Molecular and biochemical characterization of human galactokinase and its small molecule inhibitors.

Human galactokinase (GALK) is the first enzyme in the Leloir pathway, converting α-d-galactose into galactose-1-phosphate (Gal-1-P). Recently, there is increasing interest in targeting GALK as a novel therapy to ameliorate the disease manifestations in patients with Classic Galactosemia as it would, in combination with (ga-)lactose restriction reduce accumulation of Gal-1-P, a cytotoxic agent. Previously, we identified 34 small molecule compounds that inhibited GALK in vitro using experimental high-throughput screening. In order to isolate useful lead compounds, we characterized these hits with regards to their kinase selectivity profiles, potency and capability to reduce Gal-1-P accumulation in patient cell lines, and their modes of action. We found that the majority of these compounds had IC(50)s ranging from 0.7µM to 33.3µM. When tested against other members of the GHMP kinase family, three compounds (1, 4, and 24) selectively inhibited GALK with high potency. Through alignment of GALK and mevalonate kinase (MVK) crystal structures, we identified that eight amino acid residues and an L1 loop were different within the ATP-binding pockets of these two closely related kinases. By site-directed mutagenesis experiments, we identified one amino acid residue required for the inhibitory function of two of the three selective compounds. Based on these results, we generated binding models of these two compounds using a high-precision docking program. Compounds 4 and 24 inhibited GALK in a mixed model, while compound 1 exhibited parabolic competitive inhibition. Most importantly, using cells from galactosemic patients we found that selected compounds lowered Gal-1-P concentrations.

RAI1 variations in Smith-Magenis syndrome patients without 17p11.2 deletions.

BACKGROUND: Smith-Magenis syndrome (SMS) (OMIM No 182290) is a mental retardation syndrome characterised by behavioural abnormalities, including self injurious behaviours, sleep disturbance, and distinct craniofacial and skeletal anomalies. It is usually associated with deletion involving 17p11.2 and is estimated to occur in 1/25,000 births. Heterozygous frameshift mutations leading to protein truncation in retinoic acid induced 1 gene (RAI1) have been identified in individuals with phenotypic features consistent with SMS. RAI1 lies within the 17p11.2 locus, but these patients did not have 17p11.2 deletions. OBJECTIVE: Analysis of four individuals with features consistent with SMS for variations in RAI1, using a polymerase chain reaction and sequencing strategy. None of these patients carry 17p11.2 deletions. RESULTS: Two patients had small deletions in RAI1 resulting in frameshift and premature truncation of the protein. Missense mutations were identified in the other two. Orthologs across other genomes showed that these missense mutations occurred in identically conserved regions of the gene. The mutations were de novo, as all parental samples were normal. Several polymorphisms were also observed, including new and reported SNPs. The patients' clinical features differed from those found in 17p11.2 deletion by general absence of short stature and lack of visceral anomalies. All four patients had developmental delay, reduced motor and cognitive skills, craniofacial and behavioural anomalies, and sleep disturbance. Seizures, not previously thought to be associated with RAI1 mutations, were observed in one patient of the cohort. CONCLUSIONS: Haploinsufficiency of the RAI1 gene is associated with most features of SMS, including craniofacial, behavioural, and neurological signs and symptoms.

Cystathionine beta-synthase deficiency in Georgia (USA): correlation of clinical and biochemical phenotype with genotype.

Cystathionine beta-synthase (CBS) deficiency is a rare autosomal recessive disorder that is the most frequent cause of clinical homocystinuria. Patients not treated in infancy have multi-systems disorders including dislocated lenses, mental deficiency, osteoporosis, premature arteriosclerosis, and thrombosis. In this paper, we examine the relationship of the clinical and biochemical phenotypes with the genotypes of 12 CBS deficient patients from 11 families from the state of Georgia, USA. By DNA sequencing of all of the coding exons we identified mutations in the CBS genes in 21 of the 22 possible mutant alleles. Ten different missense mutations were identified and one novel splice-site mutation was found. Five of the missense mutations were previously described (G307S, I278T, V320A, T353M, and L101P), while five were novel (A226T, N228S, A231L, D376N, Q526K). Each missense mutation was tested for function by expression in S. cerevisiae and all were found to cause decreased growth rate and to have significantly decreased levels of CBS enzyme activity. The I278T and T353M mutations accounted for 45% of the mutant alleles in this patient cohort. The T353M mutation, found exclusively in four African American patients, was associated with a B(6)-nonresponsive phenotype and detection by newborn screening for hypermethioninemia. The I278T mutation was found exclusively in Caucasian patients and was associated with a B(6)-responsive phenotype. We conclude that these two mutations occurred after ethnic socialization and that the CBS genotype is predictive of phenotype.

