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Biochemistry, Molecular Biology and Molecular Genetics of Genetic Disorders,

2013: 1-23 ISBN: 978-81-7895-609-1 Editor: Rajendra Prasad

1. Biochemistry, molecular biology and

molecular genetics of galactosemia
Rajendra Prasad1, Ramandeep Singh1 and Babu Ram Thapa2
1
Department of Biochemistry; 2Department 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 -160012, India. E-mail: fateh1977@yahoo.com
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 20thcentury, the defective gene was not discovered until 1956.
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
2.7.1.6; MIM# 230200), galactose-1-phosphate uridyl transferase (GALT; EC
2.7.7.10; MIM# 230400) and uridine diphosphate galactose-4-epimerase
(GALE; EC 5.1.3.2; MIM# 230350) (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
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 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 http://www.houssay.org.ar/hh/bio/leloir.htm.

2.2. 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
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 UDP-
glucose 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 Rajendra Prasad et al.

3.1. Galactose-1-phosphate uridyl transferase (GALT) deficiency

galactosemia or type I galactosemia
Type I Galactosemia, also called Classical galactosemia (OMIM #
230400) 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.

3.1.1. 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).
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.

3.1.2. 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 86-88 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-proline-
histidine-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, http://www.genecards.org).
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.

3.1.3. Molecular genetics of GALT gene

3.1.3.1. 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
Biochemistry, molecular biology and molecular genetics of galactosemia 9

at the following addresses: http://www.ich.bris.ac.uk/galtdb/,

http://www.cc.emory.edu/PEDIATRICS/medgen/research/db.htmand,
http://arup.utah.edu/database/ 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 Zn2+ and Fe2+ 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
Co2+, Mn+2+, or Cd2+ at both sites. The metal ions most likely participate in
maintaining the active conformation of the enzyme (13).
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.

3.1.3.2. 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).

3.1.3.3. 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 0.036 (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.
Biochemistry, molecular biology and molecular genetics of galactosemia 11

Table 1. GALT mutations identified and characterized in the Indian population. Total
chromosomes – 110.

Nucleotide Detection
S. No Mutation Exon Consequence
Change 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.

3.1.4. 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 Rajendra Prasad et al.

classical galactosemia, markedly elevated concentrations of galactitol are

detected in plasma as well as in urine.

3.1.5. 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.

3.1.6. 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.
Biochemistry, molecular biology and molecular genetics of galactosemia 13

3.2. 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.

3.2.1. 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, http://www.genecards.org).
14 Rajendra Prasad et al.

3.2.2. 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).
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-His44, abuts one side of the galactose moiety
where the side chain of Glu43 and the backbone peptidic nitrogen of His44
participate in hydrogen bonding interactions with the C-6 hydroxyl group of
the sugar. In human galactokinase, Motif II is formed by 136Gly-Gly-Gly-
Leu-Ser-Ser-Ser-Ala-Ser144 and envelopes the phosphoryl moieties of the
nucleotide. Finally, Motif III, as indicated in figure 6a, is formed by 343Met-
Thr-Gly-Gly-Gly-Phe-Gly-Gly350. The backbone peptidic nitrogen of Gly346
lies within 3 Å of a γ-phosphoryl oxygen of AMPPNP. There are three cis -
prolines in human galactokinase at positions 85, 104, and 121.

3.2.3. Molecular genetics of GALK gene

3.2.3.1. 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).

3.2.3.2. 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 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 Very low or no detectable blood Protein insoluble on expression in
enzyme activity; formation of cataracts E. coli
if galactose is not omitted from the
diet
V32M No detectable blood enzyme activity; Protein insoluble on expression in
formation of cataracts if galactose is E. coli
not omitted from the diet
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 Greater probability of cataracts in late No change greater than 1.5-fold in
middle age (50+) any parameter
R239Q Reduced red blood cell enzyme Reduced activity and lower
activity 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
G349S Reduced blood enzyme activity;
no detectable blood enzyme activity
A384P Low blood enzyme activity
kcat and Km for ATP reduced; Km
for galactose unaffected
kcat reduced; modest increase in
Km for galactose; no change in
Km for ATP
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.
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 Consequence Detection

Change method
1 S79F 2 TCT TTT Serine to SSCP/DNA
Phenylalanine sequencing
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 SSCP DNA
Serine sequencing
5 A384P 8 GCC CCC Alanine to Proline SSCP DNA
sequencing

Figure 8. Different mutations in GALK subject and corresponding frequencies in

percentage.

3.2.4. 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 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.

3.2.5. 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.

3.3. UDP galactose-4-Epimerase (GALE) deficiency galactosemia

or type III galactosemia
UDP-galactose-4-epimerase (GALE: EC 5.1.3.2) deficiency
galactosemia or Type III galactosemia (MIM# 230350) 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.
Biochemistry, molecular biology and molecular genetics of galactosemia 19

3.3.1. 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 UDP-
glucose. The NAD/NADH transformation is itself stereospecific, with hydrogen
transfer exclusively to and from the B-side. However, the substrates are non-
stereospecifically 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 Rajendra Prasad et al.

3.3.2. 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, http://www.genecards.org).
Biochemistry, molecular biology and molecular genetics of galactosemia 21

Figure 11. A ribbon diagram of the polypeptide chain of UDP-galactose 4-epimerase.

3.3.3. 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.

3.3.4. 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 Rajendra Prasad et al.

3.3.5. 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.

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