A mutation in mannose‐phosphate‐dolichol utilization defect 1 reveals clinical symptoms of congenital disorders of glycosylation type I and dystroglycanopathy

Abstract Congenital disorders of glycosylation type I (CDG‐I) are inborn errors of metabolism, generally characterized by multisystem clinical manifestations, including developmental delay, hepatopathy, hypotonia, and skin, skeletal, and neurological abnormalities. Among others, dolichol‐phosphate‐mannose (DPM) is the mannose donor for N‐glycosylation as well as O‐mannosylation. DOLK‐CDG, DPM1‐CDG, DPM2‐CDG, and DPM3‐CDG are defects in the DPM synthesis showing both CDG‐I abnormalities and reduced O‐mannosylation of alpha‐dystroglycan (αDG), which leads to muscular dystrophy‐dystroglycanopathy. Mannose‐phosphate‐dolichol utilization defect 1 (MPDU1) plays a role in the utilization of DPM. Here, we report two MPDU1‐CDG patients without skin involvement, but with massive dilatation of the biliary duct system and dystroglycanopathy characteristics including hypotonia, elevated creatine kinase, dilated cardiomyopathy, buphthalmos, and congenital glaucoma. Biochemical analyses revealed elevated disialotransferrin in serum, and analyses in fibroblasts showed shortened lipid linked oligosaccharides and DPM, and reduced O‐mannosylation of αDG. Thus, MPDU1‐CDG can be added to the list of disorders with overlapping biochemical and clinical abnormalities of CDG‐I and dystroglycanopathy. Synopsis Mannose‐phosphate‐dolichol utilization defect 1 patients can have overlapping biochemical and clinical abnormalities of congenital disorders of glycosylation type I and dystroglycanopathy.


| Subjects
Plasma and fibroblasts were obtained for CDG diagnostics in the Radboudumc Expertise Center for Disorders of Glycosylation in accordance with the Declaration of Helsinki. Informed consent was obtained from patients or their legal representatives. Written informed consent was obtained for inclusion of facial images.

| Clinical description
The two patients are brother and sister from Iraqi origin. They were the third and fourth child of consanguineous parents (first cousins), and had two healthy sisters ( Figure 1A). Table 1 summarizes the clinical features of these two patients and four previously reported MPDU1-CDG patients. [7][8][9] 3.1.1 | Patient 1 Patient P1, a girl, was born spontaneously in the 37th gestational week after an uneventful pregnancy. At birth, her length was 43 cm (standard deviation score, (SDS), −2.08), weight 2390 g (SDS, −0.81), and head circumference 32 cm. Because of postnatal apneas and bradycardias with deep desaturations, followed by respiratory failure, she was resuscitated and referred to the neonatal intensive care unit. Slight dysmorphic features were noted including a smooth philtrum, retrognathia, low-set, posterior-rotated ears, and hypertelorism with megalocorneae ( Figure 1B).
At the age of 2 months, P1 showed tonic-clonic seizures with multifocal sharp waves on electroencephalography (EEG). Brain magnetic resonance imaging (MRI) revealed enlarged outer fronto-temporo-parietal cerebrospinal fluid spaces. Abdominal sonography as well as magnetic resonance cholangiopancreatography showed a vast dilatation of the intrahepatic biliary ducts, especially within the left lobe of the liver ( Figure 1C). There were no further signs of biliary duct obstruction or liver enlargement. Initially, kidney sonography showed enhanced echogenicity of the marrow pyramids without corresponding MRI abnormalities. However, at the age of 7 weeks, small renal cysts were suspected in an ultrasound scan.
Initially, electrocardiography and echocardiography were normal, except for a mild temporary pulmonary hypertension. At the age of 3 months, the patient evolved DCM with low output and a shortening fraction of at least 9%.
The megalocorneae possessed a diameter of 13 mm (normal in newborn = 9.5 mm). Further ophthalmologic examination revealed a buphthalmos with congenital glaucoma. The eye lenses were slightly cloudy and pupillary iris showed pigment epithelial cysts. A sensorineural hearing loss was identified with brainstem evoked response audiometry (BERA) evaluation. There were no abnormalities of the skin.
Prometaphase karyotype analysis showed a normal 46,XX female karyotype and a normal array-comparative genomic hybridization (array-CGH), without any microdeletion or duplication. Congenital toxoplasmosis, other (syphilis, varicella-zoster, parvovirus B19), rubella, cytomegalovirus, and herpes (TORCH) infections, peroxisomal diseases as well  as mucopolysaccharidoses were ruled out by serology and metabolic screening. The serum amino acids, acylcarnitine profile, very long chain fatty acids, 7-dehydrocholesterol, and urine organic acids were normal. In view of the clinical symptoms of multiorgan involvement with seizures, cardiomyopathy, eye, and liver abnormalities as well as thrombocytopenia and ATIII deficiency, a CDG was suspected and TIEF and ESI-MS of serum transferrin were performed. These revealed reduced tetrasialotransferrin, with increased asialo-and disialotransferrin indicative of a CDG-I ( Figure 1D,E).
In her short life, the patient did not exhibit any psychomotor development, and had to be fed parenterally or by a gastric tube. The clinical course was complicated by an increasing frequency and severity of seizures with epileptic apneas followed by respiratory insufficiency. Congestive heart failure as a consequence of the DCM led to the termination of therapeutic interventions, and she died at the age of nearly 4 months.