GALT deficiency causes UDP-hexose deficit in human galactosemic cells.

Previously we reported that stable transfection of human UDP-glucose pyrophosphorylase (hUGP2) rescued galactose-1-phosphate uridyltransferase (GALT)-deficient yeast from "galactose toxicity." Here we test in human cell lines the hypothesis that galactose toxicity was caused by excess accumulation of galactose-1-phosphate (Gal-1-P), inhibition of hUGP2, and UDP-hexose deficiency. We found that SV40-transformed fibroblasts derived from a galactosemic patient accumulated Gal-1-P from 1.2+/-0.4 to 5.2+/-0.5 mM and stopped growing when transferred from 0.1% glucose to 0.1% galactose. Control fibroblasts accumulated little Gal-1-P and continued to grow. The GALT-deficient cells had 157+/-10 micromoles UDP-glucose/100 g protein and 25+/-5 micromoles UDP-galactose/100 g protein when grown in 0.1% glucose. The control cells had 236+/-25 micromoles UDP- glucose/100 g protein and 82+/-10 micromoles UDP-galactose/100 g protein when grown in identical medium. When we transfected the GALT-deficient cells with either the hUGP2 or GALT gene, their UDP-glucose content increased to 305+/-28 micromoles/100 g protein (hUGP2-transfected) and 210+/-13 micromoles/100 g protein (GALT-transfected), respectively. Similarly, UDP-galactose content increased to 75+/-12 micromoles/100 g protein (hUGP2-transfected) and 55+/-9 micromoles/100 g protein (GALT-transfected), respectively. Though the GALT-transfected cells grew in 0.1% galactose with little accumulation of Gal-1-P (0.2+/-0.02 mM), the hUGP2-transfected cells grew but accumulated some Gal-1-P (3.1+/-0.4 mM). We found that 2.5 mM Gal-1-P increased the apparent KM of purified hUGP2 for glucose-1-phosphate from 19.7 microM to 169 microM, without changes in apparent Vmax. The Ki of the reaction was 0.47 mM. Gal-1-P also inhibited UDP-N-acetylglucosamine pyrophosphorylase, which catalyzes the formation of UDP-N-acetylglucosamine. We conclude that intracellular concentrations of Gal-1-P found in classic galactosemia inhibit UDP-hexose pyrophosphorylases and reduce the intracellular concentrations of UDP-hexoses. Reduced Sambucus nigra agglutinin binding to glycoproteins isolated from cells with increased Gal-1-P is consistent with the resultant inhibition of glycoprotein glycosylation.

Oxidation of galactose by galactose-1-phosphate uridyltransferase-deficient lymphoblasts.

The ability of EB virus-transformed lymphoblasts with undetectable galactose-1-phosphate uridyltransferase (GALT) from 15 galactosaemic patients to oxidize [1-(14)C]galactose to 14CO2 was compared to that of cells from 7 normal subjects. The oxidation of galactose but not of glucose was markedly diminished by cells from Q188R homozygous galactosaemic patients but was not absent. After 2.5 h these cells liberated 14CO2 at nearly 3% and at 5 h up to 9% of normal. Cells from patients homozygous for the S135L mutation produced much larger amounts of 14CO2 (15-17% of normal) and were distinguishable from the Q188R homozygous cells. A cell line with a homozygous deletion of the GALT gene oxidized galactose at 7% of the normal rate, suggesting that pathways(s) other than GALT exist in these cells as well as Q188R homozygous cells for oxidation of galactose to CO2. Concentration dependence studies are consistent with the presence of a pathway that is unsaturable or has a very high Km The ability of 10(7) lymphoblasts with the S135L genotype to oxidize more than 7% of the sugar to 14CO2 in 5 h suggests the presence of residual GALT despite the inability to detect the activity by enzymatic analysis.

Structure-function analyses of a common mutation in blacks with transferase-deficiency galactosemia.

