Galactose-1-phosphate Uridyltransferase Enzyme Research Articles

Measurement of temperature, pH and substrate levels on the activity of the Galactose-1-Phosphate Uridyltransferase enzyme

Galactose-1-phosphate uridyltransferase (GALT) is an important enzyme involved in galactose metabolism. Understanding the factors influencing GALT activity is critical to elucidate its physiological role and potential therapeutic implications in galactosemia. In developing new drugs, chicken intestine/liver powder can be used as an enzyme source, GALT, to treat galactosemia. Therefore, it is necessary to research the characterization of the GALT enzyme in chicken intestine and liver powder. In this study, we investigated the influence of temperature, pH, and substrate level on GALT enzyme activity using an experimental approach in vitro. The optimum pH extraction results show that the optimum pH for the extraction of the chicken intestine and liver GALT is pH 7, with activity values ​​of 0.47 units/mL and 0.3953 units/mL, respectively. The optimum temperature for the extraction of chicken intestine and liver GALT is 37℃ with substrate hydrolysis capabilities of 0.48 U/mL and 0.57 U/mL, respectively. Meanwhile, the optimum substrate content is 400x. These insights provide a valuable foundation for further research aimed at comprehensively understanding GALT function, developing targeted interventions for disorders of galactose metabolism, and possible application in the development of new drugs for galactosemia

Development, optimization and validation of LC-MS/MS method for the determination of DBS GALT enzyme activity

Galactosemia is a carbohydrate metabolism disorder often caused by galactose-1-phosphate uridyl transferase (GALT) deficiency. Detecting GALT deficiency involves measuring intra-erythrocyte enzyme activity. We aimed to create a robust liquid chromatography-mass spectrometry (LC-MS/MS) method to assess GALT activity in dried blood spot (DBS) samples. We validated this method and compared it to the fluorometric approach. We investigated the impact of K2EDTA and lithium heparin tubes on enzyme activity to identify the best sample collection tube. We also assessed the reaction-stopping method. The developed approach employed [13C6]-galactose-1-phosphate as a substrate and UDP-N-acetylglycosamine as an internal standard (IS). The mean ± SD value for GALT activity of DBS samples was determined as 6.37 ± 1.96 μmol/gHb/hour. The linear range was 0.4–50 μM (2.4–310% of normal) in the DBS method. The % coefficient of variation (%CV) values were less than 15 for intra-day and inter-day repeatability studies. Over 90% recovery was achieved in recovery studies, and no ion suppression from matrix was detected. DBS samples were quite stable for 31 days under different storage conditions. Enzyme activity results reported as <3.5 U/g Hb by fluorometric method, were quantitatively determined for even very low concentrations by LC-MS/MS method.

The Importance of Neonatal Screening for Galactosemia.

Galactosemia is an inborn metabolic disorder caused by a deficient activity in one of the enzymes involved in the metabolism of galactose. The first description of galactosemia in newborns dates from 1908, ever since complex research has been performed on cell and animal models to gain more insights into the molecular and clinical bases of this challenging disease. In galactosemia, the newborn appears to be born in proper health, having a window of opportunity before developing major morbidities that may even be fatal following ingestion of milk that contains galactose. Galactosemia cannot be cured, but its negative consequences on health can be avoided by establishing precocious diagnosis and treatment. All the foods that contain galactose should be eliminated from the diet when there is a suspicion of galactosemia. The neonatal screening for galactosemia can urge early diagnosis and intervention, preventing complications. All galactosemia types may be detected during the screening of newborns for this disorder. The major target is, however, galactose-1-phosphate uridyltransferase (GALT) deficiency galactosemia, which is diagnosed by applying a combination of total galactose and GALT enzyme analysis as well as, in certain programs, mutation screening. Most critically, infants who exhibit symptoms suggestive of galactosemia should undergo in-depth testing for this condition even when the newborn screening shows normal results. The decision to enroll global screening for galactosemia among the specific population still faces many challenges. In this context, the present narrative review provides an updated overview of the incidence, clinical manifestations, diagnosis, therapy, and prognosis of galactosemia, questioning under the dome of these aspects related to the disease the value of its neonatal monitoring.

Addition of galactose-1-phosphate measurement enhances newborn screening for classical galactosemia.