| Patient 2
This patient exhibited nearly the same clinical features and course as his affected sister (P1). He was born premature per cesarean section in the 31st gestational week because of a rupture of the placental membrane and uterine bleeding. His birth length was 38 cm (SDS, −0.38), his weight was 1420 g (SDS, −0.09), and he had a head circumference of 28 cm. He showed a smooth philtrum, retrognathia, and low-set, posterior-rotated ears. He had hypertelorism with prominent appearing eyes with enlarged and cloudy corneae ( Figure 1F). Musculature was slightly hypotonic, but otherwise the child appeared to be normal and without skin abnormalities, except a micropenis. After birth, he was cyanotic, breathless, and without muscular tonus, requiring immediate cardiopulmonary resuscitation and controlled ventilation.
Echocardiography detected a dilated aorta ascendens, a transient pulmonary hypertension, and a hypertrophic cardiomyopathy (HCM) at the age of 3 weeks, which developed in combination with an arterial hypertension.
At the age of 6 months, he had generalized seizures accompanied by desaturations, whereas EEG showed parieto-temporooccipital and multiregional spikes over both hemispheres. Sonography of the brain showed no gross abnormalities. Abdominal sonography revealed dilatation of the intrahepatic biliary duct system ( Figure 1G). Kidney sonography exhibited enhanced echogenicity of the marrow pyramids. At the age of 4 months, he developed multiple small cysts subcapsular within the renal parenchyma.
The ophthalmological examination revealed a buphthalmos with slightly opaque corneae with a diameter of 11 mm and severe congenital glaucoma, requiring prompt trabeculectomy intervention. The BERA showed a sensorineural hearing loss.
Like his sister, P2 hardly presented any psychomotor development, and had to be fed parenterally or by a gastric tube. He showed severe apneas and bradycardias which were not treatable, and he died at the age of 11 months from respiratory failure.

| Genetic analysis revealed a homozygous missense mutation in MPDU1
Whole exome sequencing revealed a homozygous missense mutation Chr17(GRCh38): g.7585994G>A; NM_004870.3 (MPDU1): c.218G>A; p.(G73E) in both patients. This mutation has been reported in two other MPDU1-CDG patients. 7,9 WES was also performed of the patients' healthy sisters and parents and confirmed parental segregation of the mutation ( Figure 1A).

| Biochemical analysis revealed elevated LLO intermediates, DPM, and reduced Omannosylation of αDG
Next, we analyzed the LLO composition in fibroblasts from patient 1. Cells were incubated with [2-3 H]mannose and [ 3 H]oligosaccharides were extracted and analyzed using HPLC. Increased levels of dolichol-linked Man 5 GlcNAc 2 and Man 9 GlcNAc 2 accompanied by reduced amounts of Glc 3 Man 9 GlcNAc 2 were detected ( Figure 2A) which indicated shortage of DPM and DPG in the ER lumen. Since TLC analysis further showed that DPM is synthesized in the patient's fibroblasts ( Figure 2B), a lack of transport of DPM from the cytosol into the ER can be assumed.
Subsequently, we analyzed the O-mannosylation of αDG in fibroblasts of patient P1 by IIH6 immunolabeling and a LO assay. We found that the signals for both IIH6 and LO were reduced in patient fibroblasts as compared to the signal of control fibroblasts ( Figure 2C), suggesting reduced glycosylation of αDG.

