1. Biochemistry, molecular biology and molecular genetics of galactosemia

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1 Transworld Research Network 37/661 (2), Fort P.O. Trivandrum Kerala, India Biochemistry, Molecular Biology and Molecular Genetics of Genetic Disorders, 2013: 1-23 ISBN: Editor: Rajendra Prasad 1. Biochemistry, molecular biology and molecular genetics of galactosemia Rajendra Prasad 1, Ramandeep Singh 1 and Babu Ram Thapa 2 1 Department of Biochemistry; 2 Department of Gastroenterology, Post Graduate Institute of Medical Education and Research, Chandigarh, India Abstract. Galactosemia is an autosomal recessive disorder of Galactose metabolism, characterized by inability to metabolize galactose. Three enzymes are principally involved in the metabolic conversion of galactose to glucose: a galactose specific kinase (GALK), galactose-1-phosphate uridyl transferase (GALT) and uridine diphosphate galactose-4-epimerase (GALE). On the basis of defective enzyme the disease is categorized into three forms viz., Type I Galactosemia due to GALT enzyme Deficiency, Type II Galactosemia due to GALK enzyme Deficiency, Type III Galactosemia due to GALE enzyme Deficiency. The GALT gene is present on the short arm of the chromosome 9. It consists of 11 exons and codes for a 379 amino acid long protein. More than 200 mutations in the GALT gene have been reported. Two common mutations viz. Q188R and K285N account for more than 70% galactosemia mutations in the Caucasian population. Recently from our laboratory characterization of underlying GALT gene lesions was performed in 55 unrelated galactosemia patients. The GALT mutational spectrum comprised 16 distinct mutations including 10 previously unreported mutations. N314D was the most common mutation with a frequency of 40% followed by Q188R at 2.7%. The novel GALT gene mutations included 6 missense mutations viz. Y89H, Q103R, P166A, S181F, K285R, R333L; one nonsense Correspondence/Reprint request: Prof. Rajendra Prasad, Department of Biochemistry, Post Graduate Institute of Medical Education and Research, Chandigarh , India. fateh1977@yahoo.com

2 2 Rajendra Prasad et al. mutation, S307X and 3silent mutations. Moreover we have also characterized spectrum of mutations in GALK gene from Indian patients. Two hundred infants with congenital cataracts were screened for galactokinase (GALK) enzyme deficiency. Total of 5 distinct mutations were identified in GALK gene in 5 different patients out of which 4 were novel mutations viz. S79F, S79Y, S205S and F275Y. Patients with galactosemia often presents with failure to thrive, jaundice, hepatosplenomegaly, cataracts, sepsis and perhaps even death within a few days of exposure to milk. In contrast to the multiple systems that are affected by GALT deficiency, cataract is usually the major manifestation of galactokinase (GALK) deficiency, also known as Type II galactosemia. Epimerase deficiency galactosemia (Type III) is a very rare disorder worldwide. The most important step in the initial management of patients with galactosemia is the immediate removal of galactose from the diet as soon as the diagnosis is suspected. 1. Introduction August von Reuss, in a 1908 publication entitled, "Sugar Excretion in Infancy," reported on a breast-fed infant with failure to thrive, enlargement of the liver and spleen, and "galactosuria" (1). By 1917, "galactosuria" was a broadly recognized inherited disorder and was treated by removal of milk products from the diet. While, the clinicians recognized galactosemia very early in the 20 th century, the defective gene was not discovered until Strikingly, Leloir established a galactose metabolism pathway in 1951 and won the Nobel Prize in 1970 for that work. The galactosemias are a group of three inborn errors of metabolism resulting in the inability to metabolize galactose. Three enzymes are principally involved in the metabolic conversion of galactose to glucose: a galactose specific kinase (GALK; EC ; MIM# ), galactose-1-phosphate uridyl transferase (GALT; EC ; MIM# ) and uridine diphosphate galactose-4-epimerase (GALE; EC ; MIM# ) (3). Deficiency of each of these three galactose-converting enzymes can result in elevated levels of galactose and its metabolites, hence termed as galactosemia. (4). The disease usually appears in the initial days of life following the ingestion of breast milk or formula. Vomiting, diarrhea, jaundice and failure to thrive are often the earliest signs of the disease. However, long term outcomes may include cataracts, speech defects, growth retardation and poor intellectual functions and ovarian failure in females. In addition to the classic form, there are two other clinical variants. Neonatal death due to E.coli sepsis may also occur. The incidence of galactosemia due to GALT deficiency varies enormously in different populations throughout the world with a frequency of approximately 1 in 30,000 40,000 in Caucasians (5,6). Type II galactosemia is an autosomal recessive genetic disorder with an incidence of 1/1,000,000 in Japan and