Galactosemia is an inborn disorder of carbohydrate metabolism of which early detection can prevent severe illness. Although the assay for galactose-1-phosphate uridyltransferase (GALT) enzyme activity has been available since the 1960s, many issues prevented it from becoming universal. In order to develop the Israeli newborn screening pilot algorithm for galactosemia, flow injection analysis tandem mass spectrometry measurement of galactose-1-phosphate in archived dried blood spots from newborns with classical galactosemia, galactosemia variants, epimerase deficiency, and normal controls, was conducted. Out of 431 330 newborns screened during the pilot study (30 months), two with classical galactosemia and four with epimerase deficiency were identified and confirmed. Five false positives and no false negatives were recorded. Following this pilot study, the Israeli final and routine newborn screening algorithm, as recommended by the Advisory Committee to the National Newborn Screening Program, now consists of galactose-1-phosphate measurement integrated into the routine tandem mass spectrometry panel as the first-tier screening test, and GALT enzyme activity as the second-tier performed to identify only newborns suspected to be at risk for classical galactosemia. The GALT enzyme activity cut-off used in the final algorithm was lowered in order to avoid false positives.

AAV-mediated expression of galactose-1-phosphate uridyltransferase corrects defects of galactose metabolism in classic galactosemia patient fibroblasts.

Classic galactosemia (CG) is a rare disorder of autosomal recessive inheritance. It is caused predominantly by point mutations as well as deletions in the gene encoding the enzyme galactose-1-phosphate uridyltransferase (GALT). The majority of the more than 350 mutations identified in the GALT gene cause a significant reduction in GALT enzyme activity resulting in the toxic buildup of galactose metabolites that in turn is associated with cellular stress and injury. Consequently, developing a therapeutic strategy that reverses both the oxidative and ER stress in CG cells may be helpful in combating this disease. Recombinant adeno-associated virus (AAV)-mediated gene therapy to restore GALT activity offers the potential to address the unmet medical needs of galactosemia patients. Here, utilizing fibroblasts derived from CG patients we demonstrated that AAV-mediated augmentation of GALT protein and activity resulted in the prevention of ER and oxidative stress. We also demonstrate that these CG patient fibroblasts exhibit reduced CD109 and TGFβRII protein levels and that these effectors of cellular homeostasis could be restored following AAV-mediated expression of GALT. Finally, we show initial in vivo proof-of-concept restoration of galactose metabolism in a GALT knockout mouse model following treatment with AAV-GALT.

Molecular analysis of GALT gene in Argentinian population: Correlation with enzyme activity and characterization of a novel Duarte-like allele

BackgroundClassical galactosemia is an autosomal recessive inherited metabolic disorder caused by mutations in the galactose-1-phosphate uridyltransferase (GALT) gene. GALT enzyme deficiency leads to the accumulation of galactose-1-phosphate in various organs, causing hepatic, renal and cerebral impairment. Over 300 mutations have been reported in the GALT gene. The aim of this study was to describe molecular characterization of GALT gene in Argentinian patients with decreased GALT activity, and to correlate molecular results with enzyme activity. Methods37 patients with enzyme activity below 6.3 μmol/h/g Hb (35% of normal value) were included. GALT activity was measured on red blood cells. DNA was extracted from peripheral blood. p.Gln188Arg mutation was studied by PCR-RFLP and, on samples negative or heterozygous, GALT gene was sequenced. In vivo splicing analysis of the GALT gene was performed on RNA extracted from leukocytes of one patient. Results14 different sequence variations were identified among 72 unrelated alleles. The two most common disease-causing mutations were p.Gln188Arg (24/72) and p.Lys285Asn (9/72). Three novel mutations were detected. One of them, c.688G>A, caused partial skipping of exon 9 of the GALT gene. Enzyme activity correlated with GALT genotype in 36 of the 37 patients. ConclusionThis is the first report of sequence variations in the GALT gene in the Argentinian population. This study highlights the contribution of the molecular analysis to the diagnosis of Galactosemia and reveals c.688G>A as a novel Duarte-like variant, with a high prevalence in our population.

