B4GALT1-CDG: Expansion of the phenotypic and molecular spectrum and review of the literature

Abstract

A congenital disorder of glycosylation due to biallelic mutations in B4GALT1 has been previously reported in only three patients with two different mutations. Through homozygosity mapping followed by segregation analysis in an extended pedigree, we identified three additional patients homozygous for a novel mutation in B4GALT1, expanding the phenotypic spectrum of the disease. The patients showed a uniform clinical presentation with intellectual disability, marked pancytopenia requiring chronic management, and novel features including pulmonary hypertension and nephrotic syndrome. Notably, affected individuals exhibited a moderate elevation of Man3GlcNAc4Fuc1 on serum N-glycan analysis, yet two of the patients had a normal pattern of transferrin glycosylation in repeated analysis. The novel mutation is the third disease-causing variant described in B4GALT1, and the first one within its transmembrane domain.

Graphical Abstract

Introduction

Congenital disorders of glycosylation (CDG) are a growing group of rare, multisystem, underdiagnosed heterogeneous diseases caused by deficient or improper synthesis or attachment of glycans to proteins and lipids¹. β-1,4-galactosyltransferase (β4GalT1, E.C.2.4.1.38) is the major enzyme responsible for the transfer of galactose residues from UDP-galactose to the terminal N-acetylglucosamine residues in glycoproteins processed in the Golgi apparatus². Deficiency of this Golgi glycosyltransferase leads to B4GALT1-CDG, caused by mutations in B4GALT1. So far, three patients with this disease have been reported, with only two mutations published to date; NM_001497.3(B4GALT1): c.1031dupC, p.(Arg345fs)^2,3 and c.579C>G, p.(Y193X)⁴. All patients had shared clinical features including hypotonia, coagulopathy, elevated serum transaminases, and a biochemical type 2 pattern on serum transferrin isoform analysis^2,3,5,6. Here, we present three new cases of B4GALT1-CDG, homozygous for a novel mutation within the B4GALT1 transmembrane domain and sharing the same extended phenotype. Notably, two of them had a normal transferrin isoform analysis.

Methods

The study was approved by Soroka Medical Center institutional review board (approval #5071G). DNA samples were obtained following written informed consent.

Whole exome sequencing (WES) was performed for patient VI-1 and his parents (V2 and V1, Figure 1A) with overall >100X coverage depth >90% covered >20X. Genome-wide homozygosity mapping of two affected individuals (VI-1, VI-4), their two healthy siblings and both parents (Figure 1A) was performed. Detailed genetics and glycosylation methods – in supplement.

Figure 1. Disease phenotype:

(A) Pedigree of the consanguineous Bedouin kindred studied. (B) Patient A1’s brain MRI, age 6 years. No pathologic findings were seen except for bilateral choroid plexus xantogranulomas. (C) Homozygosity-Mapper plot. (D) Patient A2’s brain MRI, age 17 months; no pathologic findings except signs of past bleeding. (E) Multiple sequence alignment of selected B4GALT1 orthologues. The p.(R185W) missense mutation affects a highly conserved positive residue and dramatically changes its polarity, within the predicted transmembrane region of the protein. (F) Sanger sequencing. (G) Domain analysis of B4GALT1 with known and novel variants marked.

Results

Clinical studies

Three affected individuals from a large inbred Bedouin Israëli family were studied (Figure 1A). Table 1 describes the clinical features of the affected patients.

Table 1.

Clinical features of our three patients diagnosed with B4GALT1-CDG and review of the literature.

