Galactokinase deficiency: lessons from the GalNet registry.

PURPOSE: Galactokinase (GALK1) deficiency is a rare hereditary galactose metabolism disorder. Beyond cataract, the phenotypic spectrum is questionable. Data from affected patients included in the Galactosemias Network registry were collected to better characterize the phenotype. METHODS: Observational study collecting medical data of 53 not previously reported GALK1 deficient patients from 17 centers in 11 countries from December 2014 to April 2020. RESULTS: Neonatal or childhood cataract was reported in 15 and 4 patients respectively. The occurrence of neonatal hypoglycemia and infection were comparable with the general population, whereas bleeding diathesis (8.1% versus 2.17-5.9%) and encephalopathy (3.9% versus 0.3%) were reported more often. Elevated transaminases were seen in 25.5%. Cognitive delay was reported in 5 patients. Urinary galactitol was elevated in all patients at diagnosis; five showed unexpected Gal-1-P increase. Most patients showed enzyme activities ≤1%. Eleven different genotypes were described, including six unpublished variants. The majority was homozygous for NM_000154.1:c.82C>A (p.Pro28Thr). Thirty-five patients were diagnosed following newborn screening, which was clearly beneficial. CONCLUSION: The phenotype of GALK1 deficiency may include neonatal elevation of transaminases, bleeding diathesis, and encephalopathy in addition to cataract. Potential complications beyond the neonatal period are not systematically surveyed and a better delineation is needed.

Galactokinase promiscuity: a question of flexibility?

Galactokinase catalyses the first committed step of the Leloir pathway, i.e. the ATP-dependent phosphorylation of α-D-galactose at C1-OH. Reduced galactokinase activity results in the inherited metabolic disease type II galactosaemia. However, inhibition of galactokinase is considered a viable approach to treating more severe forms of galactosaemia (types I and III). Considerable progress has been made in the identification of high affinity, selective inhibitors. Although the structure of galactokinase from a variety of species is known, its catalytic mechanism remains uncertain. Although the bulk of evidence suggests that the reaction proceeds via an active site base mechanism, some experimental and theoretical studies contradict this. The enzyme has potential as a biocatalyst in the production of sugar 1-phosphates. This potential is limited by its high specificity. A variety of approaches have been taken to identify galactokinase variants which are more promiscuous. These have broadened galactokinase's specificity to include a wide range of D- and L-sugars. Initial studies suggest that some of these alterations result in increased flexibility at the active site. It is suggested that modulation of protein flexibility is at least as important as structural modifications in determining the success or failure of enzyme engineering.

Role of galactose in cellular senescence.

Cellular senescence has been proposed to play critical roles in tumor suppression and organismal aging, but the molecular mechanism of senescence remains incompletely understood. Here we report that a putative lysosomal carbohydrate efflux transporter, Spinster, induces cellular senescence in human primary fibroblasts. Administration of d-galactose synergistically enhanced Spinster-induced senescence and this synergism required the transporter activity of Spinster. Intracellular d-galactose is metabolized to galactose-1-phosphate by galactokinase. Galactokinase-deficient fibroblasts, which accumulate intracellular d-galactose, displayed increased baseline senescence. Senescence of galactokinase-deficient fibroblasts was further enhanced by d-galactose administration and was diminished by restoration of wild-type galactokinase expression. Silencing galactokinase in normal fibroblasts also induced senescence. These results suggest a role for intracellular galactose in the induction of cellular senescence.

GALK inhibitors for classic galactosemia.

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.

Homozygosity mapping identifies a GALK1 mutation as the cause of autosomal recessive congenital cataracts in 4 adult siblings.