3 Biochemistry, molecular biology and molecular genetics of galactosemia 3 Caucasians. The disorder is characterized by elevated blood galactose levels with normal uridyl transferase activity and diminished galactokinase activity in erythrocytes. Third disorder of galactose metabolism is due to the defect in the UDP-galactose-4-epimerase. The abnormally accumulated metabolites in this disorder are very much like the GALT deficiency. There are two distinct forms of epimerase deficiency. In benign form, affected persons are healthy, and the enzyme deficiency is limited to leukocytes and erythrocytes. The second form of epimerase deficiency is severe with clinical manifestations resembling the GALT deficiency. Additional features include hypotonia and nerve deafness. Epimerase deficiency galactosemia is a very rare disorder worldwide. A lactose-galactose restricted diet during early days of life quickly resolves the complications of galactosemia disease. Thus, the prophylaxis of the disease is life saving. Moreover, galactosemia may present with a variety of clinical manifestations, most common being hepatic manifestations and neuropsychiatric disturbances but none of the clinical signs are specific or diagnostic. Therefore, in view of above facts, diagnosis of galactosemia in a patient as well as his/her family members (heterozygous/carriers) by means of clinical manifestations and biochemical investigations are not always reliable. In absence of typical data such as spectrum of clinical symptoms, differences in age of onset of clinical manifestations, family history of galactosemia and absence of spectrum of mutations in galactosemia genes in specific galactosemia population have raised the question of feasibility to make the positive diagnosis of galactosemia. Classical galactosemia is also characterized by a high allelic heterogeneity, with a typical distribution of mutations among several populations and ethnic groups. For instances, Q188R mutation in GALT gene is predominantly found Caucasian population while it is present at a very low frequency in Asian galactosemia patients. On the other hand, S135L is exclusively found among black Americans (7). The data on spectrum of mutations in any disease gene is important for better understanding of pathophysiology of the disease and its implications in diagnostic and genetic testing. 2. Major pathways of galactose metabolism There are two major pathways for galactose metabolism: 2.1. The Leloir pathway The important metabolic pathway for the conversion of galactose to glucose was elucidated initially in yeasts and bacteria between 1949 and 1953

4 4 Rajendra Prasad et al. and was named after Leloir, who had made a major contribution in the field of galactose metabolism (Figure 1). The pathway involves three enzymes: a galactose-specific kinase - galactokinase (GALK) which catalyses the phosphorylation of galactose into galactose-1-phosphate; galactose-1- phosphate (Gal-1-P) uridyltransferase (GALT), which, with UDP-glucose (UDP-Glc) as cofactor, incorporates the galactose into UDP-galactose (UDP-Gal) while releasing glucose-1-phosphate (Glc-1-P); and an epimerase, uridine diphosphate galactose-4 ' -epimerase (GALE), which catalyses epimerization of UDP-galactose at carbon-4 into UDP-glucose and thereby maintains an equilibrium between UDP-Gal and UDP-Glu, enabling galactose to make a major contribution to the energy requirements of the organism (8). Figure 1. The Leloir pathway of galactose metabolism (Inset: Federico Leloir; Source: From Leloir s Biography at Accessory pathways The accessory pathway is further divided into three sub pathways: A. The pyrophosphorylase pathway This pathway was first speculated upon by Isselbacher in 1957 (9). This alternative pathway was proposed as an explanation for the perceived ability

5 Biochemistry, molecular biology and molecular genetics of galactosemia 5 to tolerate an increasing intake of galactose with age in some cases of GALT deficiency. The pyrophosphorylase pathway involves the phosphorylation of galactose to Gal-1-P, catalyzed by GALK, followed by a UTP-dependent pyrophosphorylase reaction in which the galactose moiety is incorporated into UDP-Gal. UDP-Gal is then converted into its epimer, UDP-Glc, from which a second pyrophosphorylase reaction generates Glc-1-P and UTP. This pathway can metabolize galactose at a rate of only 1% of that of the Leloir Pathway. The activity of the pyrophosphorylase pathway increases with age in most tissues. Activity is highest in adult liver, amounting to about 5% of the liver GALT activity (10). Probably the most important function of the pyrophosphorylase pathway is the generation of UDP-galactose and UDPglucose for incorporation into glycoproteins and glycolipids. B. Reduction of galactose to galactitol The second accessory pathway is catalyzed by the enzyme aldose reductase, reducing galactose to galactitol. As galactitol cannot be further metabolized by sorbitol dehydrogenase, it is excreted in the urine. However, galactitol can also accumulate in tissues, probably contributing to the development of both the cataract and the pseudotumor cerebri observed in classical galactosemia. The significance of the metabolism of galactose to galactitol is thought to be not as an alternative means of excreting the hexose in disorders of the Leloir pathway but in the possible involvement of the hexitol in the pathogenesis of both the acute and the long-term effects of galactosemia. C. Oxidation of galactose to galactonate A third metabolic route is revealed by the observation that patients with classical galactosemia produce galactonate from galactose and excrete it in the urine (11). The exact metabolic mechanism of the production of galactonate remains unclear. Further proof for the existence of one or more alternative pathways for galactose oxidation has come from a study demonstrating galactose oxidation in a patient homozygous for a large deletion in the GALT gene. 3. Disorder of galactose metabolism: Galactosemia The galactosemias are a group of three inborn errors of metabolism resulting in the inability to metabolize galactose. Three types of galactosemia are named for and classified by the specific enzyme that is deficient or absent.