An extensive computational approach to analyze and characterize the functional mutations in the galactose-1-phosphate uridyl transferase (GALT) protein responsible for classical galactosemia

Type I galactosemia is a very rare autosomal recessive genetic metabolic disorder that occurs because of the mutations present in the galactose-1-phosphate uridyl transferase (GALT) gene, resulting in a deficiency of the GALT enzyme. The action of the GALT enzyme is to convert galactose-1-phosphate and uridine diphosphate glucose into glucose-1-phosphate (G1P) and uridine diphosphate-galactose, a crucial second step of the Leloir pathway. A missense mutation in the GALT enzyme leads to variable galactosemia's clinical presentations, ranging from mild to severe. Our study aimed to employ a comprehensive computational pipeline to analyze the most prevalent missense mutations (p.S135L, p.K285 N, p.Q188R, and p.N314D) responsible for galactosemia; these genes could serve as potential targets for chaperone therapy. We analyzed the four mutations through different computational analyses, including amino acid conservation, in silico pathogenicity and stability predictions, and macromolecular simulations (MMS) at 50 ns The stability and pathogenicity predictors showed that the p.Q188R and p.S135L mutants are the most pathogenic and destabilizing. In agreement with these results, MMS analysis demonstrated that the p.Q188R and p.S135L mutants possess higher deviation patterns, reduced compactness, and intramolecular H-bonds of the protein. This could be due to the physicochemical modifications that occurred in the mutants p.S135L and p.Q188R compared to the native. Evolutionary conservation analysis revealed that the most prevalent mutations positions were conserved among different species except N314. The proposed research study is intended to provide a basis for the therapeutic development of drugs and future treatment of classical galactosemia and possibly other genetic diseases using chaperone therapy.

Difficulties in the diagnosis of galactokinase deficiency. Urine usefulness for the diagnosis and control

Galactokinase (GALK) deficiency causes cataract leading to severe developmental consequences unless treated early. Because of the easy prevention and rapid reversibility of cataract with treatment, the Dutch Health Council advised to include GALK deficiency in the Dutch newborn screening program. The aim of this study is to establish the optimal screening method and cut-off value (COV) for GALK deficiency screening by performing a systematic review of the literature of screening strategies and total galactose (TGAL) values and by evaluating TGAL values in the first week of life in a cohort of screened newborns in the Netherlands.Systematic literature search strategies in OVID MEDLINE and OVID EMBASE were developed and study selection, data collection and analyses were performed by two independent investigators. A range of TGAL values measured by the Quantase Neonatal Total Galactose screening assay in a cohort of Dutch newborns in 2007 was evaluated.Eight publications were included in the systematic review. All four studies describing screening strategies used TGAL as the primary screening marker combined with galactose-1-phosphate uridyltransferase (GALT) measurement that is used for classical galactosemia screening. TGAL COVs of 2200 μmol/L, 1665 μmol/L and 1110 μmol/L blood resulted in positive predictive values (PPV) of 100%, 82% and 10% respectively. TGAL values measured in the newborn period were reported for 39 GALK deficiency patients with individual values ranging from 3963 to 8159 μmol/L blood and 2 group values with mean 8892 μmol/L blood (SD ± 5243) and 4856 μmol/L blood (SD ± 461). Dutch newborn screening data of 72,786 newborns from 2007 provided a median TGAL value of 110 μmol/L blood with a range of 30–2431 μmol/L blood.Based on TGAL values measured in GALK deficiency patients reported in the literature and TGAL measurements in the Dutch cohort by newborn screening we suggest to perform the GALK screening with TGAL as a primary marker with a COV of 2500 μmol/L blood, combined with GALT enzyme activity measurement as used in the classical galactosemia screening, to ensure detection of GALK deficiency patients and minimize false positive referrals.

Recommendations for newborn screening for galactokinase deficiency: A systematic review and evaluation of Dutch newborn screening data

