Association between acute complications in PMM2-CDG patients and haemostasis anomalies: Data from a multicentric study and suggestions for acute management.

OBJECTIVES: Patients with PMM2-CDG develop acute events (stroke-like episodes (SLEs), thromboses, haemorrhages, seizures, migraines) associated with both clotting factors (factor XI) and coagulation inhibitors (antithrombin, protein C and protein S) deficiencies. The aim of the study was to correlate acute events to haemostasis and propose practical guidelines. METHODS: In this multicentric retrospective study, we evaluated clinical, radiological, haemostasis and electroencephalography data for PMM2-CDG patients hospitalized for acute events. Cerebral events were classified as thrombosis, haemorrhage, SLE, or "stroke mimic" (SM: normal brain imaging or evoking a migraine). RESULTS: Thirteen patients had a total of 31 acute episodes: 27 cerebral events with 7 SLEs, 4 venous thromboses, 4 haemorrhages (3 associated with thrombosis), 15 SMs at a mean age of 7.7 years; 4 non-cerebral thromboses, one of which included bleeding. A trigger was frequently involved (infection, head trauma). Although sometimes normal at baseline state, factor XI, antithrombin and protein C levels decreased during these episodes. No correlation between haemostasis anomalies and type of acute event was found. DISCUSSION: Acute events in PMM2-CDG are not negligible and are associated with haemostasis anomalies. An emergency protocol is proposed for their prevention and treatment (https://www.filiere-g2m.fr/urgences). For cerebral events, brain Magnetic Resonance Imaging with perfusion weight imaging and diffusion sequences, electroencephalogram and haemostasis protein levels guide the treatment: anticoagulation, antithrombin or fresh frozen plasma supplementation, antiepileptic therapy. Preventing bleeding and thrombosis is required in cases of surgery, prolonged immobilization, hormone replacement therapy. CONCLUSION: Acute events in PMM2-CDG are associated with abnormal haemostasis, requiring practical guidance.

A mutation in SLC37A4 causes a dominantly inherited congenital disorder of glycosylation characterized by liver dysfunction.

SLC37A4 encodes an endoplasmic reticulum (ER)-localized multitransmembrane protein required for transporting glucose-6-phosphate (Glc-6P) into the ER. Once transported into the ER, Glc-6P is subsequently hydrolyzed by tissue-specific phosphatases to glucose and inorganic phosphate during times of glucose depletion. Pathogenic variants in SLC37A4 cause an established recessive disorder known as glycogen storage disorder 1b characterized by liver and kidney dysfunction with neutropenia. We report seven individuals who presented with liver dysfunction multifactorial coagulation deficiency and cardiac issues and were heterozygous for the same variant, c.1267C>T (p.Arg423∗), in SLC37A4; the affected individuals were from four unrelated families. Serum samples from affected individuals showed profound accumulation of both high mannose and hybrid type N-glycans, while N-glycans in fibroblasts and undifferentiated iPSC were normal. Due to the liver-specific nature of this disorder, we generated a CRISPR base-edited hepatoma cell line harboring the c.1267C>T (p.Arg423∗) variant. These cells replicated the secreted abnormalities seen in serum N-glycosylation, and a portion of the mutant protein appears to relocate to a distinct, non-Golgi compartment, possibly ER exit sites. These cells also show a gene dosage-dependent alteration in the Golgi morphology and reduced intraluminal pH that may account for the altered glycosylation. In summary, we identify a recurrent mutation in SLC37A4 that causes a dominantly inherited congenital disorder of glycosylation characterized by coagulopathy and liver dysfunction with abnormal serum N-glycans.

Novel variants and clinical symptoms in four new ALG3-CDG patients, review of the literature, and identification of AAGRP-ALG3 as a novel ALG3 variant with alanine and glycine-rich N-terminus.

ALG3-CDG is one of the very rare types of congenital disorder of glycosylation (CDG) caused by variants in the ER-mannosyltransferase ALG3. Here, we summarize the clinical, biochemical, and genetic data of four new ALG3-CDG patients, who were identified by a type I pattern of serum transferrin and the accumulation of Man5 GlcNAc2 -PP-dolichol in LLO analysis. Additional clinical symptoms observed in our patients comprise sensorineural hearing loss, right-descending aorta, obstructive cardiomyopathy, macroglossia, and muscular hypertonia. We add four new biochemically confirmed variants to the list of ALG3-CDG inducing variants: c.350G>C (p.R117P), c.1263G>A (p.W421*), c.1037A>G (p.N346S), and the intron variant c.296+4A>G. Furthermore, in Patient 1 an additional open-reading frame of 141 bp (AAGRP) in the coding region of ALG3 was identified. Additionally, we show that control cells synthesize, to a minor degree, a hybrid protein composed of the polypeptide AAGRP and ALG3 (AAGRP-ALG3), while in Patient 1 expression of this hybrid protein is significantly increased due to the homozygous variant c.160_196del (g.165C>T). By reviewing the literature and combining our findings with previously published data, we further expand the knowledge of this rare glycosylation defect.