We previously identified a missense mutation at amino acid 135 of human galactose 1-phosphate uridyltransferase (hGALT) in which a leucine (TTG) was substituted for a serine (TCG), S135L. This mutation was common in black patients with galactosemia and homozygotes (S135L/S135L) had no GALT activity or protein in their erythrocytes or lymphoblasts. However, there was residual GALT activity and protein in their leukocytes, and they had near normal total body [13C]galactose oxidation to 13CO2 in breath. To evaluate the biochemical mechanism(s) producing these effects, we overexpressed hGALT proteins with site-directed mutations in this nonconserved amino acid in a GALT-minus Escherichia coli. Enzyme activities detected in bacterial lysates overexpressing either S135 (wild type), A135, C135, H135, L135, S132-H135, T135, or Y135 were 100, 4.7, 3.0, 4.0, 2.7, 0.7, 35.4, and 1.4%, respectively. Only the threonine substitution (S135T) had significant enzyme activity in these lysates. There was also decreased abundance of all mutant proteins in the lysates exposed to bacterial proteolysis during preparation and analysis. This added the variable of bio-instability to analysis of enzyme activities in lysates. To further characterize the catalytic role of serine at amino acid 135 and to differentiate bio-instability from impaired catalysis by the leucine substitution, we purified wild-type and L135-hGALT proteins to homogeneity and analyzed identical amounts of enzyme protein. We found that the apparent Vmax of the purified L135-hGALT protein was significantly reduced from 80 +/- 5.9 to 5.8 +/- 1.8 micromol glucose 1-phosphate released/min/mg hGALT protein with no increase in KM for galactose 1-phosphate for the second displacement. The first displacement reaction, although three orders of magnitude slower, was similar between the wild type and L135-hGALT. We conclude that a hydroxyl group on amino acid 135 is required for the catalysis of uridyl transfer from UDP-glucose to UDP-galactose in the presence of galactose 1-phosphate, and plays a role in the bio-stability of hGALT.

Functional analysis of the human galactose-1-phosphate uridyltransferase promoter in Duarte and LA variant galactosemia.

Human galactose-1-phosphate uridyltransferase (hGALT) is an evolutionarily conserved enzyme central to D-galactose metabolism. The impairment of hGALT causes galactosemia. One missense mutation, an aspartate to asparagine substitution at amino acid 314 (N314D), impairs 50% activity in the homozygous state in some patients but gives near normal activity in others. The former condition is called Duarte (D) and the latter, Los Angeles (LA). The D allele is linked to hGALT polymorphisms including a deletion 5'to the translation start site (-119 to -116delGTCA), g1391G --> A and g1105G --> C. The LA allele is linked to a g1721C --> T transition. To investigate possible mechanisms for differences in hGALT activity between the D and LA alleles, we sequenced 3951 nucleotides of genomic DNA 5' to the hGALT translation start site. Using a dual-luciferase reporter system to express deletion constructs of the hGALT promoter, we noted both positive and negative regulatory regions. Two putative positive regulatory domains overlap with the naturally occurring -119 to -116delGTCA linked to Duarte. One is an E-box motif (CACGTG) at -117 to -112 bp. The second is an AP-1 motif (TCAGTCAG) at -124 to -119 bp. The delGTCA mutation confers reduced luciferase activity to transfected cell lines derived from human ovarian and liver neoplasms. Additionally, human lymphoblasts derived from patients with the Duarte allele have reduced GALT mRNA. We conclude that the human GALT gene is regulated in the first -165 bp of its promoter region by positive regulators of GALT gene expression. The -119 to -116delGTCA reduces hGALT transcription resulting in reduced GALT activity in the Duarte allele.

Decreased half-life of insulin-like growth factor I in Rabson-Mendenhall syndrome.

Rabson-Mendenhall syndrome is an autosomal recessive disorder of insulin signalling caused by mutations in the insulin receptor gene. Affected patients are insensitive to exogenous insulin. Insulin-like growth factor I (IGF-I), whose receptor is similar to the one for insulin and is not impaired in this condition, is not always effective in these patients. To understand the reason for this failure, IGF-I concentrations were measured in a patient after subcutaneous injection of 0.1 and 0.2 mg/kg of rhIGF-I. IGF-I concentrations increased only transiently because of the short half-life (1.3-3 h, compared to a normal range of 17-22 h). No correlation was found between IGF-I concentrations and glucose or insulin concentrations. The short half-life of IGF-I may contribute to treatment failure in patients with inherited insulin-resistant syndromes.

Risk factors for premature ovarian failure in females with galactosemia.