OBJECTIVE: Monogenic congenital cataract is one of the most genetically heterogeneous ocular conditions with almost 30 different genes involved in its etiology. In adult patients, genotype-phenotype correlations are troubled by eye surgery during infancy and/or long-term ocular complications. Here, we describe the molecular diagnosis of GALK1 deficiency as the cause of autosomal recessive congenital cataract in a family from Costa Rica. METHODS: Four affected siblings were included in the study. All of them underwent eye surgery during the first decade but medical records were not available. Congenital cataract was diagnosed by report. Molecular analysis included genome wide homozygosity mapping using a 250K SNP Affymetrix microarray followed by PCR amplification and direct nucleotide sequencing of candidate gene. RESULTS: Genome wide homozygosity mapping revealed a 6Mb region of homozygosity shared by two affected siblings at 17q25. The GALK1 gene was included in this interval and direct sequencing of this gene revealed a homozygous c.1144C>T mutation (p.Q382) in all four affected subjects. CONCLUSIONS: This work demonstrates the utility of homozygosity mapping in the retrospective diagnosis of a family with congenital cataracts in which ocular surgery at early age, the lack of medical records, and the presence of long term eye complications, impeded a clear clinical diagnosis during the initial phases of evaluation.

Early cataract formation due to galactokinase deficiency: impact of newborn screening.

BACKGROUND AND AIMS: Galactokinase (GALK) deficiency is an autosomal recessive disorder causing cataract formation that can be prevented or mitigated by early diagnosis and galactose-restricted diet. The aim of this retrospective study was to explore whether GALK-deficiency meets the criteria for neonatal mass screening programs. METHODS: From 2000 until 2010, the Screening Laboratory Hannover performed newborn screening in 1,950,927 infants from Germany for galactosemia by measuring galactose-1-phosphate-uridyl-transferase and total galactose concentration (free galactose plus galactose-1-phosphate), including automatic screening for GALK deficiency. RESULTS: Eleven cases were found with elevated galactose levels accompanied by normal transferase activity. Nine of 11 cases were informative; the diagnosis was established by demonstrating deficient activity of the GALK enzyme in erythrocytes. To our knowledge, screening did not produce any false negative results. All patients were treated with a galactose-restricted diet from the neonatal period or infancy. Three of nine patients suffered from congenital cataracts or eventual development of cataracts, despite normal galactose concentrations in blood. CONCLUSIONS: Newborn screening for GALK deficiency prevents or at least mitigates cataract formation. As screening for GALK deficiency is technically simple, it seems to be reasonable to include this disorder in routine screening programs by simultaneous determination of transferase activity and quantification of galactose plus galactose-1-phosphate in dried blood spots.

Ultra fast and sensitive liquid chromatography tandem mass spectrometry based assay for galactose-1-phosphate uridylyltransferase and galactokinase deficiencies.

The diagnosis of transferase and galactokinase deficiency galactosemia usually involves the measurement of erythrocyte galactose-1-phosphate uridylyltransferase (GALT) and galactokinase (GALK) enzyme activity, respectively. The current gold standard assays for these enzymes are radioactive assays, which are laborious and/or incapable of measuring low enzyme activities. To further our knowledge of genotype-phenotype relationships, we had developed an assay for GALT activity alone using LC-MS/MS. In this study we generated a robust and sensitive LC-MS/MS based GALT and GALK assay using a novel normal phase chromatographic condition. We improved upon our earlier assay by drastically reducing the instrument run time and eliminating the use of an ion pairing reagent. Stable isotope labeled substrates were utilized in the GALT and GALK assays. The enzymatic products ([(13)C(6)]-uridine diphosphate galactose in GALT assay and [(13)C(6)]-galactose-1-phosphate in GALK assay) were quantified in a 3 min LC-MS/MS run. The assays were sensitive enough to allow for the quantification of enzyme activities as low as 0.2% and 0.3% of normal control values in the GALT and GALK assays, respectively. Thirty-three samples from non-galactosemic patients were assayed to have erythrocyte GALT activity of 23.4±4.2 and GALK activity of 1.8±0.47 (mean±SD) µmol⋅(g Hgb)(-1) h(-1). Erythrocyte GALT activities in a cohort of 16 patients with classic or severe galactosemia were measured: 4 patients had GALT activity less than 1% of normal control values and the remaining 12 had no detectable GALT activity. No GALK activity was detected in a GALK deficient sample we analyzed. Lastly, we tested the feasibility of adapting this LC-MS/MS based GALT/GALK assay as a newborn screening (NBS) test.

Diagnosis of inherited disorders of galactose metabolism.