Mutations in SLC2A2 gene reveal hGLUT2 function in pancreatic ß cell development.

The structure-function relationships of sugar transporter-receptor hGLUT2 coded by SLC2A2 and their impact on insulin secretion and ß cell differentiation were investigated through the detailed characterization of a panel of mutations along the protein. We studied naturally occurring SLC2A2 variants or mutants: two single-nucleotide polymorphisms and four proposed inactivating mutations associated to Fanconi-Bickel syndrome. We also engineered mutations based on sequence alignment and conserved amino acids in selected domains. The single-nucleotide polymorphisms P68L and T110I did not impact on sugar transport as assayed in Xenopus oocytes. All the Fanconi-Bickel syndrome-associated mutations invalidated glucose transport by hGLUT2 either through absence of protein at the plasma membrane (G20D and S242R) or through loss of transport capacity despite membrane targeting (P417L and W444R), pointing out crucial amino acids for hGLUT2 transport function. In contrast, engineered mutants were located at the plasma membrane and able to transport sugar, albeit with modified kinetic parameters. Notably, these mutations resulted in gain of function. G20S and L368P mutations increased insulin secretion in the absence of glucose. In addition, these mutants increased insulin-positive cell differentiation when expressed in cultured rat embryonic pancreas. F295Y mutation induced ß cell differentiation even in the absence of glucose, suggesting that mutated GLUT2, as a sugar receptor, triggers a signaling pathway independently of glucose transport and metabolism. Our results describe the first gain of function mutations for hGLUT2, revealing the importance of its receptor versus transporter function in pancreatic ß cell development and insulin secretion.

Identification of mutations in TMEM5 and ISPD as a cause of severe cobblestone lissencephaly.

Cobblestone lissencephaly is a peculiar brain malformation with characteristic radiological anomalies. It is defined as cortical dysplasia that results when neuroglial overmigration into the arachnoid space forms an extracortical layer that produces agyria and/or a "cobblestone" brain surface and ventricular enlargement. Cobblestone lissencephaly is pathognomonic of a continuum of autosomal-recessive diseases characterized by cerebral, ocular, and muscular deficits. These include Walker-Warburg syndrome, muscle-eye-brain disease, and Fukuyama muscular dystrophy. Mutations in POMT1, POMT2, POMGNT1, LARGE, FKTN, and FKRP identified these diseases as alpha-dystroglycanopathies. Our exhaustive screening of these six genes, in a cohort of 90 fetal cases, led to the identification of a mutation in only 53% of the families, suggesting that other genes might also be involved. We therefore decided to perform a genome-wide study in two multiplex families. This allowed us to identify two additional genes: TMEM5 and ISPD. Because TMEM has a glycosyltransferase domain and ISPD has an isoprenoid synthase domain characteristic of nucleotide diP-sugar transferases, these two proteins are thought to be involved in the glycosylation of dystroglycan. Further screening of 40 families with cobblestone lissencephaly identified nonsense and frameshift mutations in another four unrelated cases for each gene, increasing the mutational rate to 64% in our cohort. All these cases displayed a severe phenotype of cobblestone lissencephaly A. TMEM5 mutations were frequently associated with gonadal dysgenesis and neural tube defects, and ISPD mutations were frequently associated with brain vascular anomalies.

Cobblestone lissencephaly: neuropathological subtypes and correlations with genes of dystroglycanopathies.