6 6 Rajendra Prasad et al Galactose-1-phosphate uridyl transferase (GALT) deficiency galactosemia or type I galactosemia Type I Galactosemia, also called Classical galactosemia (OMIM # ) is caused by absence or deficient activity of the enzyme galactose-1- phosphate uridyl transferase (GALT), which, in turn, is caused by mutations at the GALT gene. In this defect neonates were screened and treated within days of birth by a galactose restricted diet, older children had an unexpected poor outcome. Dysfunctions included ovarian failure, verbal dyspraxia and neurological signs of cortical and extrapyramidal tract impairment Biochemistry of type I galactosemia or GALT deficiency The essential function of the transferase in the metabolism of galactose is to incorporate the uridine nucleotide moiety into the substrate thereby producing UDP-galactose and glucose-1-p from UDP-glucose and galactose- 1-P. Glucose-1-P enters glucose metabolism and UDP-galactose undergoes epimerization to UDP-glucose. In this way, each turnover of the transferase produces phosphorylated glucose for metabolism and introduces the catalytically essential uridylyl moiety in UDP-galactose for epimerization into UDP-glucose, which in turn provides the uridylyl moiety for another cycle of uridylyltransfer and epimerization (Figure 2). The normal GALT enzyme exhibits ping-pong, bi-bi kinetics which denotes that one domain of the enzyme binds uridine diphosphate (UDP) glucose to form a GALT enzyme UDP glucose intermediate. Glucose-1- phosphate is released whereas uridine monophosphate remains bound to glucose. Galactose-1-phosphate then binds with the GALT-uridine monophosphate complex form GALT-UDP galactose, UDP-galactose is released and GALT is freed for the next round of reactions (Figure 2). Figure 2. Galctose-1-phosphate uridyltransferase exhibits ping pong, bi-bi kinetics. Enz Enzyme; UDP Glu UDP glucose; Glu-1-P Glucose-1-phosphate; Gal-1-P Galctose-1-phosphate; UDP-Gal UDP-galactose (12).

7 Biochemistry, molecular biology and molecular genetics of galactosemia 7 The essential aspect of the reaction kinetics is the binding of the uridyl (UMP) group to a histidine molecule within the active site of the enzyme. In E. coli the binding occurs on a histidine at position 166 of the GALT gene, which is part of a His-Pro-His sequence Molecular biology of GALT gene GALT gene is located at chromosome 9p13, spans about 4.3 kb of DNA arranged into 11 exons (Figures 3). The cdna is 1295 bases in length and encodes a polypeptide of 379 amino acids (Figure 4). The active protein is a dimer with an estimated molecular mass of kda. The promoter region of the human GALT gene contains two GC-rich Sp1 sites, three AP-1 sequences, and a CCAAT sequence. There is no consensus TATA box. Thus, the gene may be expressed as a housekeeping gene, but the regulatory domains could play a role in tissue-specific and development-specific gene expression. His-Pro-His sequence is highly conserved in evolution and is found in the human GALT enzyme. The site of particular interest is histidine-prolinehistidine-cysteine-glutamine at amino acids 184 to 188 in human GALT protein (Figure 5). This important site resides on a β-sheet at the core of the catalytic site as revealed in its three dimensional crystal structure. The critical role of histidine at position 186 in catalysis was confirmed from experiments Figure 3. Location of GALT gene on short (p) arm of chromosome 9 (source Human Gene Compendium,

8 8 Rajendra Prasad et al. Figure 4. cdna sequence of Human GALT gene with corresponding deduced amino acids. of site directed mutagenesis in the equivalent of E.coli GALT protein. Because the most common G (galactosemia) allele in the white population is an arginine substituted for a glutamine at position 188 (Q188R), the role of glutamine in these catalytic reactions is of considerable interest Molecular genetics of GALT gene Worldwide most common mutations and polymorphisms Till date, more than 200 different base changes have been documented. An up-to-date information is accessible on three websites