IntroductionGalactokinase (GALK) deficiency causes cataract leading to severe developmental consequences unless treated early. Because of the easy prevention and rapid reversibility of cataract with treatment, the Dutch Health Council advised to include GALK deficiency in the Dutch newborn screening program. The aim of this study is to establish the optimal screening method and cut-off value (COV) for GALK deficiency screening by performing a systematic review of the literature of screening strategies and total galactose (TGAL) values and by evaluating TGAL values in the first week of life in a cohort of screened newborns in the Netherlands. MethodsSystematic literature search strategies in OVID MEDLINE and OVID EMBASE were developed and study selection, data collection and analyses were performed by two independent investigators. A range of TGAL values measured by the Quantase Neonatal Total Galactose screening assay in a cohort of Dutch newborns in 2007 was evaluated. ResultsEight publications were included in the systematic review. All four studies describing screening strategies used TGAL as the primary screening marker combined with galactose-1-phosphate uridyltransferase (GALT) measurement that is used for classical galactosemia screening. TGAL COVs of 2200 μmol/L, 1665 μmol/L and 1110 μmol/L blood resulted in positive predictive values (PPV) of 100%, 82% and 10% respectively. TGAL values measured in the newborn period were reported for 39 GALK deficiency patients with individual values ranging from 3963 to 8159 μmol/L blood and 2 group values with mean 8892 μmol/L blood (SD ± 5243) and 4856 μmol/L blood (SD ± 461). Dutch newborn screening data of 72,786 newborns from 2007 provided a median TGAL value of 110 μmol/L blood with a range of 30–2431 μmol/L blood. ConclusionBased on TGAL values measured in GALK deficiency patients reported in the literature and TGAL measurements in the Dutch cohort by newborn screening we suggest to perform the GALK screening with TGAL as a primary marker with a COV of 2500 μmol/L blood, combined with GALT enzyme activity measurement as used in the classical galactosemia screening, to ensure detection of GALK deficiency patients and minimize false positive referrals.

GALT Deficiency Galactosemia.

Galactosemia is an inborn error of galactose metabolism that results from a deficiency in one of three enzymes, uridine diphosphate galactose 4'epimerase, galactokinase, or galactose-1-phosphate uridyltransferase (GALT). This article focuses on classical, clinical variant, and biochemical variant (Duarte) galactosemias caused by GALT enzyme deficiency. A brief overview of galactosemia and newborn screening is presented, followed by detailed information about each of the conditions. Confirmatory testing, acute and long-term management, and outcome for these galactosemia types are discussed as well as the importance of genetic counseling and testing for the infant and family to refine reproductive risk.

Galactose‐1‐phosphate uridyltransferase deficiency: A literature review of the putative mechanisms of short and long‐term complications and allelic variants

Galactosemia type 1 is an autosomal recessive disorder of galactose metabolism, determined by a deficiency in the enzyme galactose-1-phosphate uridyltransferase (GALT). GALT deficiency is classified as severe or variant depending on biochemical phenotype, genotype and potential to develop acute and long-term complications. Neonatal symptoms usually resolve after galactose-restricted diet; however, some patients, despite the diet, can develop long-term complications, in particular when the GALT enzyme activity results absent or severely decreased. The mechanisms of acute and long-term complications are still discussed and several hypotheses are presented in the literature like enzymatic inhibition, osmotic stress, endoplasmic reticulum stress, oxidative stress, defects of glycosylation or epigenetic modification. This review summarizes the current knowledge of galactosemia, in particular the putative mechanisms of neonatal and long-term complications and the molecular genetics of GALT deficiency.

The adult galactosemic phenotype

Classic galactosemia is an autosomal recessive disorder due to galactose-1-phosphate uridyltransferase (GALT) deficiency. Newborn screening and early treatment do not completely prevent tremor, speech deficits, and diminished IQ in both sexes and premature ovarian insufficiency (POI) in women. Data on how individuals with galactosemia fare as adults will improve our ability to predict disease progression. Thirty-three adults (mean age = 32.6 ± 11.7 years; range = 18-59) with classic galactosemia, confirmed by genotype and undetectable GALT enzyme activity, were evaluated. Analyses assessed associations among age, genotype, clinical features and laboratory measures. The sample included 17 men and 16 women. Subjects exhibited cataracts (21%), low bone density (24%), tremor (46%), ataxia (15%), dysarthria (24%), and apraxia of speech (9%). Subjects reported depression (39%) and anxiety (67%). Mean full scale IQ was 88 ± 20, (range = 55-122). All subjects followed a dairy-free diet and 75-80% reported low intake of calcium and vitamin D. Mean height, weight and body mass were within established norms. All female subjects had been diagnosed with POI. One woman and two men had had children. Logistic regression analyses revealed no associations between age, genotype or gender with IQ, tremor, ataxia, dysarthria, apraxia of speech or anxiety. Each 10- year increment of age was associated with a twofold increase in odds of depression. Taken together, these data do not support the hypothesis that galactosemia is a progressive neurodegenerative disease. However, greater attention to depression, anxiety, and social relationships may relieve the impact of this disorder in adults.