Galactose metabolism occurs through an evolutionarily conserved pathway in which galactose and uridine diphosphoglucose are converted to glucose-1-phosphate and uridine diphosphogalactose through the action of three sequential enzymes: galactokinase (GALK, EC 2.7.1.6), galactose-1-phosphate uridyltransferase (GALT, EC 2.7.7.12), and uridine phosphogalactose 4'-epimerase (GALE, EC 5.1.3.2). Inborn errors of galactose metabolism occur with impaired activity for each of the enzymes. Classical galactosemia is the most common and the most severe of these diseases and is caused by deficiency of the GALT enzyme, affecting from approximately 1 in 10,000 to 1 in 30,000 live births. Deficiency of GALE is the rarest of the three diseases. Assays for galactitol and galactose-1-phosphate and methods for assaying enzyme activities of GALT, GALK, and GALE are provided here. Interpretation of diagnostic results for screen-positive newborns or symptomatic patients, as well as therapeutic interventions based on biochemical phenotype and molecular genotype, are also included as decision trees.

Molecular and biochemical characterization of the GALK1 gene in Korean patients with galactokinase deficiency.

Galactokinase (GALK) deficiency is an autosomal recessive disorder characterized by elevation of blood galactose concentration and diminished galactose-1-phosphate, leading to the production of galactitol. To investigate the molecular defects of GALK1 gene and the biochemical characteristics of their mutant proteins, PCR-direct sequencing and in vitro expression analysis in Cos7 cells were performed in five Korean patients with GALK deficiency galactosemia. Four missense mutations (p.G137R, p.R256W, p.R277Q, and p.V281M) and one small insertion (c.850_851insG) were identified. Among four patients with severely reduced GALK activity, two were found to be homozygotes for p.R256W and the other two were compound heterozygotes for different molecular defects (p.G137R/p.R277Q and p.V281M/c.850_851insG). One Patient with moderately decreased GALK activity was heterozygous for p.R256W. Expression analysis in Cos7 cells confirmed that each of the mutations resulted in reduction of GALK activity and caused GALK deficiency.

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

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.

A system for studying genetic changes in Candida albicans during infection.

Candida albicans is a diploid yeast with a dimorphic life history. It exists commensally in many healthy humans but becomes a potent pathogen in immunocompromised hosts. The underlying genetic mechanisms by which C. albicans switches from a commensal to a pathogenic form in the host are not well understood. To study the evolution of virulence in mammalian hosts, we used GAL1 as selectable marker system that allows for both positive and negative selection in selective media. We show that the deletion of one or both copies of GAL1 in the C. albicans genome does not change virulence in a systemic mouse model. We obtained estimates for the frequency of mitotic recombination at the GAL1 locus during systemic infection. Our observations suggest that genetic changes such as mitotic recombination and gene conversion occur at a high enough frequency to be important in the transition of C. albicans from a commensal to a pathogenic organism.

[Mutation P28T in gene GK1 as the cause of a familial galactokinase deficiency]. / La mutación P28T del gen GK1 como causa de una deficiencia familiar de galactoquinasa.

OBJECTIVE/METHOD: To alert about galactokinase deficiency (GK) as a possible cause of infantile cataracts, and even presenile cataracts in heterozygous carriers. Diagnosis by enzyme and galactitol determination would lead to the introduction of a galactose-free diet which completely prevents the damage. RESULT/CONCLUSIONS: We report on a highly consanguineous Spanish family of gypsy ethnia, with three females of different sibships affected by GK deficiency. The deficiency was due to their homozygosis for mutation P28T in gene GK1. P28T mutation in european Romani gypsies, is also present in Spanish gypsies. It is important to bear in mind that GK deficiency may be an important cause of blindness in that endogamous group.

Pathways for lactose/galactose catabolism by Streptococcus salivarius.

Galactokinase and beta-galactosidase-deficient strains of Streptococcus salivarius were constructed to define the pathways for lactose and galactose catabolism. It was found that S. salivarius does not possess a lactose-specific phosphoenolpyruvate phosphotransferase system (PTS), that intracellular lactose was hydrolyzed by beta-galactosidase, and that galactose is catabolized exclusively through the Leloir pathway. The lack of a high-affinity PTS for lactose may reflect the higher availability of the substrates to soft tissue organisms, such as S. salivarius, compared to dental plaque bacteria.