UNLABELLED: The risk for premature ovarian failure (POF) in females with galactosemia can be predicted by analyzing 3 areas of risk pathology: the patient's molecular genotype for galactose-1-phosphate uridyltransferase (GALT), alternate pathways for galactose metabolism, and the patient's environment at diagnosis and during treatment. STUDY DESIGN: Retrospective cross-sectional information was collected on 53 females with classic galactosemia, and their ovarian function was analyzed by determination of serum follicle-stimulating hormone and luteinizing hormone levels and by clinical observation. The associations were analyzed between POF and the mutations in GALT, the highest erythrocyte galactose-1-phosphate (Gal-1-P) level at diagnosis, the age at which dietary treatment was initiated, mean erythrocyte Gal-1-P level during treatment, and whole-body carbon 13-labeled galactose oxidation to (13)CO(2). RESULTS: The most prevalent mutation, Q188R, had a significant effect of genotype category (Q188R/Q188R, Q188R/Other, Other/Other) on POF (P =.04, Fisher exact test and an odds ratio of 8.3). Mean erythrocyte Gal-1-P level during treatment was a significant risk factor for POF (P =.04). Also, all patients studied with less than 5% total body oxidation of galactose to (13)CO(2) had POF, whereas those with more than 5% did not have POF (P =.008, Fisher exact test). CONCLUSION: The development of POF in females with galactosemia is more likely if the patient's genotype is Q188R/Q188R, if the mean erythrocyte Gal-1-P is >3.5 mg/dL during therapy, and if the recovery of (13)CO(2) from whole-body (13)C-galactose oxidation is reduced below 5% of administered (13)C-galactose.

Galactose breath testing distinguishes variant and severe galactose-1-phosphate uridyltransferase genotypes.

A galactose breath test that quantitates [1-(13)C]galactose conversion to 13CO2 provides information on the whole body galactose oxidative capacity. As there is little information on the relationship between whole body oxidation and the genotype in patients with galactosemia, we measured the 13CO2 excretion for 2 h after administration of [1-(13)C]galactose in 37 patients (3-48 y old) with galactose-1-phosphate uridyltransferase (GALT) deficiency and 20 control subjects (3-37 y old). Eleven patients with the common Q188R/Q188R genotype and no detectable erythrocyte GALT activity eliminated <2% of a bolus of [1-(13)C]galactose as 13CO2 compared with 8.47 to 28.23% in controls. This defines a severe metabolic phenotype. Seven patients with one Q188R allele and a second mutant allele such as L195P, E308K, V151A, M142K, or Q344K and one patient with a K285N/unknown genotype also released <2% as 13CO2 in 2 h. The presence of N314D or S135L as the second mutant allele does not impair total body galactose oxidation, as individuals with the GALT genotype of Q188R/N314D, K285N/N314D, and Q188R/S135L had normal 2-h galactose breath tests. Subjects with S135L/S135L, N314D/N314D, S135L/deltaT2359 as well as other rarer genotypes such as R258C/Y209C, E203K/IVSC-N314D, K285N/T138M, Q188R/D113N, S135L/F171S, R148W/N314D, and IVSC-N314D/N314D oxidized galactose comparable to controls. The dissociation of residual erythrocyte GALT activity and whole body galactose oxidative capacity is exemplified by blacks with a S135L/S135L genotype and absent erythrocyte GALT activity. An oral 2-h [1-(13)C]galactose breath test distinguishes severe and variant GALT genotypes and enables delineation of the extent of impaired galactose metabolism in an array of patients who possess diverse GALT mutations. It may prove to be useful in establishing whether a patient is capable of manifesting disease similar to patients with a Q188R/Q188R genotype.

Impact of a camp experience on phenylalanine levels, knowledge, attitudes, and health beliefs relevant to nutrition management of phenylketonuria in adolescent girls.