9 Biochemistry, molecular biology and molecular genetics of galactosemia 9 at the following addresses: galactosemia/galt_welcome.php. The most common mutations reported in GALT gene are Q188R, K285N, S135L and N314D, of which only S135L is at a CpG hypermutable site. Q188R is the most frequently occurring mutation in European populations or those of predominantly European descent. Taking Europe as a whole, Q188R accounts for 64% of more than 700 mutant chromosomes; however, there is considerable variation in individual populations. The highest frequencies are in the Republic of Ireland and Great Britain, and the frequency decreases in continental Europe moving through populations in an eastern and southern direction. Globally, K285N is much rarer but, like Q188R, there are large differences in its relative frequency in different European populations. In many populations, it is the second most common disease-causing mutation, and in some countries of east/central Europe, it accounts for 25 40% of Figure 5. A ribbon diagram of the polypeptide chain in galactose-l-p uridylyltransferase from E. coli. Shown is a ribbon diagram of one subunit in the dimeric unit of galactose-i-p uridylyltransferase from E. coli. UDP binds to the active site, and its location is illustrated in the diagram. The two metal ions Zn 2+ and Fe 2+ are shown in their preferential binding sites. Neither metal ion appears to be located within the active site. At least one of the metal ions is required for enzymatic activity, or both may be required. Enzymatic activity is supported by either metal at both sites or by Co 2+, Mn +2+, or Cd 2+ at both sites. The metal ions most likely participate in maintaining the active conformation of the enzyme (13).

10 10 Rajendra Prasad et al. mutant chromosomes. This finding suggests that the centre of diffusion for K285N lies farther to the east than does that for Q188R. These two mutations are rare in galactosemic individuals whose ancestries are non-european. In African Americans relative frequencies of Q188R vary from 12% to 21%, however, mutations found almost exclusively in African Americans are S135L and F171S, which account for 50% and 4% of mutant chromosomes, respectively. Q188R and K285N are rare in individuals of Indian, Pakistani, Jewish, or Arabic ancestry N314D and Duarte alleles The Duarte variant (D1 or D2) has been defined biochemically by reduced enzyme activity and by an isoform distinguishable by native gel electrophoresis and isoelectric focusing. The Duarte allele is common in many populations and neonatal screening programmes have established its prevalence with 5-6% in the non-galactosemic population of North America (14) Spectrum of mutations in GALT gene identified in Indian population Our laboratory has established the first report on clinical and molecular spectrum of galactosemia from Indian population (15). Table 1, summarizes all the mutations identified in GALT deficiency galactosemia patients (n=55). In this study, 110 GALT chromosomes were first analyzed for most common GALT gene mutations. Three alleles were found to be present at a frequency of (Q 188R), 0.40 (N314D) and 0.39 (D2) in Indian population. Notwithstanding D1 alleles were absent which suggest the presence of milder form of galactosemia, which can be well managed by early diagnosis and dietary management. Besides Q188R and N314D, 14 different mutations were identified in GALT gene using SSCP and RFLP followed by DNA sequencing. These included 9 missense mutations (Y89H, Q103R, P166A, F171S, S181F, P185L, K285R, H319Q, and R333L), one nonsense mutation (S307X) and 4 silent mutations (Q103Q, K210K, L218L and H319H). In total, 22 of the 62 chromosomes (35%) were identified as mutated chromosomes (16). Taken together, 16 different mutations were identified in 70 of 110 GALT chromosomes analyzed with a detection rate of 64%. Further, these 16 mutations were detected in 49 patients out of 55 galactosemic patients screened who constituted 89% of GALT population.

11 Biochemistry, molecular biology and molecular genetics of galactosemia 11 Table 1. GALT mutations identified and characterized in the Indian population. Total chromosomes 110. S. No Mutation Exon Nucleotide Change Consequence Detection method 1 Y89H 3 TAC CAC Tyrosine to Histidine SSCP 2 Q103Q 3 CAG CAA Silent SSCP 3 Q103R 3 CAG CGG Glutamine to Arginine SSCP 4 P166A 5 CCT GCT Proline to Alanine SSCP 5 F171S 6 TTT TCT Phenylalanine to Serine SSCP 6 S181F 6 TCT TTT Serine to Phenylalanine SSCP 7 Q188R 6 CAG CGG Glutamine to Arginine RFLP 8 P185L 6 CCC CTC Proline to Leucine SSCP 9 L218L 7 CTA TTA Silent RFLP 10 K210K 7 AAG AAA Silent SSCP 11 K285R 9 AAG AGG Lysine to Arginine SSCP 12 N314D 10 AAC GAC Asparagine to Aspartate RFLP 13 S307X 10 TCA TAA Serine to Stop Codon SSCP 14 H319Q 10 CAC CAA Histidine to Glutamine SSCP 15 H319H 10 CAC CAT Silent SSCP 16 R333L 10 CGG CTG Arginine to Leucine SSCP Novel mutations are shown in red. Blue indicates most common mutation. Green indicates second most common mutation Metabolic aberrations in GALT deficiency Complete deficiency of GALT has a profound effect on many metabolic pathways particularly when normal galactose intake continues. Normal infants rapidly metabolize galactose to glucose. In patients with classical galactosemia, however, galactose accumulates and is excreted in high concentrations in the urine. Blood galactose-1-phosphate (G-1-P) is the most commonly measured metabolite in galactosemia patients. As a result of the GALT deficiency, galactose-1-phosphate cannot be further metabolized and accumulates in red blood cells as well as in many other cells and tissues. Similarly to the case for Gal-1-P, numerous tissues from a galactosemic infant have been shown to accumulate galactitol. In untreated patients with

