase (GALK1) or galactose-1 phosphate uridylyltransferase (GALT) gene expression would control the growth of cultured hepatoma cells. Our results showed small interfering RNA (siRNA) against GALK1 or GALT inhibited the growth of HepG2 cells in culture. Western blot analysis revealed simultaneous down-regulation of multiple players of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) growth signaling pathway, as well as heat-shock protein 90 (HSP90) and poly ADP ribose polymerase (PARP). Reverse transcription-polymerase chain reaction (RT-PCR) data, however, showed no significant mRNA reduction of the encoded genes. Our study thus not only supports GALK1 and GALT as being possible novel targets for treating HCC, but also uncovers new post-transcriptional regulatory mechanisms that link the galactose metabolic pathway to protein expression of the PI3K/AKT pathway in hepatoma.
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http://dx.doi.org/10.21873/anticanres.11221DOI Listing
December 2016

Assessment of ataxia phenotype in a new mouse model of galactose-1 phosphate uridylyltransferase (GALT) deficiency.

J Inherit Metab Dis 2017 01 25;40(1):131-137. Epub 2016 Oct 25.

Department of Pediatrics, University of Utah School of Medicine, 295 Chipeta Way, Salt Lake City, UT, 84108, USA.

Despite adequate dietary management, patients with classic galactosemia continue to have increased risks of cognitive deficits, speech dyspraxia, primary ovarian insufficiency, and abnormal motor development. A recent evaluation of a new galactose-1 phosphate uridylyltransferase (GALT)-deficient mouse model revealed reduced fertility and growth restriction. These phenotypes resemble those seen in human patients. In this study, we further assess the fidelity of this new mouse model by examining the animals for the manifestation of a common neurological sequela in human patients: cerebellar ataxia. The balance, grip strength, and motor coordination of GALT-deficient and wild-type mice were tested using a modified rotarod. The results were compared to composite phenotype scoring tests, typically used to evaluate neurological and motor impairment. The data demonstrated abnormalities with varying severity in the GALT-deficient mice. Mice of different ages were used to reveal the progressive nature of motor impairment. The varying severity and age-dependent impairments seen in the animal model agree with reports on human patients. Finally, measurements of the cerebellar granular and molecular layers suggested that mutant mice experience cerebellar hypoplasia, which could have resulted from the down-regulation of the PI3K/Akt signaling pathway.
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http://dx.doi.org/10.1007/s10545-016-9993-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5203948PMC
January 2017

Galactose-1 phosphate uridylyltransferase (GalT) gene: A novel positive regulator of the PI3K/Akt signaling pathway in mouse fibroblasts.

Biochem Biophys Res Commun 2016 Jan 8;470(1):205-212. Epub 2016 Jan 8.

Division of Medical Genetics, Department of Pediatrics, University of Utah School of Medicine, USA. Electronic address:

The vital importance of the Leloir pathway of galactose metabolism has been repeatedly demonstrated by various uni-/multicellular model organisms, as well human patients who have inherited deficiencies of the key GAL enzymes. Yet, other than the obvious links to the glycolytic pathway and glycan biosynthetic pathways, little is known about how this metabolic pathway interacts with the rest of the metabolic and signaling networks. In this study, we compared the growth and the expression levels of the key components of the PI3K/Akt growth signaling pathway in primary fibroblasts derived from normal and galactose-1 phosphate uridylyltransferase (GalT)-deficient mice, the latter exhibited a subfertility phenotype in adult females and growth restriction in both sexes. The growth potential and the protein levels of the pAkt(Thr308), pAkt(Ser473), pan-Akt, pPdk1, and Hsp90 proteins were significantly reduced by 62.5%, 60.3%, 66%, 66%, and 50%, respectively in the GalT-deficient cells. Reduced expression of phosphorylated Akt proteins in the mutant cells led to diminished phosphorylation of Gsk-3β (-74%). Protein expression of BiP and pPten were 276% and 176% higher respectively in cells with GalT-deficiency. Of the 24 genes interrogated using QIAGEN RT(2) Profiler PCR Custom Arrays, the mRNA abundance of Akt1, Pdpk1, Hsp90aa1 and Pi3kca genes were significantly reduced at least 2.03-, 1.37-, 2.45-, and 1.78-fold respectively in mutant fibroblasts. Both serum-fasted normal and GalT-deficient cells responded to Igf-1-induced activation of Akt phosphorylation at +15 min, but the mutant cells have lower phosphorylation levels. The steady-state protein abundance of Igf-1 receptor was also significantly reduced in mutant cells. Our results thus demonstrated that GalT deficiency can effect down-regulation of the PI3K/Akt growth signaling pathway in mouse fibroblasts through distinct mechanisms targeting both gene and protein expression levels.
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http://dx.doi.org/10.1016/j.bbrc.2016.01.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728015PMC
January 2016

Structure activity relationships of human galactokinase inhibitors.