OBJECTIVE: To evaluate the effectiveness of an education intervention in a summer camp setting on knowledge, attitudes, and health beliefs regarding metabolic control of phenylketonuria and dietary compliance. DESIGN: An observational study of a weeklong metabolic camp for adolescent girls with phenylketonuria (PKU) who were followed up over the course of 1 year. Observations also were made in 3 subsequent years of camp. INTERVENTION: The camp experience consisted of diet and disease education, sessions on reproductive development, and recreation. Group discussions on attitudes and perceptions about PKU related to dietary compliance were held with nutritionists and a pediatric psychologist. OUTCOME MEASURES: Biochemical and psychological data were collected on the first and last days of the camp to assess short-term effects of the intervention, then at quarterly intervals during the year to determine the long-term impact of the camp. Precamp and Postcamp plasma amino acid data for the subsequent 3 years were also collected. SUBJECTS/SETTING: Analyses were based on 13 adolescent girls with PKU in the first year of a camp at Emory University in Atlanta, Ga, and compared with data from 11 additional campers enrolled the second year, 8 in the third year, and 7 in the fourth year. Mean age +/- standard deviation of first-year campers was 13 +/- 2 years, mean IQ +/- standard deviation was 98 +/- 16, and 9 of 13 girls had menstruated. STATISTICAL ANALYSES PERFORMED: Short-term effects of the intervention were computed by comparing mean levels of response from the baseline period to those from the last day of camp using t tests for dependent samples. Repeated-measures analysis of variance was used to assess the long-term effects of the camp experience over the course of a year at regular quarterly intervals. RESULTS: Short-term effects of the education intervention were significant reductions in dietary phenylalanine intake, plasma phenylalanine levels, and perceived isolation. However, these effects progressively returned to baseline levels over the course of a year. The significant short- and long-term effects of increased knowledge of diet and disease persisted throughout the study period. APPLICATIONS/CONCLUSIONS: Short-term effects of the education intervention resulted in improved metabolic control associated with improved attitudes, increased knowledge of diet and disease, increased perceived support, and decreased barriers to dietary compliance in a camp setting.

Overexpression of human UDP-glucose pyrophosphorylase rescues galactose-1-phosphate uridyltransferase-deficient yeast.

To better understand the pathophysiology of galactose-1-phosphate uridyltransferase (GALT) deficiency in humans, we studied the mechanisms by which a GALT-deficient yeast survived on galactose medium. Under normal conditions, GALT-deficient yeast cannot grow in medium that contains 0.2% galactose as the sole carbohydrate, a phenotype of Gal(-). We isolated revertants from a GALT-deficient yeast by direct selection for growth in galactose, a phenotype of Gal(+). Comparison of gene expression profiles among wild-type and revertant strains on galactose medium revealed that the revertant down-regulated genes encoding enzymes including galactokinase, galactose permease, and UDP-galactose-4-epimerase (the GAL regulon). By contrast, the revertant strain up-regulated the gene for UDP-glucose pyrophosphorylase, UGP1. There was reduced accumulation of galactose-1-phosphate in the galactose-grown revertant cells when compared to the GALT-deficient parent cells. In vitro biochemical analysis showed that UDP-glucose pyrophosphorylase had bifunctional properties and could catalyze the conversion of galactose-1-phosphate to UDP-galactose in the presence of UTP. To test if augmented expression of this gene could produce a Gal(+) phenotype in the GALT-deficient parent cells, we overexpressed the yeast UGP1 and the human homolog, hUGP2 in the mutant strain. The Gal(-) yeast transformed with either UGP1 or hUGP2 regained their ability to grow on galactose. We conclude that revertant can grow on galactose medium by reducing the accumulation of toxic precursors through down-regulation of the GAL regulon and up-regulation of the UGP1 gene. We speculate that increased expression of hUGP2 in humans could alleviate poor outcomes in humans with classic galactosemia.

Cancer genetics in primary care. When is genetic screening an option and when is it the standard of care?

In some families, predisposition to cancer is caused by a germline mutation in a single gene, which produces an autosomal dominant pattern of cancer transmission. Several hereditary cancer syndromes have been identified and for many of them, genetic testing is clinically available. Determining which patients are at risk for hereditary cancer begins by taking a complete family history. In particular, a three-generation family history and pedigree can provide valuable information for both patients and primary care physicians. When the history supports the possibility of hereditary cancer, a qualified genetic professional can assist with specific advice about testing and treatment options for the entire family. If pedigree analysis substantiates a heritable form of cancer, patients should be thoroughly informed about potential benefits, limitations, and risks of genetic screening and offered testing as appropriate. The ultimate goal is to reduce cancer morbidity and mortality through interventions that decrease cancer risk or increase early detection.

Gender verification of female athletes.

