GALE variants associated with syndromic manifestations, macrothrombocytopenia, bleeding, and platelet dysfunction

Abstract GALE gene encodes the uridine diphosphate [UDP]-galactose-4-epimerase, which catalyzes the bidirectional interconversion of UDP-glucose to UDP-galactose, and UDP-N-acetyl-glucosamine to UDP-N-acetyl-galactosamine. In that way, GALE balances, through reversible epimerization, the pool of four sugars that are essential during the biosynthesis of glycoproteins and glycolipids. GALE-related disorder presents an autosomal recessive inheritance pattern, and it is commonly associated with galactosemia. Peripheral galactosemia generally associates with non-generalized forms or even asymptomatic presentations, while classical galactosemia may be related to complications such as learning difficulties, developmental delay, cardiac failure, or dysmorphic features. Recently, GALE variants have been related to severe thrombocytopenia, pancytopenia, and in one patient, to myelodysplastic syndrome. Plain Language Summary What is the context? GALE gene encodes for the UDP-Galactose 4-Epimerase, an enzyme involved in the Leloir pathway of galactose catabolism and protein glycosylation. Homozygous or compound heterozygous GALE variants associate with the disorder known as galactosemia type III. Three types of galactosemia can be distinguished: the peripheral, the intermediate, and the generalized form, which associate with different clinical symptoms and GALE genetic variants. Peripheral form is considered benign, while the intermediate and the generalized form is associated with severe and syndromic manifestations, including learning difficulties, delayed growth, sensorineural hearing loss, and early-onset cataracts, among others. What is new? In the last few years, GALE variants have been linked to hematological manifestations, such as anemia, febrile neutropenia, and severe thrombocytopenia. To date, the only GALE variants described in patients presenting hematological disorders are GALE p.Arg51Trp, p.Lys78ValfsX32, p.Val128Met, p.Thr150Met, p.Leu223Pro, and p.Gly237Asp. The thrombocytopenia observed in GALE patients is associated with reduced GPIbα and β1 integrin glycosylation and externalization to the megakaryocyte and platelet surface, disrupting the actin cytoskeleton remodeling. What is the impact? GALE is an essential protein for the correct megakaryocyte and platelet glycosylation.


Plain Language Summary
What is the context?
• GALE gene encodes for the UDP-Galactose 4-Epimerase, an enzyme involved in the Leloir pathway of galactose catabolism and protein glycosylation.• Homozygous or compound heterozygous GALE variants associate with the disorder known as galactosemia type III.
• Three types of galactosemia can be distinguished: the peripheral, the intermediate, and the generalized form, which associate with different clinical symptoms and GALE genetic variants.• Peripheral form is considered benign, while the intermediate and the generalized form is associated with severe and syndromic manifestations, including learning difficulties, delayed growth, sensorineural hearing loss, and early-onset cataracts, among others.
What is new?
• In the last few years, GALE variants have been linked to hematological manifestations, such as anemia, febrile neutropenia, and severe thrombocytopenia.• To date, the only GALE variants described in patients presenting hematological disorders are GALE p.Arg51Trp, p.Lys78ValfsX32, p.Val128Met, p.Thr150Met, p.Leu223Pro, and p.Gly237Asp.• The thrombocytopenia observed in GALE patients is associated with reduced GPIbα and β1 integrin glycosylation and externalization to the megakaryocyte and platelet surface, disrupting the actin cytoskeleton remodeling.
What is the impact?
• GALE is an essential protein for the correct megakaryocyte and platelet glycosylation.606953; ENSG00000117308) is a ~ 5 kb gene with 11 exons mapped at chromosome 1p36.11,which encodes for 348 amino acids (aa) protein with a molecular weight of 38 kDa.GALE protein contains a NAD+ binding site, a substrate binding site, and a proton acceptor site (Figure 1).*On behalf of "Grupo Español de Alteraciones Plaquetarias Congénitas (GEAPC)", Sociedad Española de Trombosis y Hemostasia (SETH).# Equal authors′ contributions: JMB and JR share senior authorship.
Correspondence: José María Bastida, Unidad de Trombosis y Hemostasia, Hospital Universitario de Salamanca, Paseo de San Vicente, 58, Salamanca 37007, Spain.Email: jmbastida@saludcastillayleon.esThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The human enzyme UDP-Galactose 4-Epimerase is a homodimer of two monomers, and a member of the shortchain dehydrogenase/reductase (SDR) family.The catalytic mechanism requires a tightly bound NAD+ cofactor that transiently oxidizes the sugar in the proton acceptor site, and then it is reduced in a non-stereospecific form leading to NADH [1].
This enzyme catalyzes the reversible epimerization of UDPglucose (Glu) to UDP-galactose (Gal) and the reversible epimerization of UDP-N-acetylglucosamine (GluNAc) to UDP-N-acetylgalactosamine (GalNAc), by binding of any of the four molecules into the substrate binding site.The conversion of UDP-Gal is the final step in the Leloir pathway of galactose catabolism in which the galactose is converted to the glycolytic intermediate glucose 6-phosphate, thus, contributing to the catabolism of dietary galactose [2,3].
The crucial interconversions of all four of these UDP-hexoses also serve as sugar donors for glycosyltransferase enzymes, so GALE also influences the biosynthesis of glycoproteins and glycolipids in human cells [4].GALE mediates the protein glycosylation by providing the moieties into the branches during the N-and O-glycosylation in the endoplasmic reticulum (Figure 2), allowing the formation of mature and functional proteins [5].