Patient	Family A		Family B	Patient 1 Peters et al; Hanßke et al; (Guillard et al)	Patient 2 Guillard et al	Patient 3 Pérez-Cerdá et al (Medrano et al)
	A1	A2	B
Age at presentation	Birth	Birth	Birth	Birth	NA	4m (diagnosis)
Gestational age at birth	Term	Preterm 35 weeks	Preterm 34 weeks	Term	Term	Premature
Gastrointestinal	Hepatosplenomegaly with transient cholestatic jaundice treated with ursodeoxycholic acid.	-	Hepatosplenomegaly with transient cholestatic jaundice treated with ursodeoxycholic acid.	Transient cholestasis. Persistently elevated elevated serum aspartate aminotransferase/transaminases.	Mild hepatomegaly. Recurrent diarrhea. Elevated serum aspartate aminotransferase/transaminases.	Elevated serum aspartate aminotransferase/transaminases.
Respiratory	Severe pulmonary hypertension necessitated aggressive treatment for 4 days. No sequela.	Severe pulmonary hypertension necessitated aggressive treatment for 9 days. No sequela.	Severe pulmonary hypertension necessitated assisted ventilation for the first week of life. No sequela.	-	-	-
Central Nervous System	Motor and language developmental delay. Moderate global intellectual disability. Marked hypotonia. Refractory epilepsy. Brain imaging presented in Figure 1.	Motor and language developmental delay. Moderate global intellectual disability. Marked hypotonia. Refractory epilepsy. Brain imaging presented in Figure 2.	Motor and language developmental delay. Moderate-severe global intellectual disability. Movement disorder consistent of choreoathetosis and dystonia. High muscle tone Refractory epilepsy.	Macrocephaly due to hydrocephalus. Dandy-Walker malformation. Marked muscular hypotonia that improved over time. Myopathic EMG.	Transient axial hypotonia.	Hypotonia.
Hematology	Bone marrow failure with pancytopenia.	Bone marrow failure with pancytopenia. Single episode of hemolytic anemia.	Thrombocytopenia.	Decreased clotting factors.	Decreased colotting factors. Borderline thrombocytopenia.	Decreased clotting factors.
Nephrology	-	Transient nephrotic syndrome resistant to steroid treatment. Renal biopsy: membranoproliferative glomerulonephritis.	-	-	-	-
Other	Mild facial dysmorphism: prominent maxillary incisors.	-	Mild facial dysmorphism: prominent maxillary incisors.	Mild facial dysmorphism: broad nose, low-set ears. Myopia	Facial dysmorphism: hypertelorism, broad nasal bridge, long philtrum, thin upper lip, low-set ears. Myopia.	Facial dysmorphism. Inverted nipples. Dermatitis. Hypothyroidism
Laboratory investigation	Recurrent bone marrow aspiration, urine organic acids, plasma amino acids and acylcarnitines were normal. Normal TIEF	Diagnosed prenatally by amniocenthesis. Normal TIEF	Recurrent bone marrow aspirations, urine organic acids, plasma amino acids and acylcarnitines were normal.	Normal neonatal screening including normal acylcarnitines, organic and amino acids in urine. Elevated creatine kinase. Type 2 TIEF.	Type 2 TIEF.	Elevated creatine kinase. Type 2 TIEF.
Genetic diagnosis	NM_001497.3 (B4GALT1): c.61C>T, p.(Arg21Trp)	NM_001497.3 (B4GALT1): c.61C>T, p.(Arg21Trp)	NM_001497.3 (B4GALT1): c.61C>T, p.(Arg21Trp)	NM_001497.3 (B4GALT1): c.1031dupC, p.(Arg345fs)	NM_001497.3 (B4GALT1): c.1031dupC, p.(Arg345fs)	NM_001497.3 (B4GALT1): c.579C>G, p.(Y193X)

Abbreviations: NA-not available; TIEF-transferrin isoelectric focusing; EMG-electromyography