Bioorg Med Chem Lett 2015 Feb 13;25(3):721-7. Epub 2014 Dec 13.

National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, USA. Electronic address:

Classic Galactosemia is a rare inborn error of metabolism that is caused by deficiency of galactose-1-phosphate uridyltransferase (GALT), an enzyme within the Leloir pathway that is responsible for the conversion of galactose-1-phosphate (gal-1-p) and UDP-glucose to glucose-1-phosphate and UDP-galactose. This deficiency results in elevated intracellular concentrations of its substrate, gal-1-p, and this increased concentration is believed to be the major pathogenic mechanism in Classic Galactosemia. Galactokinase (GALK) is an upstream enzyme of GALT in the Leloir pathway and is responsible for conversion of galactose and ATP to gal-1-p and ADP. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of human GALK would act to prevent the accumulation of gal-1-p and offer a novel entry therapy for this disorder. Herein we describe a quantitative high-throughput screening campaign that identified a single chemotype that was optimized and validated as a GALK inhibitor.
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http://dx.doi.org/10.1016/j.bmcl.2014.11.061DOI Listing
February 2015

GALK inhibitors for classic galactosemia.

Future Med Chem 2014 Jun;6(9):1003-15

Division of Medical Genetics, Department of Pediatrics, University of Utah, 50 N Medical Drive, Salt Lake City, UT 84132, USA.

Classic galactosemia is an inherited metabolic disease for which, at present, no therapy is available apart from galactose-restricted diet. However, the efficacy of the diet is questionable, since it is not able to prevent the insurgence of chronic complications later in life. In addition, it is possible that dietary restriction itself could induce negative side effects. Therefore, there is a need for an alternative therapeutic approach that can avert the manifestation of chronic complications in the patients. In this review, the authors describe the development of a novel class of pharmaceutical agents that target the production of a toxic metabolite, galactose-1-phosphate, considered as the main culprit for the cause of the complications, in the patients.
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http://dx.doi.org/10.4155/fmc.14.43DOI Listing
June 2014

Subfertility and growth restriction in a new galactose-1 phosphate uridylyltransferase (GALT) - deficient mouse model.

Eur J Hum Genet 2014 Oct 19;22(10):1172-9. Epub 2014 Feb 19.

Division of Medical Genetics, Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT, USA.

The first GalT gene knockout (KO) mouse model for Classic Galactosemia (OMIM 230400) accumulated some galactose and its metabolites upon galactose challenge, but was seemingly fertile and symptom free. Here we constructed a new GalT gene-trapped mouse model by injecting GalT gene-trapped mouse embryonic stem cells into blastocysts, which were later implanted into pseudo-pregnant females. High percentage GalT gene-trapped chimera obtained were used to generate heterozygous and subsequently, homozygous GalT gene-trapped mice. Biochemical assays confirmed total absence of galactose-1 phosphate uridylyltransferase (GALT) activity in the homozygotes. Although the homozygous GalT gene-trapped females could conceive and give birth when fed with normal chow, they had smaller litter size (P=0.02) and longer time-to-pregnancy (P=0.013) than their wild-type littermates. Follicle-stimulating hormone levels of the mutant female mice were not significantly different from the age-matched, wild-type females, but histological examination of the ovaries revealed fewer follicles in the homozygous mutants (P=0.007). Administration of a high-galactose (40% w/w) diet to lactating homozygous GalT gene-trapped females led to lethality in over 70% of the homozygous GalT gene-trapped pups before weaning. Cerebral edema, abnormal changes in the Purkinje and the outer granular cell layers of the cerebellum, as well as lower blood GSH/GSSG ratio were identified in the galactose-intoxicated pups. Finally, reduced growth was observed in GalT gene-trapped pups fed with normal chow and all pups fed with high-galactose (20% w/w) diet. This new mouse model presents several of the complications of Classic Galactosemia and will be useful to investigate pathogenesis and new therapies.
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http://dx.doi.org/10.1038/ejhg.2014.12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4169538PMC
October 2014

Formal Synthesis of 4-diphosphocytidyl-2-C-methyl D-erythritol From D-(+)-Arabitol.