| DISCUSSION
Here, we describe two patients with mutations in MPDU1, causing MPDU1-CDG (CDG-1f) with overlapping symptoms of CDG-I and dystroglycanopathy. MPDU1-CDG is the fifth disorder related to DPM biosynthesis or utilization that bridges CDG-I and the O-mannosylation disorders.
So far, seven MPDU1-CDG patients have been described. All patients showed psychomotor retardation and most patients had hypotonia, facial dysmorphism, eye defects, apnea, and skin abnormalities such as ichthyosis. [7][8][9] Including the two patients described here, four patients have been described with the same G73E substitution. 7,9 Hypertelorism, dysphagia, small renal cysts, thrombocytopenia, cardiomyopathy, and respiratory problems characterized these patients, whereas these symptoms were not reported in the other three MPDU1-CDG patients. In addition, all G73E patients died at an early age (<11 months), whereas the other MPDU1-CDG patients at least reached their teenage years. Taken together, this suggests that the G73E substitution affects MPDU1 function more severely, and the additional clinical features can aid in the prediction of the disease progression when new MPDU1-CDG patients are identified.
Interestingly, the two siblings described here showed a very similar clinical pattern, including very similar facial features, whereas siblings with other CDG disorders do not necessarily share as many clinical characteristics, for example, in ALG3-CDG. 16 The MPDU1-CDG siblings shared the following abnormalities: massive dilatation of the intrahepatic biliary duct system, small renal cysts, buphthalmos with glaucoma, DCM, thrombocytopenia, elevated CK, and low ATIII. MPDU1 has been associated with the flipping of DPM over the ER membrane and thereby is part of the DPM biosynthesis defects. 6 DPM is required for multiple glycosylation pathways. Thus, different symptoms can be caused by dysfunction of different glycosylation pathways. Buphthalmos, glaucoma, DCM, and elevated CK are clinical features that overlap with the disease spectrum of the dystroglycanopathies. Buphthalmos has so far not been described in any of the other DPM disorders, but is associated with Walker-Warburg syndrome (WWS) and muscle-eye-brain (MEB) disease, which are severe variants of dystroglycanopathy. 17 Glaucoma is a common feature of WWS and MEB, and has also been reported in SRD5A3-CDG. 17,18 Unfortunately, the role of glycosylation in cardiac disease is not completely understood. Therefore, there is no clear explanation why patient 1 shows DCM, whereas patient 2 developed HCM. HCM has been reported in PMM2-CDG, ATP6V1A-CDG, and ATP6V1E1-CDG. 19 Recent studies have shown that abnormal glycosylation, for example, abnormal sialylation or reduced hybrid/complex N-glycosylation, is related to heart disease. [19][20][21] However, future investigations are required to understand the clinical relevance of these findings. So far, DCM has been described in FKRP-CDG, FKTN-CDG, POMT1-CDG, POMT2-CDG, DOLK-CDG, DPM3-CDG, and PGM1-CDG. 19,22 With the exception of PGM1-CDG, these CDGs are associated with abnormal O-mannosylation of αDG. Hence, the cardiac pathomechanism in MPDU1-CDG patient 1 could be related to abnormal O-mannosylation, although studies in heart biopsies are warranted to study this. In line with the clinical symptoms overlapping with dystroglycanopathies, we showed reduced O-mannosylation of αDG in patient fibroblasts.
In addition, we reported biliary duct abnormalities and renal cysts in our MPDU1-CDG patients. Biliary duct abnormalities have also been observed in patients with mutations in mannose-6-phosphate isomerase (MPI), which interconverts fructose-6-phosphate to mannose-6-phosphate. 23 Sabry et al 24 reported a dehydrodolichyl diphosphate synthase (DHDDS-CDG) patient with dilations of the biliary duct and renal failure, and Schenk et al 7 reported renal cysts in a MPDU1-CDG patient with the same G73E substitution. Biliary duct abnormalities and renal cysts are clinical symptoms associated with Caroli syndrome. In some cases, this syndrome has been associated with PKHD1 mutations, which are also known to cause autosomal recessive polycystic kidney disease. PKHD1 is extensively glycosylated, 25 and it is tempting to speculate that abnormal glycosylation of PKHD1 causes the biliary duct abnormalities and renal cysts.
In summary, we reported on two newly identified MPDU1-CDG patients and showed reduced N-glycosylation and O-mannosylation in patient material. Together with the muscular, eye, and heart abnormalities, this adds MPDU1-CDG to the list of DPM disorders causing biochemical and clinical abnormalities overlapping CDG-I and dystroglycanopathy. Identification of additional patients with defects in DPM availability, and extensive O-mannosylation and N-glycosylation analysis of patient material is required to further increase our understanding of the pathophysiology of the DPM disorders.