Patient A-1 (born at term, 4025g), presented at birth with marked hypotonia, hepatosplenomegaly, severe pulmonary hypertension, cholestatic jaundice and severe pancytopenia. Pulmonary hypertension and jaundice resolved within a month. Bone marrow failure persisted, with severe neutropenia treated with granulocyte colony stimulating factor (G-CSF) and thrombocytopenia requiring weekly treatment with romiplostim (thrombopoietin analogue). Bone marrow biopsy demonstrated grade 2 reticular fibrosis. Seizures began at age 15 months. EEG at 5 years consisted of diffuse epileptiform discharges. At 6 months, marked developmental delay, hypotonia and lack of age-appropriate milestones were noted. Independent walking ensued at 3.5 years. Currently (age 11 years) he is seizure-free on three antiepileptic drugs, has moderate intellectual disability and behavioral problems. Head circumference is at the 50th percentile, with mild dysmorphism with prominent maxillary incisors and an open mouth. He has ataxic gait, is hardly able to lift his feet, exhibiting dysmetria and mild hand tremor when reaching out. Muscle tone is low with intact strength. Brain MRI was normal at 2 years of age, yet at 6 years disclosed choroid plexus xantogranulomas in the lateral ventricles with no parenchymal changes (Figure 1B). Urine organic acids, plasma amino acids and acylcarnitines were normal.

Patient A-2 (born 35 weeks, 2355g) presented at birth with hypotonia, pancytopenia and severe pulmonary hypertension; the latter resolved within the first month of life. Bone marrow failure continued, requiring chronic G-CSF and weekly treatment with romiplostim. Bone marrow biopsy was normal. At age 4 months, he presented with an episode of hemolytic anemia of unknown etiology that resolved with immunoglobulins, and developed steroid-resistant nephrotic syndrome that resolved at one year. Viral panel and parvovirus B19 testing, toxoplasmosis, hepatitis A, B, and C serology, complement activity and ADAMTS13 levels, were normal. Renal biopsy demonstrated membranoproliferative glomerulonephritis (MPGN). Seizures began at age 13 months. EEG showed bursts of diffuse spike/polyspike and wave with or without a myoclonic jerk correlate on video. He is currently (age 2.5 years) on three antiepileptic drugs without full control of seizures. Developmental delay was noted at age of 2 months. At 2 years his development was equivalent to that of a 6-month-old, with poor communication skills. Physical examination revealed an open mouth with drooling, head circumference ~25th percentile, poor eye contact, normal eye movements, low muscle tone and brisk deep tendon reflexes with a spread and cross adduction, bilateral clonus, and flexor plantar reflexes bilaterally. Brain MRI (18 months) was normal except for signs of remote bleeding (Figure 1D). Notably, two transferrin isoelectric focusing analyses of both siblings performed ages 2–11 years demonstrated a normal pattern of transferrin isoforms. Transferrin and apolipoprotein CIII analyses by LC/MS were normal in patients A-1 and A-2, and their parents (not shown). Serum total N-glycan analysis revealed moderate elevation of Man3GlcNAc4Fuc1, m/z 1836) in both affected siblings; both parents had a normal profile (Figure 2).

Figure 2. Glycosylation studies:

Serum total N-glycan analysis for patients A-1 (A), A-2 (B), their mother (C) and father (D). Moderate elevation of Man3GlcNAc4Fuc1 (m/z 1836) evident in both affected individuals.

Patient B1 (born 34 weeks, 2150g) presented at birth with marked hypotonia, severe pancytopenia, hepatosplenomegaly, cholestatic jaundice and severe pulmonary hypertension during the first week of life. Developmental delay was noted at 4 months, with marked hypotonia. At age 11 years he remains wheelchair bound, has moderate to severe intellectual disability, and a complex movement disorder including chorea, athetosis and dystonia. He has mild dysmorphism (prominent widely spaced maxillary incisors), drooling, increased limb muscle tone with decreased truncal tone and muscle wasting. Deep tendon reflexes are brisk with a spread and cross adduction, bilateral clonus, and flexor plantar reflexes. Seizures began at 2 years of age. At age 11 years he is still having infrequent seizures despite polypharmacy. Brain MRI (22 months) showed mild ventriculomegaly with periventricular nonspecific white matter changes. He continues to suffer from thrombocytopenia, treated with hexacaprone and platelets during acute bleeding episodes. Repeated bone marrow aspirations, urine organic acids, plasma amino acids and acylcarnitines tests were normal.