Tetrahedron 2012 Oct;68(43):8937-8941

Division of Medical Genetics, Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT 84132, U.S.A.

2-C-methyl-D-erythritol-4-phosphate (MEP) is a key chemical intermediate of the non-mevalonate pathway for isoprenoid biosynthesis employed by many pathogenic microbes. MEP is also the precursor for the synthesis of 4-diphosphocytidyl-2-C-methyl D-erythritol (CDP-ME), another key intermediate of the non-mevalonate pathway. As this pathway is non-existent in higher animals, including humans, it represents great opportunities for novel antimicrobial development. To facilitate the in-depth studies of this pathway, we reported here a formal synthesis of CDP-ME through a new synthesis of 2-C-Methyl-D-erythritol-4-phosphoric acid from D-(+)-arabitol.
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http://dx.doi.org/10.1016/j.tet.2012.08.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3462025PMC
October 2012

Correlation assessment among clinical phenotypes, expression analysis and molecular modeling of 14 novel variations in the human galactose-1-phosphate uridylyltransferase gene.

Hum Mutat 2012 Jul 30;33(7):1107-15. Epub 2012 Apr 30.

Division of Medical Genetics, Department of Pediatrics, University of Utah, Salt Lake City, UT, USA.

Galactose-1-phosphate uridylyltransferase (GALT) catalyzes the conversion of galactose-1-phosphate to UDP-galactose, a key step in the galactose metabolism. Deficiency of GALT activity in humans caused by deleterious variations in the GALT gene can cause a potentially lethal disease called classic galactosemia. In this study, we selected 14 novel nucleotide sequence changes in the GALT genes found in galactosemic patients for expression analysis and molecular modeling. Several variants showed decreased levels of expression and decreased abundance in the soluble fraction of the Escherichia coli cell extracts, suggesting altered stability and solubility. Only six variant GALT enzymes had detectable enzymatic activities. Kinetic studies showed that their V(max) decreased significantly. To further characterize the variants at molecular level, we performed static and dynamic molecular modeling studies. Effects of variations on local and/or global structural features of the enzyme were anticipated for the majority of variants. In-depth studies with molecular dynamic simulations on selected variants predicted the alteration of the protein structure even though static models apparently did not highlight any perturbation. Overall, these studies offered new insights on the molecular properties of GALT enzyme, with the aim of correlating them with the clinical outcome. Hum Mutat 33:1107-1115, 2012. © 2012 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/humu.22093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431212PMC
July 2012

Galactose toxicity in animals.

IUBMB Life 2009 Nov;61(11):1063-74

Division of Medical Genetics, Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.

In most organisms, productive utilization of galactose requires the highly conserved Leloir pathway of galactose metabolism. Yet, if this metabolic pathway is perturbed due to congenital deficiencies of the three associated enzymes, or an overwhelming presence of galactose, this monosaccharide which is abundantly present in milk and many non-dairy foodstuffs, will become highly toxic to humans and animals. Despite more than four decades of intense research, little is known about the molecular mechanisms of galactose toxicity in human patients and animal models. In this contemporary review, we take a unique approach to present an overview of galactose toxicity resulting from the three known congenital disorders of galactose metabolism and from experimental hypergalactosemia. Additionally, we update the reader about research progress on animal models, as well as advances in clinical management and therapies of these disorders.
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http://dx.doi.org/10.1002/iub.262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2788023PMC
November 2009

High-throughput screening for human galactokinase inhibitors.

J Biomol Screen 2008 Jun 19;13(5):415-23. Epub 2008 May 19.

Department of Pediatrics, Leonard M. Miller School of Medicine, University of Miami, Miami, Florida 33101, USA.

Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) can result in a potentially lethal disorder called classic galactosemia. Although the neonatal lethality associated with this disease can be prevented through early diagnosis and a galactose-restricted diet, the lack of effective therapy continues to have consequences: developmental delay, neurological disorders, and premature ovarian failure are common sequelae in childhood and adulthood. Several lines of evidence indicate that an elevated level of galactose-1-phosphate (gal-1-p), the product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in patients with classic galactosemia. The authors hypothesize that elimination of gal-1-p production by inhibiting GALK will relieve GALT-deficient cells from galactose toxicity. To test this hypothesis, they obtained human GALK using a bacterial expression system. They developed a robust, miniaturized, high-throughput GALK assay (Z' factor = 0.91) and used this assay to screen against libraries composed of 50,000 chemical compounds with diverse structural scaffolds. They selected 150 compounds that, at an average concentration of 33.3 microM, inhibited GALK activity in vitro more than 86.5% and with a reproducibility score of at least 0.7 for a confirmatory screen under identical experimental conditions. Of these 150 compounds, 34 were chosen for further characterization. Preliminary results indicated that these 34 compounds have potential to serve as leads to the development of more effective therapy of classic galactosemia.
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http://dx.doi.org/10.1177/1087057108318331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2705177PMC
June 2008

Use of hyperspectral imaging to distinguish normal, precancerous, and cancerous cells.

Cancer 2008 Feb;114(1):13-21

Department of Pathology, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505, USA.

Background: The objective of the current study was to test the hypothesis that the cytologic diagnosis of cancer cells can be enhanced by the technique of hyperspectral imaging (HSI).

Methods: As a proof of principle, HSI was employed to obtain hyperspectrum from a normal human fibroblast, as well as its telomerase-immortalized and SV40-transformed derivatives. Novel algorithms were developed to differentiate among these cell models based on spectral and spatial differences. Using the same technique with modified algorithms, the authors were able to differentiate among normal and precancerous (low-grade [LG] and high-grade [HG]) cervical cells and squamous cell carcinoma (SCC) on liquid-based Papanicolaou (Pap) test slides.

Results: The specificity for identifying normal fibroblast cell type based on spatial and spectral algorithms was 74.2%. The sensitivity for identifying telomerase-immortalized and SV40-transformed cells was 100% and 90.3%, respectively. The system identified normal cervical cells with a specificity of 95.8%. With regard to LG precancerous cells and HG precancerous cells, the sensitivity was 66.7% and 93.5%, respectively. The sensitivity detected for SCC was 98.6%.

Conclusions: HSI can be utilized in prescreening liquid-based Pap test slides to improve efficiency in Pap test diagnoses with the goal of ultimately reducing the mortality from cervical cancer while reducing health care costs.
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http://dx.doi.org/10.1002/cncr.23286DOI Listing
February 2008

Involvement of endoplasmic reticulum stress in a novel Classic Galactosemia model.

Mol Genet Metab 2007 Sep-Oct;92(1-2):78-87. Epub 2007 Jul 20.

The Dr. John T. Macdonald Foundation Center for Medical Genetics, Department of Pediatrics, The Leonard M. Miller School of Medicine, University of Miami, P.O. Box 016820 (D-820), Miami, FL 33101, USA.

Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) activity in humans leads to a potentially lethal disorder called Classic Galactosemia. It is well known that patients often accumulate high levels of galactose metabolites such as galactose-1-phosphate (gal-1-p) in their tissues. However, specific targets of gal-1-p and other accumulated metabolites remain uncertain. In this study, we developed a new model system to study this toxicity using primary fibroblasts derived from galactosemic patients. GALT activity was reconstituted in these primary cells through lentivirus-mediated gene transfer. Gene expression profiling showed that GALT-deficient cells, but not normal cells, responded to galactose challenge by activating a set of genes characteristic of endoplasmic reticulum (ER) stress. Western blot analysis showed that the master regulator of ER stress, BiP, was up-regulated at least threefold in these cells upon galactose challenge. We also found that treatment of these cells with galactose, but not glucose or hexose-free media reduced Ca2+ mobilization in response to activation of Gq-coupled receptors. To explore whether the muted Ca2+ mobilization is related to reduced inositol turnover, we discovered that gal-1-p competitively inhibited human inositol monophosphatase (hIMPase1). We hypothesize that galactose intoxication under GALT-deficiency resulted from accumulation of toxic galactose metabolite products, which led to the accumulation of unfolded proteins, altered calcium homeostasis, and subsequently ER stress.
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http://dx.doi.org/10.1016/j.ymgme.2007.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2141683PMC
November 2007

A different spectrum of DMD gene mutations in local Chinese patients with Duchenne/Becker muscular dystrophy.

Chin Med J (Engl) 2006 Jul;119(13):1079-87

Clinical Genetic Service, Department of Health, Hong Kong, China.

