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

Figures & data

Figure 1. Schematic representation of GALE cDNA and protein. (a) Schematic representations of GALE cDNA and UDP-galactose 4-epimerase (GALE protein). GALE encodes a 348 amino acids (aa) protein containing multiple sites for NAD+ binding (blue) (aa from Gly12 to Ile14 (Gly12-Ile14), Asp33-Asn37, Asp66-Ile67, Phe88, Lys92, Lys161, and Tyr185), substrate UDP-glucose binding (green) (aa Ser132-Thr134, Tyr185-Asn187, Asn206-Leu208, Asn224-Phe226, Arg239, and Arg300-Asp304), and a proton acceptor site (yellow) (aa Tyr157). (b) Structural 3D representation of GALE (Rcsb:1ek6). The protein is colored gray, with NAD+ binding sites in light blue, UDP-glucose-binding sites in green, and the proton acceptor site in yellow. NADH molecule and UDP-glucose are represented in dark blue and light green, respectively. Image was created using ChimeraX 1.3 software.

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 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 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.

Table I. Molecular, clinical, and laboratory presentation of patients with GALE variants and generalized galactosemia.

Variant	Compound Het/Hom.	% of enzymatic activity vs. control	Syndromic manifestations	Hematological alterations			Bone marrow aspirate	Age at diagnosis	Functional characterization /in vitro models	Ref.
				RBC and Hb	WBC	Platelets
c.101A>G; p.Asn34Ser	Compound het. with p.Leu183Pro	0%	Delay in gross motor development, mild to moderate mental retardation. Transient hyperbilirubinemia. Galactosemia.	n/a	n/a	n/a	n/a	5 years	Reduced protein stability, increased proteolysis, slight kinetic impairment, normal dimerization.	[13–15]
c.151C>T; p.Arg51Trp	Hom	40%	Consanguineous family. Increased bleeding tendency (intracranial hemorrhages). No galactosemia, neither other systemic manifestation.	Mild anemia (RBC and Hb counts not reported)	Febrile neutropenia (neutrophils: 1.00 × 10^9/L)	Severe thrombocytopenia (P: 44 × 10^9/L). Big and pale platelets in blood film.	Normal Mk cellularity but dysplastic with hypolobated nuclei and hypogranularity	29, 19, 16, 24, 17 and 5 years	Impaired binding to NAD+, thermal instability, defects in megakaryocyte differentiation.	[16]
	Compound het. with p.Gly237Asp	Severely decreased	Mitral and tricuspid regurgitation, pyloric stenosis, recurrent infections, orbital cellulitis, and mild splenomegaly. Galactosemia.	Slight anemia (Hb: 9.2 g/dL)	Febrile leukopenia (WBC:1.9 x 10^9/L, neutrophils: 0.86× 10^9/L)	Severe thrombocytopenia (P: 13.9 x 10^9/L). Size not reported.	Increased myelomonocytic population with immature features, erythroid precursors with nuclear irregularities and megaloblastic changes. Megakaryocytic hyperplasia with small hypolobated forms.	2 years	Reduced NAD + cofactor binding, thermal protein instability.	[9]
c.230_231 insTGTT; p.Lys78ValfsX32	Compound het. with p.Thr150Met	15%	Mental retardation, mitral valve prolapse and mitral insufficiency, hyperbilirubinemia, hepatomegaly, retinal disease, cataracts, and severe bleeding tendency (mucocutaneous, epistaxis, minor wounds, conjunctival and vitrectomy, menorrhagia, surgery). One patient was splenectomized.	Slight anemia (Hb: 12.1 g/dL and 11.5 g/dL)	Slight leukopenia (WBC: 9.9 x 10^9/L and 3.5 x 10^9/L, neutrophils: 5.1 x 10^9/L and 1.6 x 10^9/L)	Severe macrothrombocytopenia (P: 12.7 x 10^9/L and 5.3 x 10^9/L, MPV: 17.4 fL and 19.2 fL, respectively) Grey and giant platelets in blood film.	Erythroid hyperplasia and increased Mks.	45 and 38 years	Severely reduced alpha and dense granules secretion. Impaired platelet aggregation with all agonists. Severely reduced/absent GPIb-IX-V complex and β1 integrin. Impaired proplatelet formation, impaired megakaryocyte and platelet glycosylation, and presence of apoptotic platelets.	[5]
280G>A; p.Val94Met	Hom	1–5%	Consanguineous family. Developmental delay, nerve deafness, moderate learning difficulties, vomiting, hypotonia, hyperbilirubinemia, and hepatomegaly. Galactosemia and galactosuria.	n/a	n/a	n/a	n/a	5 days	Severe kinetic impairment, normal dimerization and stability. Alteration in the active site dynamics: cofactor binding not stable.	[12,17–19]