An unexpectedly high frequency of hypergalactosemia in an immigrant Bosnian population revealed by newborn screening.

In galactokinase (GALK) deficiency, galactose cannot be phosphorylated into galactose-1-phosphate, which leads to cataract formation. Neonatal screening for hypergalactosemia in Berlin has been performed by thin-layer chromatography since 1978, which detects classical galactosemia and GALK deficiency. Until 1991, GALK deficiency has not been identified in a total of approximately 260,000 samples. In contrast, from 1992 to 1999, nine patients were detected in a total of approximately 240,000 screened newborns. One Turkish patient was homozygous for two novel S142I/G148C GALK mutations in close proximity to the putative ATP-binding site of the enzyme. The other eight children were born to five families belonging to the Bosnian refugee population consisting of approximately 30,000 individuals who have arrived in Berlin since 1991. In two of these families, GALK deficiency was subsequently diagnosed in siblings who had cataract surgery at 4 and 5 y of age, respectively. In all these 10 Bosnian patients, a homozygous P28T mutation located near the active center of the enzyme was identified. We propose that neonatal screening of populations with a significant proportion of Bosnians and possibly other southeastern Europeans, e.g. Romani, should be particularly directed toward GALK deficiency, an inborn error of metabolism that is readily amenable to effective treatment.

Clinical features of galactokinase deficiency: a review of the literature.

Galactokinase deficiency (McKusick 230200) is a rare autosomal recessive inborn error of galactose metabolism. Cataract and, rarely, pseudotumor cerebri caused by galactitol accumulation seem to be the only consistently reported abnormalities in this disorder. We performed a literature search to obtain information on the clinical spectrum of galactokinase deficiency. A total of 25 publications were traced describing 55 galactokinase-deficient patients. Cataract was reported in most patients. Clinical abnormalities other than cataract were reported in 15 (35%) out of 43 cases on which information was available. However, all symptoms were reported infrequently and a causal relationship with the galactokinase deficiency is unlikely. As cataract and pseudotumor cerebri appear to be the sole complications of galactokinase deficiency, the outcome for patients with galactokinase deficiency is much better than for patients with classical galactosaemia (McKusick 230400), a more common autosomal recessive disorder of galactose metabolism caused by galactose-1-phosphate uridyltransferase (GALT; EC 2.7.7.12) deficiency. Long-term follow-up of patients with this disorder has shown that, in spite of a severely galactose-restricted diet, most patients develop abnormalities such as a disturbed mental and/or motor development, dyspraxia and hypergonadotropic hypogonadism. Endogenous production of galactose has been considered an important aetiological factor. Although damage may well occur in utero, available evidence suggests that damage will continue after birth. Inhibition of galactokinase may then be a promising approach for controlling damage in GALT-deficient patients.

Novel mutations in the GALK1 gene in patients with galactokinase deficiency.

Galactokinase deficiency is an inborn error of galactose metabolism whose major clinical manifestation is the development of cataracts during the first months of life. Only 20 mutations have been reported to date and understanding of the functionally important domains of the galactokinase protein is still limited. Here we report four novel mutations in GALK1 that were identified in two unrelated patients with galactokinase deficiency. Three of these were amino acid substitutions: 1569C-->T in exon 2 (R68C); 7093C-->T in exon 6 (T288M) and 7538G-->C in exon 8 (A384P). In addition, a single base-pair deletion was found in exon 5 (2833delC), predicted to result in a shift of the reading frame and a premature termination codon at position 263. Some differences with the GALK1 sequence deposited in Genbank are also reported.

Unusual presentation of galactosemia in a 4-month-old child.

Galactosemia is a rare inborn error of metabolism, which if detected can be treated effectively. Galactosemia can occur due to the deficiency of either galatose-1-phosphate uridyl transferase (GLUT) or galactokinase. Both these deficiencies have their characteristic presentation. In this case report we describe a 4-month-old infant who presented with clinical symptoms highly suggestive of GLUT deficiency but on investigation turned out to be galactokinase deficiency.