Deficiency of UDP-galactose 4-epimerase: clinical and functional phenotype associated with genetic variants
Genetic variants in GALE gene leading to reduced expression or impaired enzyme activity are associated with the inherited metabolic disorder known as epimerase-deficiency galactosemia or galactosemia type III (OMIM number 230350).Three forms of this autosomal recessive disorder have been distinguished, associated with clinical phenotypes that range from nearly benign to life-threatening: the peripheral, the intermediate, and the generalized form.
Patients with the peripheral epimerase-deficiency galactosemia exhibit enzyme deficiency that is restricted to the circulating blood cells, and it is considered as clinically benign.It has been described as more frequent in some ethnic groups, such as African-Americans [6].The common phenotype of these patients is galactosemia, caused by the accumulation of galactose metabolites, and the treatment is mainly based on the restriction of lactose and derivatives.Nevertheless, patients with reduced enzymatic activity without galactosemia had also been described [7].Otherwise, the generalized form of galactosemia III associates with epimerase deficiency in all tissues, and it is more severe and less common than the peripheral form.Several patients worldwide have been described with syndromic manifestations that may include learning difficulties, delayed growth, sensorineural hearing loss, and early-onset cataracts, and, less frequently, cardiac failure and hepatomegaly [2,[8][9][10] (Figure 3).Despite the removal of lactose from diet, it is not sufficient to prevent long-term complications [11].In the last decade, some patients with an intermediate form of galactosemia have been reported some patients with an intermediate form, which present with syndromic manifestations, less marked than in the generalized form, due to an enzyme activity that is markedly deficient in circulating blood cells, but it is higher than 50% compare to normal levels in other cell types [12].
To date, around 30 variants in GALE have been described in patients, commonly in compound heterozygosis rather than homozygosis, with different phenotypes (Figure 4, Table 1).
a consanguineous family presenting galactosemia, developmental delay, moderate learning difficulties, and hepatomegaly among other clinical symptoms [17].The same variant was found in a homozygous state in two additional patients with similar clinical features [18].This variant does not change the overall stability but alters the active site dynamics causing that the substrate, NAD+ cofactor, and binding are less stable [19].Moreover, Timson DJ characterized several GALE variants in E. Coli, unraveling that the specific activity of the mutant protein p.Val94Met was nearly absent regarding UDP-galactose interconversion, and severely reduced to UDP-N-acetylgalactosamine [13].This study also demonstrated that variants p.Asn34Ser, p.Gly90Glu, p. Asp103Gly, and p.Leu183Pro were more susceptible to proteolysis than the wild-type protein [13].However, only p.Asn34Ser and p.Leu183Pro variants have been reported to associate with syndromic manifestations, such as delay in gross motor development and mild-to-moderate mental retardation [14,15] (Table 1, Figure 4).Of mention, none of the nine variants that have been characterized showed alterations in the enzyme dimerization [13].
Studies in 293T cell lines demonstrated that several GALE variants, such as p.Glu165Lys and p.Trp336*, lead to unstable proteins with reduced half-life and to the presence of aggregates  which are partly degraded by the proteasome complex.Moreover, variants p.Arg239Trp and Gly302Asp have no detectable enzyme activity.Authors stated that the altered protein stability is due to its misfolding, and the impaired enzymatic activity is responsible for the molecular defects underlying GALE deficiency [21].