Quantification of Galactose-1-Phosphate Uridyltransferase Enzyme Activity by Liquid Chromatography–Tandem Mass Spectrometry

The diagnosis of galactosemia usually involves the measurement of galactose-1-phosphate uridyltransferase (GALT) activity. Traditional radioactive and fluorescent GALT assays are nonspecific, laborious, and/or lack sufficient analytical sensitivity. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay for GALT enzyme activity measurement. Our assay used stable isotope-labeled alpha- galactose-1-phosphate ([(13)C(6)]-Gal-1-P) as an enzyme substrate. Sample cleanup and separation were achieved by reversed-phase ion-pair chromatography, and the enzymatic product, isotope-labeled uridine diphosphate galactose ([(13)C(6)]-UDPGal), was detected by MS/MS at mass transition (571 > 323) and quantified by use of [(13)C(6)]-Glu-1-P (265 > 79) as an internal standard. The method yielded a mean (SD) GALT enzyme activity of 23.8 (3.8) mumol x (g Hgb)(-1) x h(-1) in erythrocyte extracts from 71 controls. The limit of quantification was 0.04 micromol x (g Hgb)(-1) x h(-1) (0.2% of normal control value). Intraassay imprecision was determined at 4 different levels (100%, 25%, 5%, and 0.2% of the normal control values), and the CVs were calculated to be 2.1%, 2.5%, 4.6%, and 9.7%, respectively (n = 3). Interassay imprecision CVs were 4.5%, 6.7%, 8.2%, and 13.2% (n = 5), respectively. The assay recoveries at the 4 levels were higher than 90%. The apparent K(m) of the 2 substrates, Gal-1-P and UDPGlc, were determined to be 0.38 mmol/L and 0.071 mmol/L, respectively. The assay in erythrocytes of 33 patients with classical galactosemia revealed no detectable activity. This LC-MS/MS-based assay for GALT enzyme activity will be useful for the diagnosis and study of biochemically heterogeneous patients with galactosemia, especially those with uncommon genotypes and detectable but low residual activities.

Frequencies of the Los Angeles and Duarte galactose-1-phosphate uridyltransferase variant alleles in the Hungarian population

Classical galactosaemia is an autosomal recessively inherited disorder caused by deficient activity of the enzyme galactose-1-phosphate uridyltransferase (GALT), which can be detected by newborn screening. The p.N314D mutation defines two variant forms of the GALT enzyme, the Los Angeles and Duarte, depending on the presence of additional base changes. The aim of our study was to analyze a healthy Hungarian population for the frequencies of the Los Angeles and Duarte galactose-1-phosphate uridyltransferase variant alleles. DNA samples from 100 subjects were analyzed by polymerase chain reaction, followed by digestion with restriction endonucleases. The frequencies of the p.N314D, the Los Angeles and the Duarte variants were 11.5%, 2.5% and 9%, respectively. The allele frequencies of the Los Angeles and Duarte variant alleles in the Hungarian population correlate well with the allele frequencies in other healthy Caucasian populations.