12 12 Rajendra Prasad et al. classical galactosemia, markedly elevated concentrations of galactitol are detected in plasma as well as in urine Diagnosis Galactose is a reducing sugar that is readily excreted in the urine. Although determination of reducing substances in the urine can be used as a first simple screening test for classical galactosemia, however this test should not be used either to confirm or to reject a diagnosis. No galactose will be present in the urine if the child is on intravenous fluids, as will be often the case during a neonatal crisis. In addition, galactosuria is frequently found in patients with liver disease. Moreover, other reducing sugars (glucose and fructose) also gives a positive test. This test, accordingly, should always be accompanied by a glucose dipstick test. The gold standard for diagnosis of classical galactosemia is the measurement of galactose-1-phosphate uridyltransferase enzyme activity in erythrocytes. Most newborn-screening programs for galactosemia in the US monitor blood spot galactose concentrations with a fluorescence assay as a first-line screen and follow up with a fluorometric blood spot enzyme assay for GALT, known as the Beutler test. However, Beutler test like any quantitative enzyme assays may give false negative results following blood transfusion. In such situations, the diagnosis can usually be confirmed by DNA analysis for most common mutations like N314D, Q188R, K285N and S135L. Also, red blood cell examination should include testing for the enzymes and by-products of galactose metabolism, specifically gal-1-p. Decreased GALT enzyme levels and elevated gal-1-p levels are considered diagnostic for galactosemia Treatment/Management The most important step in the initial management of patients with classical galactosemia is the immediate removal of galactose from the diet as soon as the diagnosis is suspected. Additional therapies may be indicated in the case of complications such as sepsis, liver failure with clotting abnormalities or hyper bilirubinemia. Breast feeding and cow s milk formulas must therefore be stopped. Vitamin K and fresh-frozen plasma may be necessary to correct clotting abnormalities. Most infants will tolerate enteral feeding, in which case breast milk or cow s milk formula should be completely replaced by soy milk formula. Infant formula based on casein hydrolysates and dextrin maltose as carbohydrate source is also used in the initial management.

13 Biochemistry, molecular biology and molecular genetics of galactosemia Galactokinase (GALK) deficiency galactosemia or type II galactosemia Galactokinase is the first enzyme in the Leloir pathway of galactose metabolism and it catalyzes the phosphorylation of galactose to galactose- 1- phosphate. GALK deficiency, first described in 1965 (17), is an autosomal recessive genetic disorder with an incidence of 1/1,000,000 in Japan and Caucasians (18). Heterozygotes for the condition have approximately 50 percent of normal enzyme activity in their red cells. Studies in rat liver and in human red cells have indicated that GALK is inhibited by both its substrate and its product. This may limit the accumulation of Gal-1-P in GALT or GALE deficiency Molecular biology of GALK gene The human galactokinase (GALK) gene (Figure 6) localized at 17q24 and consists of eight exons spanning 7.3 kb across human genome cdna encoding human GLAK gene was cloned and functionally characterized in. It is 1.35 kb long and encodes peptide of 392 amino acids. Figure 6. Showing the location of GALK gene on chromosome 17 (Source: Human Gene Compendium,

14 14 Rajendra Prasad et al Structure of human galactokinase As indicated in Figure 7, the structure of human galactokinase enzyme, with overall dimensions of ~44 Å 56 Å 63 Å, can be envisioned as two domains. The N-terminal region initiates with a long α helix of 15 residues, which flanks one side of a six-stranded mixed β sheet. A four helical bundle Figure 7. Molecular architecture of human galactokinase. A ribbon representation of one subunit of the enzyme is shown in a with the bound-d-galactose and Mg- AMPPNP ligands displayed in ball-and-stick representations. The green sphere indicates the position of the bound magnesium ion. The locations of the conserved structural motifs in the GHMP superfamily are indicated by the labels MI, MII, and MIII. The four molecules contained within the crystalline asymmetric unit pack as dimers with local 2-fold rotational axes. One of these dimers, viewed down the local dyad, is shown in b. In both dimers a disulfide bond is formed between neighboring Cys391 side chains (19).