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive, allelic disorders. This study was conducted to look into the spectrum of DMD gene mutations in Hong Kong Chinese patients with Duchenne or Becker muscular dystrophy (DMD/BMD), and to study genotype-phenotype correlation.

Methods: A retrospective review of 67 patients.

Results: Twenty-three (34.3%) patients had exon deletions; whereas 5 (7.5%) patients had exon duplications. Twenty-three (34.3%) patients had small mutations, including 17 point mutations and 6 small insertions or deletions. No correlation was found between the type of mutation and the muscle phenotype or mental retardation. Significantly fewer maternal carriers were found in patients with exon deletions, and a positive family history was more common in those with small mutations. DMD phenotype was significantly less common in patients with exon deletions/duplications at the 5' hotspot, whereas all 4 small mutations associated with mental retardation were located in the 3' end of the gene.

Conclusions: The percentage of DMD exon deletions in local Chinese patients was significantly lower than the commonly quoted 60%. This indicated an ethnic or regional difference in predisposition to DMD exon deletions.
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July 2006

Prevention of a molecular misdiagnosis in galactosemia.

Genet Med 2006 Mar;8(3):178-82

Dr. John T. Macdonald Foundation Center for Medical Genetics, Department of Pediatrics and Biochemistry, Miller School of Medicine, University of Miami, FL 33136, USA.

Purpose: The polymerase chain reaction is generally used for mutational analysis of the galactose-1-phosphate uridyl transferase (GALT) gene in the diagnosis of galactosemia. This method is problematic when used in families of Ashkenazi Jewish descent.

Methods: We amplified the GALT gene from leukocyte DNA followed by allele specific oligonucleotide hybridization, DNA sequencing and Southern Blot analysis to determine the mutant alleles causing galactosemia in a representative Jewish family.

Results: The proband's diagnosis of galactosemia was confirmed by high levels of erythrocyte galactose-1-phosphate, absence of erythrocyte GALT activity and impaired total body oxidation of galactose to expired CO2. Initial molecular analysis of GALT alleles in the family showed homozygosity for a K285N missense mutation in the proband, homozygosity for N314D in the mother and heterozygosity for N314D and K285N in the father. These results contradicted Mendelian logic. Southern blot hybridization with GALT cDNA proved the presence of a complex 5 kb GALT deletion in the proband and her mother's DNA enabling a corrected genotype.

Conclusions: Since a deletion of the GALT gene is a common mutation causing galactosemia among Ashkenazim Jewish families, this deletion should be suspected and tested for by genomic hybridization or by using primers specific for the 5 kb deletion.
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http://dx.doi.org/10.1097/01.gim.0000204019.54509.40DOI Listing
March 2006

High rate of detection of subtelomeric aberration by using combined MLPA and subtelomeric FISH approach in patients with moderate to severe mental retardation.

Clin Biochem 2006 Mar 23;39(3):196-202. Epub 2006 Feb 23.

Clinical Genetic Service, Department of Health, HKSAR Government, Cheung Sha Wan, Kowloon, Hong Kong.

Objectives: (1) To evaluate the prevalence of subtelomeric deletion in moderate to severe mental retardation population, (2) to assess the feasibility and cost-effectiveness of combined methodology in routine workup of this sub-population.

Method: Twenty unrelated patients using strict selection criteria were recruited for the study from the Clinical Genetic Service. Patients were initially screened by Multiplex Ligation-dependent Probe Amplification (MLPA) for subtelomeric imbalance followed by FISH analysis for anatomical integrity. This is then followed by parental subtelomeric FISH analysis.

Results: Three subtelomeric deletions were identified. They were Deletion 1p36, Deletion 1q44 and Deletion 10q26; these were previously unidentified by conventional technique.

Conclusions: The prevalence of subtelomeric deletion in our cohort of moderate to severe mental retardation patients is consistent with published findings of around 10%. The figure is on the higher side if more stringent criteria is used. The combination of strict clinical criteria, MLPA and selective subtelomeric FISH was shown to be feasible and cost-effective.
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http://dx.doi.org/10.1016/j.clinbiochem.2006.01.003DOI Listing
March 2006

Detecting exon deletions and duplications of the DMD gene using Multiplex Ligation-dependent Probe Amplification (MLPA).

Clin Biochem 2006 Apr 17;39(4):367-72. Epub 2006 Jan 17.

Clinical Genetic Service, Department of Health, Hong Kong SAR, 3/F, Cheung Sha Wan Jockey Club Clinic, Kwong Lee Road, Shumshipo, Kowloon, Hong Kong.