Negative screening tests in classical galactosaemia caused by S135L homozygosity

Classical galactosaemia is relatively common in Ireland due to a high carrier rate of the Q188R GALT mutation. It is screened for using a bacterial inhibition assay (BIA) for free galactose. A Beutler assay on day one of life is performed only in high risk cases (infants of the Traveller community and relatives of known cases). A 16-month-old Irish-born boy of Nigerian origin was referred for investigation of developmental delay, and failure to thrive. He had oral aversion to solids and his diet consisted of cow's milk and milk-based cereal mixes. He was found to have microcephaly, weight <2nd percentile, hepatomegaly and bilateral cataracts. Coagulation screen was normal and transaminases were slightly elevated. His original newborn screen was reviewed and confirmed to have been negative; urinary reducing substances on three separate occasions were negative. Beutler assay demonstrated "absent" red cell galactose-1-phosphate uridyltransferase (GALT) activity. GALT enzyme activity was <0.5 gsubs/h per gHb confirming classical galactosaemia. Gal-1-P was elevated at 1.88 micromol/gHb. Mutation analysis of the GALT gene revealed S135L homozygosity. S135L/S135L galactosaemia is associated with absent red cell GALT activity but with approximately 10% activity in other tissues such as the liver and intestines, probably explaining the negative screening tests and the somewhat milder phenotype associated with this genotype. The patient was commenced on galactose-restricted diet; on follow-up at 2 years of age, growth had normalized but there was global developmental delay. In conclusion, galactosaemia must be considered in children who present with poor growth, hepatomegaly, developmental delay and cataracts and GALT enzyme analysis should be a first line test in such cases. Non-enzymatic screening methods such as urinary reducing substances and BIA for free galactose are not reliable in S135L homozygous galactosaemia.

Newborn screening for classic galactosaemia and primary congenital hypothyroidism in the Nkangala district of Mpumalanga province, South Africa

Objectives: The main objective of this work was to establish the newborn incidence of classic galactosemia and congenital hypothyroidism within the Nkangala district of Mpumalanga. In the process a cost effective protocol for newborn screening of both diseases was developed. Study design and setting: Blood spot specimens were collected from over 30 % of newborn infants in the Nkangala district of Mpumalanga Province in a six month period from June to November 2005. The specimens were subsequently screened for classic galactosemia and hypothyroidism using metabolite quantification assays. GALT (galactose-1-phosphate uridyltransferase) enzyme activity assays were also performed to confirm the reliability of the total galactose assays. Real-time PCR was used to detect commonly occurring mutations in the GALT gene that cause galactosemia. Subjects and outcome measures: Informed consent was obtained from the parents of the newborn infants prior to commencement of screening. Total galactose levels of above 0,9 mg/L and TSH concentrations of above 25,1 mU/L were considered to be indicative of galactosemia and hypothyroidism, respectively. A decrease in the total financial input on the screening protocol was evaluated for significance in cost reduction. Results: The incidence of hypothyroidism was found to be 0.1 % while none of the newborns presented with classic galactosemia. There was up to 20 % reduction in direct input costs of screening when our protocol is applied. Conclusion: Cost effective newborn screening is possible when classic galactosemia and congenital hypothyroidism are screened, simultaneously. Cumulative disease frequency plots confirm the already established fact that hypothyroidism tends to prevail in higher frequencies than classic galactosemia

Combination of enzyme analysis, allele‐specific PCR and sequencing to detect mutations in the GALT gene

Newborn screening can identify patients with classical galactosaemia, and their diagnosis needs to be confirmed with assay of the activity of galactose-1-phosphate uridyltransferase (GALT). Unfortunately, in many cases the results can be ambiguous and further testing is required. Here we report a combination of biochemical analysis of GALT enzyme activity and mutation analysis of the most common mutations in the corresponding gene. Samples (n = 243) submitted for confirmatory testing for classical galactosaemia were analysed simultaneously for GALT enzyme activity and allele-specific PCR/fragment analysis for seven mutations and two polymorphisms in the GALT gene (mutations IVS2-2A>G, p.S135L, p.T138M, p.L195P, p.K285N, p.Q188R, p.Y209C; polymorphisms p.N314D, p.L218L). Mutation detection accorded with biochemical analysis in 93% of samples. Subsequently, a total of 34 samples with either discordant results between the above methods or low enzyme activity were fully sequenced, identifying previously reported pathogenic mutations and seven novel variations (p.P185H, p.R201C, p.E220K, p.R223S, p.I278N, p.L289F and p.L218X) in the GALT gene. This approach further increased concordance between genetic and biochemical analysis to 99% of all alleles tested. Our results indicate that DNA testing can help to verify biochemical enzymatic data and improve distinction of borderline enzyme activities where a patient may still benefit from treatment.

The N314D polymorphism of the GALT gene is not associated with congenital absence of the uterus and vagina.