Deficiency of UDP-galactose 4-epimerase and hematological characteristics
Few GALE variants had been associated with hematological alterations.The first evidence of GALE deficiency associated with hematological alterations was reported in 1995 by Rosoff PM, who described a 4-year-old girl with UDP-galactose 4-epimerase deficiency presenting with bruising, thrombocytopenia, and platelet function defect.Moreover, dysplastic cells in the peripheral film and bone marrow changes in the biopsy were observed, so the patient was diagnosed with a myelodysplastic syndrome (MDS).However, no molecular diagnosis was performed and the underlying GALE variants are unknown [22].The first association of inherited thrombocytopenia with molecular variants in GALE was reported in 2019, in 6 members from a consanguineous family carrying the variant p.Arg51Trp in homozygosis, all of them affected by anemia, febrile neutropenia, and severe thrombocytopenia, associating increased bleeding tendency, without symptoms of systemic galactosemia [16].One year later, the same variant was identified in compound heterozygosis with p.Gly237Asp in a patient with pancytopenia, cardiac problems, recurrent infections, and splenomegaly [9].Moreover, in 2021, it was reported a patient with mild macrocytic anemia, leukopenia, thrombocytopenia, mild epistaxis, and increased bilirubin levels, who carried the variant p.Thr150Met in homozygosis [10].
Of mention, none of the patients with pancytopenia and bone marrow dysplasia (Table 1) have so far developed MDS.However, this patient with p.Thr150Met in homozygosis presented mild megaloblastic changes, which could evolve in the future to SMD [10].
Very recently, we have identified the p.Thr150Met variant in compound heterozygosis with the novel one p.Lys78ValfsX32 in a non-consanguineous family affected with mental retardation, mitral valve prolapse, hyperbilirubinemia, and severe bleeding tendency and macrothrombocytopenia.One of the affected patients had slight leukopenia and mild anemia, while another one presented with erythroblast in blood film since the patient was splenectomized [5].In addition, we also characterized a second unrelated family with a similar phenotype, carrying the novel variants p.Val128Met and p.Leu223Pro.In this study, we unveiled that these GALE variants are associated with reduced GPIbα and β1 integrin glycosylation and externalization to the megakaryocyte and platelet surface, disrupting the F-actin and filamin A distribution in megakaryocytes, thus affecting the platelet production.Moreover, hypoglycosylated and unfunctional platelets were reported, which were prone to apoptosis, highlighting the key role of GALE in platelet glycosylation, production, and function [5].Similar to the p.Glu165Lys and p.Trp336* variants described by Bang YL, et al. [21], both p.Val128Met and p.Leu223Pro seem to have an unstable folding leading to reduced protein levels in platelets [5].Considering that the most GALE variants associated with the generalized forms of the disease are not found in the NAD+ or substrate binding sites (Figure 4), it is expected that the pathogenicity is due to an abnormal folding and function of the protein.
Overall, loss of GALE activity explains the abnormal glycosylation patterns also seen in some cell cultures and animal models of type III galactosemia [23].Disease-associated variants have lower activity than the wild-type in red blood cells, and the activity reduction in other tissues is generally greater in variants associated with severe forms of the disease [13,21,24] (Table 1).Although the role of GALE in galactose metabolism has been recognized for decades, the biochemical bases of pathophysiology and clinical variability in epimerase deficiency remain unclear, and the nature and severity of the symptoms depend upon a variety of factors that may include environmental factors, such as the dietary restriction of galactose.To date, the mechanisms associated with thrombocytopenia, platelet dysfunction, and  GALE variants causing syndromic thrombocytopenia 7 bleeding have been unraveled [5], though it is still unknown why some patients with serious syndromic disorders present hematologic disorders, while others do not.Considering the serious syndromic manifestations that patients present, an early diagnosis is essential for the correct treatment, including the withdrawal of dairy milk from the diet, and a periodic review with different medical specialists.

Figure 3 .
Figure 3. Schematic illustration of the clinical phenotype in patients with peripheral, intermediate, and generalized galactosemia III. in peripheral galactosemia, GALE dysfunction is restricted to the circulating blood cells, especially red blood cells, and the common clinical manifestation is galactosemia.In intermediate/generalized galactosemia, GALE dysfunction can be detected in multiple tissues, and therefore, patients present with syndromic manifestations, that may include neurological disorders, learning difficulties, delay growth, sensorineural hearing loss, early-onset cataracts, cardiac failure, hepatomegaly and hyperbilirubinemia, and pancytopenia associated with increased bleeding tendency.Image resource: Servier Medical Art (https://smart.servier.com/).

Figure 2 .
Figure 2. Role of the UDP-galactose 4-epimerase in O-linked and N-linked glycosylation.GALE protein allows the interconversion of UDP-glucose and UDP-galactose, and that of UDP-N-acetyl-galactosamine and UDP-N-acetyl-glucosamine.These four molecules are essential in the glycosylation process, by serving as substrates for other enzymes incorporating the carbohydrates of interest and releasing UDP.Branches of carbohydrates are essential in both N-glycosylation and O-glycosylation processes.

Figure 4 .
Figure 4. Variants associated to peripheral, intermediate, and generalized forms of the disease.Reported variants among the GALE protein.Variants marked in red indicate variants causing generalized forms of the disease associated with hematological manifestations.NAD+ binding is represented in blue, substrate UDP-glucose binding in green, and the proton acceptor site in yellow.