c.382G>A; p.Val128Met	Compound het. with p.Leu223Pro	7%	Mental retardation, mitral valve prolapse and mitral insufficiency, hyperbilirubinemia, hepatomegaly, and bleeding tendency (Mucocutaneus and gastrointestinal).	Normal (Hb: 13 g/dL)	Normal (WBC: 8.8 x 10^9/L, neutrophils: 5.6 x 10^9/L)	Severe macrothrombocytopenia (P: 17 x 10^9/L, MPV: 20 fL). Giant platelets in blood film.	Not done	38 years	Severely reduced alpha and dense granules secretion. Impaired platelet aggregation with all agonists. Severely reduced/absent GPIb-IX-V complex and β1 integrin. Impaired proplatelet formation, impaired megakaryocyte and platelet glycosylation, and presence of apoptotic platelets.	[5]
c.449C>T; p.Thr150Met	Hom	40%	Growth retardation, hyperbilirubinemia, galactosemia and mild bleeding tendency (epistaxis and intermittent gingival bleeding). No recurrent or severe infections were diagnosed.	Mild macrocytic anemia (Hb: 11.2 g/dL)	Febrile leukopenia and lymphopenia (WBC: 4.78 x 10^9/L, neutrophils: 3.83 x 10^9/L, lymphocytes: 0.39 x 10^9/L)	Thrombocytopenia (P: 50 x 10^9/L). Size not reported.	Normocellular (90%) with mild dysmegakaryopoiesis and mild megaloblastic changes	17 years	No	[10]
	Compound het. with p.Lys78ValfsX32	15%	Mental retardation, mitral valve prolapse and mitral insufficiency, hyperbilirubinemia, hepatomegaly, retinal disease, cataracts, and severe bleeding tendency (mucocutaneous, epistaxis, minor wounds, conjunctival and vitrectomy, menorrhagia, surgery). One patient was splenectomized.	Slight anemia (12.1 g/dL and 11.5 g/dL)	Slight leukopenia (WBC: 9.9 x 10^9/L and 3.5 x 10^9/L, neutrophils: 5.1 x 10^9/L and 1.6 x 10^9/L)	Severe macrothrombocytopenia (P:12.7 x 10^9/L and 5.3 x 10^9/L, MPV: 17.4 fL and 19.2 fL, respectively) Grey and giant platelets in blood film.	Erythroid hyperplasia and increased Mks.	45 and 38 years	Severely reduced alpha and dense granules secretion. Impaired platelet aggregation with all agonists. Severely reduced/absent GPIb-IX-V complex and β1 integrin. Impaired proplatelet formation, impaired megakaryocyte and platelet glycosylation, and presence of apoptotic platelets	[5]
c.548T>C; p.Leu183Pro	Compound het. with p.Asn34Ser	0%	Delay in gross motor development, mild to moderate mental retardation. Transient hyperbilirubinemia. Galactosemia.	n/a	n/a	n/a	n/a	5 years	Reduced stability, severe proteolytic degradation, severe kinetic impairment, normal dimerization.	[13–15]
c.658C>T; p.Arg220Trp	Hom	30%	Galactosemia, galaonset hyperbilirubinemia, elevated liver transaminases, gastroesophageal reflux, and failure to thrive. Amino aciduria with echogenic kidneys and interstitial nephritis. Cardiac murmur and mild supravalvar pulmonary stenosis. Vitamin D deficiency. Hypotonia, brisk deep tendon reflexes, and language and gross motor delays. Recurrent infections.	n/a	n/a	n/a	n/a	5 days and 5 days (twins)	Mild kinetic impairment	[20]
c.668T>C; p.Leu223Pro	Compound het. with p.Val128Met	7%	Mental retardation, mitral valve prolapse and mitral insufficiency, hyperbilirubinemia, hepatomegaly, and bleeding tendency (Mucocutaneus and gastrointestinal).	Normal (Hb: 13 g/dL)	Normal (WBC: 8.8 x 10^9/L, neutrophils: 5.6 x 10^9/L)	Severe macrothrombocytopenia (P: 17 x 10^9/L, MPV: 20 fL). Giant platelets in blood film.	Not done	38 years	Giant platelets. Severely reduced alpha and dense granules secretion. Impaired platelet aggregation with all agonists. Severely reduced/absent GPIb-IX-V complex and β1 integrin. Impaired proplatelet formation, impaired megakaryocyte and platelet glycosylation, and presence of apoptotic platelets.	[5]
c.710G>A; p.Gly237Asp	Compound het. with p.Arg51Trp	Severely decreased	Mitral and tricuspid regurgitation, pyloric stenosis, recurrent infections, orbital cellulitis, and mild splenomegaly. Galactosemia.	Slight anemia (Hb: 9.2 g/dL)	Febrile leukopenia (WBC:1.9 x 10^9/L, neutrophils: 0.86× 10^9/L)	Severe thrombocytopenia (P: 13.9 x 10^9/L). Size not reported.	Increased myelomonocytic population with immature features, erythroid precursors with nuclear irregularities and megaloblastic changes. Megakaryocytic hyperplasia with small hypolobated forms.	2 years	Loss of catalytic site, altered substrate binding	[9]

Het: heterozygous variant; Hom: homozygous variant; % of Enzymatic Activity measured in red blood cells. RBC: red blood cells; Hb: hemoglobin; WBC: white blood cells; P: platelets; MPV: mean platelet volume. n/a: not reported.

Normal range: Hemoglobin: 12–16 g/dL, WBC: 3.8–11 x 10⁹/L, neutrophils: 1.4–6.5 x 10⁹/L, P: 150–450 x 10⁹/L, MPV: 7.2–11.1 fL.

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