The International Olympic Committee (IOC) officially mandated gender verification for female athletes beginning in 1968 and continuing through 1998. The rationale was to prevent masquerading males and women with "unfair, male-like" physical advantage from competing in female-only events. Visual observation and gynecological examination had been tried on a trial basis for two years at some competitions leading up to the 1968 Olympic Games, but these invasive and demeaning processes were jettisoned in favor of laboratory-based genetic tests. Sex chromatin and more recently DNA analyses for Y-specific male material were then required of all female athletes immediately preceding IOC-sanctioned sporting events, and many other international and national competitions following the IOC model. On-site gender verification has since been found to be highly discriminatory, and the cause of emotional trauma and social stigmatization for many females with problems of intersex who have been screened out from competition. Despite compelling evidence for the lack of scientific merit for chromosome-based screening for gender, as well as its functional and ethical inconsistencies, the IOC persisted in its policy for 30 years. The coauthors of this manuscript have worked with some success to rescind this policy through educating athletes and sports governors regarding the psychological and physical nature of sexual differentiation, and the inequities of genetic sex testing. In 1990, the International Amateur Athletics Federation (IAAF) called for abandonment of required genetic screening of women athletes, and by 1992 had adopted a fairer, medically justifiable model for preventing only male "impostors" in international track and field. At the recent recommendation of the IOC Athletes Commission, the Executive Board of the IOC has finally recognized the medical and functional inconsistencies and undue costs of chromosome-based methods. In 1999, the IOC ratified the abandonment of on-site genetic screening of females at the next Olympic Games in Australia. This article reviews the history and rationales for fairness in female-only sports that have led to the rise and fall of on-site, chromosome-based gender verification at international sporting events.

Outcomes analysis of verbal dyspraxia in classic galactosemia.

PURPOSE: This study evaluates a genotype/phenotype relationship between developmental verbal dyspraxia (DVD) and the common, missense mutation of the galactose-1-phosphate uridyltransferase gene, Q188R, in patients with classic galactosemia (G/G). METHODS: As part of this study, we devised a questionnaire for "speech problems" to be completed by the patient\'s clinician. To validate the questionnaire and determine its accuracy in detecting DVD, we analyzed questionnaire responses for 21 patients by testing them independently and directly for DVD through a speech pathologist blinded to the patients' genotype. RESULTS: We found that the questionnaire had a sensitivity of 0.56 and a specificity of 0.75. We then calculated the prevalence of DVD for a larger set of 113 patients with G/G galactosemia whose biochemical phenotype, molecular genotypes, and clinical status were known. The prevalence of "speech problems" from raw data were 50 of 113 (44.2%). After adjusting for misclassification, 43 (38.1%) were classified as cases of DVD. Using multivariate, logistic, regression analyses we found a significant interaction between genotype and mean red blood cell (RBC) galactose-1-phosphate (Gal-1-P). When corrected, using mean RBC Gal-1-P < h 3.28 mg%, the Q188R/Q188R genotype was the best predictor of DVD. There was a significant risk (odds ratio = 9.6, p = 0.0504) of having DVD associated with homozygosity for Q188R compared with other genotypes. CONCLUSIONS: We conclude that homozygosity for Q188R mutations in the GALT gene is a significant risk factor for DVD. However, poor metabolic control obviates this relationship as indicated by RBC Gal-1-P greater than 3.28 mg%.

Classical galactosemia and mutations at the galactose-1-phosphate uridyl transferase (GALT) gene.

Classical galactosemia is caused by a deficiency in activity of the enzyme galactose-1-phosphate uridyl transferase (GALT), which, in turn, is caused by mutations at the GALT gene. The disorder exhibits considerable allelic heterogeneity and, at the end of 1998, more than 150 different base changes were recorded in 24 different populations and ethnic groups in 15 countries worldwide. The mutations most frequently cited are Q188R, K285N, S135L, and N314D. Q188R is the most common mutation in European populations or in those predominantly of European descent. Overall, it accounts for 60-70% of mutant chromosomes, but there are significant differences in its relative frequency in individual populations. Individuals homoallelic for Q188R tend to have a severe phenotype and this is in keeping with the virtually complete loss of enzyme activity observed in in vitro expression systems. Globally, K285N is rarer, but in many European populations it can be found on 25-40% of mutant chromosomes. It is invariably associated with a severe phenotype. S135L is found almost exclusively in African Americans. In vitro expression results are discrepant, but some individuals carrying S135L appear to exhibit GALT activity in some tissues. Duarte 1 (or Los Angeles) and Duarte 2 (or Duarte) variants carry the same amino acid substitution, N314D, even though D1 is associated with increased erythrocyte GALT activity and D2 with reduced activity. N314D is in linkage disequilibrium with other base changes that differ on the D1 and D2 alleles. N314D does not impair GALT activity in in vitro expression systems. However, there are differences in the abundance of GALT protein in lymphoblastoid cells lines from D2 and D1 individuals. It is unclear whether the specific molecular changes that distinguish the D1 and D2 alleles account for the different activities. The considerable genetic heterogeneity documented to date undoubtedly contributes to the phenotypic heterogeneity that is observed in galactosemia. The additional effects of nonallelic variation and other constitutional factors on phenotypic variability remain to be elucidated.