Objectives: To evaluate the efficacy of Multiplex Ligation-dependent Probe Amplification (MLPA) technique in comparison with the traditional multiplex PCR assay in detection of exon deletions and duplications of the DMD gene.

Design And Methods: The sensitivity and accuracy of MLPA were assessed and compared with the multiplex PCR in a total of 63 subjects including 43 subjects with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) and 20 female carriers.

Results: MLPA was able to detect all the known deletions and duplications; it detected four additional mutations that had been missed by multiplex PCR. In addition, the extent of the deletions and duplications could be more accurately defined which in turn facilitated a genotype-phenotype correlation.

Conclusions: MLPA is superior to multiplex PCR. It should be the method of choice for the detection of exon deletions and duplications of the DMD gene in patients with DMD or BMD, as well as in female carriers.
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http://dx.doi.org/10.1016/j.clinbiochem.2005.11.019DOI Listing
April 2006

Intracellular galactose-1-phosphate accumulation leads to environmental stress response in yeast model.

Mol Genet Metab 2005 Nov;86(3):360-71

The Dr. John T. Macdonald Foundation Center for Medical Genetics, Department of Pediatrics, University of Miami Miller School of Medicine, P.O. Box 016820D-20, Miami, FL 33101, USA.

In humans, deficiency of galactose-1-phosphate uridyltransferase (GALT) can lead a metabolic disorder Classic Galactosemia. Although the biochemical abnormalities associated with this disease have been described in detail, few attempts have been made to characterize the pathogenic mechanisms of this disorder at the molecular level. Here we report the use of high-throughput DNA microarray to examine how galactose affects gene expression in isogenic yeast models that are deficient in either galactokinase (GALK) or GALT, two enzymes which are essential for normal galactose metabolism. We confirmed that the growth of our GALT-deficient, but not GALK-deficient yeast strain ceased 4 h after challenge with 0.2% galactose. Such inhibition was not associated with a reduction of ATP content and was reversible after removal of galactose from medium. We compared the gene expression profiles of the GALT-deficient and GALK-deficient cells in the presence/absence of galactose. We revealed that in the absence of galactose challenge, a subset of genes involved in RNA metabolism was expressed at a level 3-fold lower in the GALT-deficient cells. Upon galactose challenge, significantly more genes involved in various aspects of RNA metabolism and almost all ribosomal protein genes were downregulated in the GALT-deficient, but not GALK-deficient cells. Remarkably, genes involved in inositol biosynthesis and turnover were exclusively induced at high level in the galactose-intoxicated GALT-deficient cells. Our data thus suggested that RNA metabolism, ribosome biogenesis, and inositol metabolism were likely targets for galactose-1-phosphate, a toxic intermediate that is uniquely accumulated under GALT-deficiency.
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http://dx.doi.org/10.1016/j.ymgme.2005.08.002DOI Listing
November 2005

Alternative pathways of galactose assimilation: could inverse metabolic engineering provide an alternative to galactosemic patients?

Metab Eng 2004 Jul;6(3):239-44

The Dr. John T. Macdonald Foundation Center for Medical Genetics, Department of Pediatrics, University of Miami School of Medicine, FL 33101, USA.

The galactose assimilation pathway has been extensively studied as an example of a genetic regulatory switch. Besides the importance of this pathway as a tool in basic biological research, unraveling its structure and regulation is also of major medical importance. Impairment of galactose assimilation is the cause of the genetic metabolic disease known as "galactosemia", while the in vivo activity of the pathway affects the production of glycans. The latter have been connected to tumor metastasis, anti-cancer drug resistance and various cardiovascular diseases. Despite the vast amount of studies, however, galactose assimilation and its interaction with other parts of the metabolic network have not been fully elucidated yet. In yeast and higher eukaryotes, it is still being studied as comprising only the linear Leloir pathway. Recent observations, however, indicate that alternative pathways of galactose assimilation identified in prokaryotes and fungi might also be present in yeast. Such a result is valuable per se, because it could lead to the discovery of these pathways in humans. Even more importantly, these pathways provide alternative phenotypes with known genetic fingerprints that can be used in the context of classical and inverse metabolic engineering to examine and treat the mechanisms of defects of galactose assimilation.
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http://dx.doi.org/10.1016/j.ymben.2004.01.001DOI Listing
July 2004