Genetic analysis

Homozygosity mapping (Figure 1C) with WES trio analysis (patient A-1 and parents) identified three loci (Table s.1), harboring only three predicted deleterious homozygous variants (Table s.2), segregating as expected for recessive heredity in B4GALT1, GNE, OTOG; (Figure 1F, s.1). The homozygous c.61C>T, p.(Arg21Trp) variant (rs1065764) in B4GALT1 (NM_001497.3) is rare (no homozygous individuals in gnomAD⁷; allele count 3:253,694) and was not found in 236 ethnically-matched exomes. This variant, predicted by several algorithms to be probably damaging, affects an evolutionary conserved amino acid (Figure 1E), predicted by SMART sequence analysis⁸ to constitute the first amino acid within B4GALT’s transmembrane region (Figure 1E,G).

Discussion

Genetic studies of the affected kindred identified a disease-associated B4GALT1 mutation and 2 co-segregating variants: OTOG variant that was evidently non-pathogenic as none of the individuals had hearing loss; and a previously-described pathogenic GNE (NM_001128227.2; c.1676T>G) mutation (Table s.2), known to cause a progressive myopathy that typically presents in early adulthood^9,10. The patients were pre-symptomatic for the myopathy. The B4GALT1 p.(Arg21Trp) variant is predicted to dramatically affect the protein’s structure and function. Serum total N-glycan analysis revealed moderate elevation of Man3GlcNAc4Fuc1, a glycan produced by fucosylation of Man3GlcNAc4 by fucosyltransferase 8.¹¹

B4GALT1-CDG is a multisystem disease characterized by central nervous system (CNS) involvement, coagulopathy, and liver disease. Until now, only three patients with B4GALT1-CDG had been reported, mainly characterized by marked hypotonia with myopathic features, resolving over time (Table 1). Two of the previously described patients exhibited normal development at 9 and 11 years of age³ (Table 1). The combination of CNS and hepatocellular liver disease is common in CDG. However, while two of our patients presented at birth with prolonged cholestatic jaundice that resolved within the first year of life, none of them had hepatocellular disease at any point in time.

β4-galactosylation of glycoproteins is important for protein conformation, stability, transport and clearance from circulation. Therefore, deficiency of β4GalT1 would lead to aberrant glycosylation and different disease states. IgA nephropathy is the most common form of glomerulonephritis worldwide.¹² Focal and diffuse mesangial proliferative glomerulonephritis are the most common reported histologic lesions in renal biopsies from patients with IgA nephropathy^13,14, although in some cases an MPGN-like pattern is seen.^15,16 Deficiency of β4GalT1 in mice leads to complete absence of β4-galactosylation of N-glycans in serum IgA.¹² Additionally, β4GalT1 is known to play an important role in the clearance and initial deposition of IgA.¹⁷ Immunofluorescence on renal biopsy from one of our patients was not optimal; however, a few deposits were suspected to represent IgA. Thus, it is tempting to suggest that the nephropathy seen in patient A-2 was related to his underlying diagnosis of B4GALT1-CDG.

Decreased coagulation factors were described in the three previously reported patients, with borderline thrombocytopenia in one. However, this is the first description of severe pancytopenia that requires treatment for all cell lines, with profound thrombocytopenia that required treatment with recurrent blood product transfusions.

Persistent pulmonary hypertension of the newborn (PPHN), to our knowledge, has not been described so far as part of other glycosylation defects. All three patients had severe PPHN, which resolved with no residual pulmonary disease - possibly a consequence of their neonatal anemia.¹⁸

In summary, we report three patients homozygous for the same B4GALT1 mutation, presenting with PPHN, cholestasis, severe pancytopenia and significant neurologic involvement, with nephrotic syndrome in one of the patients. The novel B4GALT1 mutation is the third described to date and the first one within its transmembrane domain, representing a founder mutation in the Bedouin population.