15 Biochemistry, molecular biology and molecular genetics of galactosemia 15 surrounds the other side of this β sheet. The C-terminal domain is dominated by six α helices and two layers of anti-parallel β sheet, each containing four strands. The location of the three structural motifs characteristic of the GHMP superfamily are indicated in Figure 7a. Motif I, having the sequence 38 Val-Asn-Leu-Ile-Gly-Glu-His 44, abuts one side of the galactose moiety where the side chain of Glu 43 and the backbone peptidic nitrogen of His 44 participate in hydrogen bonding interactions with the C-6 hydroxyl group of the sugar. In human galactokinase, Motif II is formed by 136 Gly-Gly-Gly- Leu-Ser-Ser-Ser-Ala-Ser 144 and envelopes the phosphoryl moieties of the nucleotide. Finally, Motif III, as indicated in figure 6a, is formed by 343 Met- Thr-Gly-Gly-Gly-Phe-Gly-Gly 350. The backbone peptidic nitrogen of Gly 346 lies within 3 Å of a γ-phosphoryl oxygen of AMPPNP. There are three cis - prolines in human galactokinase at positions 85, 104, and Molecular genetics of GALK gene Worldwide prevalence of mutation in GALK gene Thus far ~25 mutations including base substitutions, base deletions and larger deletions have been identified in human galactokinase gene which resulted in the Type II galactosemia (Table 2) (20) Spectrum of mutation in GALK gene from India Recently in our laboratory we have established spectrum of mutations in GALK gene from galactokinase deficient Indian patients (21). This is the first available report in India. In this study, 32 unrelated GALK chromosomes were analyzed for the presence of known and unknown mutations in the GALK gene. Table 3 shows the mutations identified in the GALK gene from Type II galactosemia patients. A total of 5 different mutations were identified. These included 4 missense mutations (S79F, S79Y, F275Y and A384P) and one silent mutation (S205S). S79F, S79Y and A384P were present in the homozygous state while S205S and F275Y in heterozygous state. By SSCP, 8 of the 32 GALK chromosomes (25%) were identified as mutated chromosomes. Taken together, 5 mutations were identified in 5 out of 16 patients constituting around 31% of the Type II galactosemia population. All the mutations were novel except A384P exon 8 of GALK gene. Figure 8 shows the various mutations identified in GALK subjects with corresponding frequencies. Mutations S79F, S79Y and A384P were present in a homozygous state at a frequency of 6.25% each in our population while S205S and F275Y were present in a heterozygous state at a frequency of

16 16 Rajendra Prasad et al. Table 2. Human galactokinase mutations associated with Type II galactosemia[20]. Mutation Disease phenotype Functional consequences M1I Reduced blood enzyme activity Not tested. Presumed to abolish initiation codon. P28T V32M Very low or no detectable blood enzyme activity; formation of cataracts if galactose is not omitted from the diet No detectable blood enzyme activity; formation of cataracts if galactose is not omitted from the diet Protein insoluble on expression in E. coli Protein insoluble on expression in E. coli G36R No detectable blood enzyme activity Protein insoluble on expression in E. coli H44Y No detectable blood enzyme activity Reduction in kcat; increase in Km for both substrates R68C Low blood enzyme activity Small reductions in kcat and Km for galactose; increase in Km for ATP S131I Low blood enzyme activity Not tested G137C Low blood enzyme activity Not tested A198V R239Q Greater probability of cataracts in late middle age (50+) Reduced red blood cell enzyme activity No change greater than 1.5-fold in any parameter Reduced activity and lower thermal stability R256W Reduced blood enzyme activity Not tested T288M Reduced blood cell enzyme activity Protein insoluble on expression in E. coli T344M Reduced blood enzyme activity Not tested G346S No detectable blood enzyme activity kcat and Km for ATP reduced; Km for galactose unaffected G349S Reduced blood enzyme activity; no detectable blood enzyme activity kcat reduced; modest increase in Km for galactose; no change in Km for ATP A384P Low blood enzyme activity Protein insoluble on expression in E. coli 3.1%. Out of 16 patients with GALK deficiency, 4 were also found to have reduced GALT enzyme activity. Two of these 4 composite cases of Type I and Type II galactosemia were identified with the presence of N314D mutation in exon 10 of GALT gene along with GALK gene mutations, the final genotypes of these patients being S205S/U//N314D/U and A384P/A384P//N314D/N314D.

17 Biochemistry, molecular biology and molecular genetics of galactosemia 17 Table 3. GALK mutations identified and characterized in the Type II galactosemia patients. Total Chromosomes 32. S. No Mutation Exon Nucleotide Change Consequence 1 S79F 2 TCT TTT Serine to Detection method SSCP/DNA sequencing Phenylalanine 2 S79Y 2 TCT TAT Serine to Tyrosine SSCP/DNA sequencing 3 S205S 5 TCC TCA Silent SSCP/ DNA sequencing 4 F275Y 6 TTC TAC Phenylalanine to Serine SSCP DNA sequencing 5 A384P 8 GCC CCC Alanine to Proline SSCP DNA sequencing Figure 8. Different mutations in GALK subject and corresponding frequencies in percentage Clinical presentation and diagnosis The only consistent clinical finding in galactokinase deficiency is cataract. Other abnormalities have been described in individual patients that include macular deposits, mental retardation, complement deficiency, and