The aetiology of anomalous embryonic and fetal development of the female reproductive tract, ranging from common uterine abnormalities to the somewhat rare congenital absence of the uterus and vagina (CAUV), is unknown. Some have proposed that abnormal galactose metabolism might cause CAUV. An association between CAUV and the N314D allele of the galactose-1-phosphate uridyl transferase (GALT) gene has been proposed as aetiological. We tested this hypothesis further by performing a case-control molecular study analysing 32 patients with CAUV for the presence of the N314D allele. These patients were compared with 138 normal controls. No association between CAUV and the N314D polymorphism was found (P = 0.32). It is unlikely that either maternal or fetal GALT enzyme activity could affect paramesonephric duct development, because neither galactosaemic subjects nor their children have an increased incidence of uterine anomalies.

Analysis of Common Mutations in the Galactose-1-Phosphate Uridyl Transferase Gene: New Assays to Increase the Sensitivity and Specificity of Newborn Screening for Galactosemia

Classical galactosemia is a genetic disease caused by mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. Prospective newborn screening for galactosemia is routine and utilizes the universally collected newborn dried blood specimen on filter paper. Screening for galactosemia is achieved through analysis of total galactose (galactose and galactose-1-phosphate) and/or determining the activity of the GALT enzyme. While this approach is effective, environmental factors and the high frequency of the Duarte D2 mutation (N314D) does lead to false positive results. Using DNA derived from the original newborn dried blood specimen and Light Cycler technology a panel of five assays able to detect the four most frequently encountered classical galactosemia alleles (Q188R, S135L, K285N, and L195P) and the N314D Duarte variant mutation are described. The five assays are performed simultaneously using common conditions. Including DNA preparation, set-up, amplification, and analysis the genotype data for all five loci is obtained in less than 2 hours. The assays are easily interpreted and amenable to high-throughput newborn screening. Mutational analysis is useful to reduce false positive results, differentiate D/G mixed heterozygotes from classical galactosemia, and to clearly identify a very high percentage of those affected by classical galactosemia.

Galactose-1-phosphate uridyl transferase (GALT) genotype and phenotype, galactose consumption, and the risk of borderline and invasive ovarian cancer (United States).

Previous studies have suggested that high levels of galactose consumption and/or low levels of galactose-I-phosphate uridyl transferase (GALT) activity may result in an increased risk of epithelial ovarian cancer. Similarly, some have reported that carriers of the N314D (asparagine at codon 314 replaced by aspartate) GALT polymorphism, which can be associated with low GALT activity, may have a higher risk of ovarian cancer. We examined these issues as part of a large case-control study of ovarian cancer conducted in Los Angeles between 1992 and 1998. A total of 1,439 histologically confirmed borderline and invasive ovarian cancer cases among English-speaking non-Asian women were ascertained through the population-based cancer registry for Los Angeles County and completed in-person interviews were obtained from 689 of these (78% of cases approached). Controls consisted of 645 English-speaking non-Asian women with at least one intact ovary matched to cases on race/ethnicity (African-American, Latina, non-Latina White), date of birth (+/-3 years), and neighborhood of residence. Interviewer-administered questionnaires included information on reproductive factors, exogenous hormone use, medical history, and diet. Dietary information for the year before each case's diagnosis (and the same period for her matched control) was obtained using a self-administered food-frequency questionnaire. Blood samples were obtained from 452 controls, 136 cases with borderline ovarian cancer, and 312 cases with invasive ovarian cancer. The N314D polymorphism was characterized using PCR-RFLP and GALT enzyme activity, and was determined for a sample of the subjects with GALT genotype using an erythrocyte-based radioactive enzyme assay. We found no effect of N314D GALT genotype on the risk of borderline ovarian cancer (odds ratio (OR)=0.91; 95% confidence interval (CI)=0.54-1.6) or invasive ovarian cancer (OR=0.78; 95% CI= 0.53-1.2). Neither did we observe a relationship between GALT activity or lactose/galactose intake and risk of borderline or invasive ovarian cancer. Among N314D carriers, galactose consumption was associated with an increased risk of borderline (OR = 2.7, p = 0.01), but not invasive (OR = 1.2, p = 0.34), ovarian cancer; however, this result was based on only 24 N314D-positive borderline cases. Differences in galactose intake and GALT metabolism do not contribute significantly to the risk of ovarian cancer. There is some evidence that galactose intake may play a role in the development of borderline ovarian cancer among women who carry the uncommon GALT N314D polymorphism. More data are needed if this latter suggestion is to be definitively addressed.