18 18 Rajendra Prasad et al. seizures with neurologic deterioration, these are most likely to be coincidental. Cataract is the result of osmotic phenomena caused by the accumulation of galactitol in the lens. The galactitol is synthesized from the reduction of galactose by aldose reductase. It has been suspected that this cataract formation is caused by hypergalactosemia due to the presence of a partial or complete enzyme deficiency in the galactose metabolic pathway, and/or high adult jejunal lactase activity, and/or high consumption of lactose. The need for assaying red blood cell galactokinase arises when deficiency of this enzyme is suspected on clinical grounds, i.e. in patients afflicted with a juvenile type of clouding of eye lenses Treatment Cataracts due to GALK deficiency may be reversible provided a galactose-free diet is started in early infancy. After a few months of age, permanent changes occurs to lens that cannot be corrected by diet UDP galactose-4-epimerase (GALE) deficiency galactosemia or type III galactosemia UDP-galactose-4-epimerase (GALE: EC ) deficiency galactosemia or Type III galactosemia (MIM# ) is an autosomal recessive disorder of galactose metabolism and is diagnosed in newborn screening by an increase in galactose or galactose-1-phosphate levels and a decrease in GALE enzyme activity (22). Epimerase deficiency galactosemia is the most poorly understood form of galactosemia. Which was First hypothesized by Kalckar (23), epimerase-deficiency galactosemia was originally described as a benign condition in which human GALE (hgale) impairment was restricted to the circulating red blood cells (RBC) and white blood cells Fibroblasts, liver, phytohemagglutinin (PHA)-stimulated leukocytes, and Epstein Barr virus (EBV) transformed lymphoblasts from these patients all demonstrated normal or near-normal levels of hgale activity, which lead to the designation of this condition as peripheral epimerase deficiency. Another form of epimerase deficiency galactosemia was identified in patients who, despite normal GALT activity, presented with symptoms reminiscent of classic galactosemia and demonstrated severely impaired GALE activity in both RBCs and fibroblasts. The patients, whose acute clinical symptoms responded to dietary restriction of galactose, was said to have generalized epimerase deficiency. Together, these reports supported the conclusion that epimerase deficiency galactosemia was a binary condition, with a benign peripheral form occurring at a population frequency of 1/6,700 to < 1/60,000, depending on the racial group.

19 Biochemistry, molecular biology and molecular genetics of galactosemia Biochemistry of GALE deficiency The main function of uridine diphosphate galactose 4-epimerase (GALE), is usually seen as being the last enzyme in the Leloir pathway is inverting the hydroxyl and hydrogen groups at the 4-carbon in the galactose molecule. The enzyme occurs widely in microorganisms and mammalian cells, human cells in particular. However, epimerase is essentially a reversible enzyme. This is important in situations in which galactose is not available and UDP-Gal cannot be synthesized via the Leloir pathway, because both UDP-Gal and UDP-Glc are necessary cofactors for the incorporation of galactose and glucose into essential complex polysaccharides. The chemical mechanism by which UDP-galactose-4 epimerase catalyzes the inter-conversion of UDP-galactose and UDP-glucose was worked out in the 1970s and has been reviewed (24). This mechanism is illustrated in figure 9. NAD + participates as a redox cofactor by reversibly and non-stereospecfically dehydrogenating carbon-4 in the pyranosyl rings of UDP-galactose and UDPglucose. The NAD/NADH transformation is itself stereospecific, with hydrogen transfer exclusively to and from the B-side. However, the substrates are nonstereospecifically oxidized and reduced by way of the UDP-4-ketohexopyranose intermediate. Strong binding of the UDP group and weak binding of the hexopyranosyl group allows hydride transfer between the pyridine nucleotide and glycosyl C-4 to take place nonstereospecifically. Nonstereospecificity requires weak binding of either the nicotinamide or the 4-ketosugar, so that hydride transfer to either face of the sugar can take place. Figure 9. The mechanism of the interconversion of UDP-galactose and UDP-glucose by UDP-galactose 4-epimerase (13).

20 20 Rajendra Prasad et al Molecular biology of the GALE gene The human GALE gene was mapped to chromosome 1 using somatic cell hybrids and fluorescent in situ hybridization has subsequently localized the gene more precisely to the short arm at 1p36 (Figure 10). The GALE cdna is 1488 bp in length and predicts a protein of 348 amino acids with a molecular weight of 38 kda. The gene consists of 11 exons extending over about 4 kb of genomic DNA. Both the yeast and E. coli enzymes are dimeric (125K and 79K, respectively) and composed of identical subunits. The 3-dimensional structure of E. coli epimerase is depicted in figure 13 by a ribbon diagram, showing the locations at which NAD and the inhibitor UDP- phenol are bound. NAD is bound to a typical Rossmann fold in the lower half of figure 11 that appears similar to the NAD binding domains of pyridine nucleotide dependent oxidoreductases. UDP-phenol binds to a smaller domain in the upper half of figure 11 that forms a cleft with the larger NAD domain. The uridine-5 -diphosphoryl portion of the inhibitor does not contact NAD, a fact that becomes significant in connection with chemical and spectroscopic observations (13). Figure 10. The GALE gene is located on the short (p) arm of chromosome 1 between positions 36 and 35. (Source: Human Gene Compendium,

21 Biochemistry, molecular biology and molecular genetics of galactosemia 21 Figure 11. A ribbon diagram of the polypeptide chain of UDP-galactose 4-epimerase Molecular genetics of GALE gene Till date, only 20 mutations have been identified in the GALE gene worldwide. Most mutations are individually rare, having been reported only on single alleles [24, 25]. With respect to UDP-Gal as substrate, the mutations associated with an almost complete loss of enzyme activity were V94M, G90E, and L183P. D103G, L313M, and N34S, on the other hand, demonstrated near-normal activity (12). No such report is available from Indian population Clinical presentation and diagnosis GALE deficiency inhibits UDP glucose regeneration preventing the formation of glucose-1-phosphate and leading to the accumulation of galactose and galactose-1-phsphate. GALE deficiency also perturbs glycolipid and glycoprotein biosynthesis due to the decreased production of UDP-GalNAc and UDP-GlcNAc. Symptoms of congenital Type III galactosemia includes- infantile jaundice, infantile hypotonia, dysmorphic features, sensorineural hearing loss, impaired growth, cognitive deficiencies, depletion of cerebellar purkinje cells, ovarian failure and hypertrophic hypogonadism, liver failure, splenomegaly, cataracts. Diagnosis for GALE deficiency galactosemia can be done by screening the suspected cases for deficiency of GALE enzyme followed by the GALE gene mutation studies.

22 22 Rajendra Prasad et al Treatment No treatment appears to be required for peripheral epimerase deficiency. Treatment for children with generalized epimerase deficiency, as for those with GALT deficiency, consists of restriction of dietary galactose. References 1. Von, Reuss, A. 1908, WIEN Med Wochenschr, 58: Leloir, L.F. 1951, Arch Biochem., 33: Barisic, K., Rumora. L., Grdic, M., Juretic, D. 2008, Croat Chem Acta., 1: Reichardt, J.K., Belmont, J., Levy, H., Woo, S. 1992a Genomics, 12: Murphy, M., McHugh, B., Tighe, O., Mayne, P., O Neill C., Naughten, E. 1997, Eur J Hum Genet., 7: Tyfield L, Reichardt J, Fridoviuch-Keil J, Croke DT, Elsas LJ, Strobl W. 1999, Hum Mutat., 13, Lai, K., Langley, S., Singh, R., Dembure, P., Hjelm. L., Elsas, L. 1996, J. Pediatr., 128, Goldstein, N., Cohen, Y., Pode-Shakked, B., Sigalov, E., Vilensky, B., Peleg, L. 2011, Mol. Genet. Metab., 102, Isselbache, K.J. 1957, Science, 126, Shin, Y., Niedermeier, H., Endres, W., Schaub, J., Weidinger, S. Clin. Chim. Acta. 1987, 166, Wehrli, S.L., Berry, G.T., Palmieri M., Mazur A., Elsas L., Segal S. 1997, Pediatr Res., 42, Holton, J.B., Walter, J.H., Tyfield, L.A. Galactosemia the Metabolic and Molecular Basis of Inherited Disease, 8th edn. 2001; New York: McGraw-Hill, Frey, P.A., 1996, FASEB J.,10, Elsas L, Dembure P, Langley S, Paulk E, Hjelm L, Fridovich-Keil J. A common mutation associated with the Duarte galactosemia allele. Am. J. Hum. Genet. 1994, 54, Sing, R., Thapa, B.R., Kaur, G., Prasad, R. 2012, Biochem. Genet., 50, Singh, R., Thapa,B.R., Kaur, G., Prasad, R. 2012, Clin. Chim. Acta.,414, Asada, M., Okano, Y., Imamura, T., Suyama, I., Hase, Y., Ishikki G. 1999, J. Hum. Genet., 44, Oki, K., Wada, Y.1988, Acta. Paediatr. Jpn. 30: Segal S, Berry G. Disorders of galactose metabolism. In: Scriver C, Beaudet A, Sly W, Valle D (eds) The metabolic and molecular bases of inherited disease, 7th edn, 1995; McGraw-Hill, New York, pp Thoden, J.B., Timson, D.J., Reece, R., Holden, H. 2005, Journal of Biological Chemistry, 280, Singh, R., Ram, J., Kaur, G., Prasad, R. 2012, Crr. Eye Res., 37, Park, H.D., Park, K., Kim, J., Shin, C., Yang, S., Lee, D.,H. 2005, Genet Med 7,646.

23 Biochemistry, molecular biology and molecular genetics of galactosemia Kalckar, H.,M. 1965, Science, 150, Frey, P.A. Complex pyridine nucleotide-dependent transformations. Pyridine Nucleotide Coenzyrnes: Chemical, Biochemical and Medical Aspects (Dolphin D, Poulson R, and Avramovic O. eds) vol. 2B, 1987; John Wiley & Sons, New York, pp Maceratesi, P., Daude, N., Dallapiccola, B., Novelli, G., Allen, R., Okano, Y. 1998, Mol. Genet. Metab., 63, 26.