Cobblestone lissencephaly represents a peculiar brain malformation with characteristic radiological anomalies, defined as cortical dysplasia combined with dysmyelination, dysplastic cerebellum with cysts and brainstem hypoplasia. Cortical dysplasia results from neuroglial overmigration into the arachnoid space, forming an extracortical layer, responsible for agyria and/or 'cobblestone' brain surface and ventricular enlargement. The underlying mechanism is a disruption of the glia limitans, the outermost layer of the brain. Cobblestone lissencephaly is pathognomonic of a continuum of autosomal recessive diseases with cerebral, ocular and muscular deficits, Walker-Warburg syndrome, muscle-eye-brain and Fukuyama muscular dystrophy. Mutations in POMT1, POMT2, POMGNT1, LARGE, FKTN and FKRP genes attributed these diseases to α-dystroglycanopathies. However, studies have not been able to identify causal mutations in the majority of patients and to establish a clear phenotype/genotype correlation. Therefore, we decided to perform a detailed neuropathological survey and molecular screenings in 65 foetal cases selected on the basis of histopathological criteria. After sequencing the six genes of α-dystroglycanopathies, a causal mutation was observed in 66% of cases. On the basis of a ratio of severity, three subtypes clearly emerged. The most severe, which we called cobblestone lissencephaly A, was linked to mutations in POMT1 (34%), POMT2 (8%) and FKRP (1.5%). The least severe, cobblestone lissencephaly C, was linked to POMGNT1 mutations (18%). An intermediary type, cobblestone lissencephaly B, was linked to LARGE mutations (4.5%) identified for the first time in foetuses. We conclude that cobblestone lissencephaly encompasses three distinct subtypes of cortical malformations with different degrees of neuroglial ectopia into the arachnoid space and cortical plate disorganization regardless of gestational age. In the cerebellum, histopathological changes support the novel hypothesis that abnormal lamination arises from a deficiency in granule cells. Our studies demonstrate the positive impact of histoneuropathology on the identification of α-dystroglycanopathies found in 66% of cases, while with neuroimaging criteria and biological values, mutations are found in 32-50% of patients. Interestingly, our morphological classification was central in the orientation of genetic screening of POMT1, POMT2, POMGNT1, LARGE and FKRP. Despite intensive research, one-third of our cases remained unexplained; suggesting that other genes and/or pathways may be involved. This material offers a rich resource for studies on the affected neurodevelopmental processes of cobblestone lissencephaly and on the identification of other responsible gene(s)/pathway(s).

Congenital muscular dystrophy type 1D (MDC1D) due to a large intragenic insertion/deletion, involving intron 10 of the LARGE gene.

Mutation of the LARGE gene is the rarest of the six known genetic causes of α-dystroglycanopathy. We report further a family with MDC1D due to a complex genomic rearrangement that was not apparent on standard sequencing of LARGE. Two sisters in a consanguineous family had moderate mental retardation and cerebellar malformations, together with dystrophic changes and markedly reduced α-dystroglycan glycosylation staining on muscle biopsy. There was homozygous linkage to the LARGE locus but sequencing of LARGE coding regions was normal. Analysis of LARGE cDNA showed an abnormal sequence inserted between exons 10 and 11, in most of the transcripts, predicted to introduce a premature stop codon. The abnormal sequence mapped to a spliced EST (DA935254) of unknown function, normally located at 100 kb centromeric of LARGE on chromosome 22q12.3. Quantitative PCR analysis of the EST and adjacent regions showed twice the normal copy number in patients' genomic DNA samples, consistent with a large intra-chromosomal duplication inserted into intron 10 of LARGE in a homozygous state. This insertion was associated with deletion of a central region of intron 10, but the exact break points of the deletion/duplication were not found, suggesting that an even more complex rearrangement may have occurred. The exact function of LARGE, a golgi protein, remains uncertain. POMT and POMGnT enzyme activities were normal in patients' lymphoblast cells, suggesting that defects in LARGE do not affect the initiation of O-mannosyl glycans.

Protein O-mannosyltransferase activities in lymphoblasts from patients with alpha-dystroglycanopathies.

Defects in O-mannosylation of alpha-dystroglycan cause some forms of congenital muscular dystrophy (CMD), the so-called alpha-dystroglycanopathies. Six genes are responsible for these diseases with overlapping phenotypes. We investigated the usefulness of a biochemical approach for the diagnosis and investigation of the alpha-dystroglycanopathies using immortalized lymphoblasts prepared from genetically diagnosed and undiagnosed CMD patients and from control subjects. We measured the activities of protein O-mannose beta1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) and protein O-mannosyltransferase (POMT). Lymphoblasts from patients harbouring known mutations in either POMGNT1 or POMT1 showed a marked decrease in POMGnT1 or POMT activity, respectively, compared to controls. Furthermore, we identified pathogenic mutations in POMGNT1, POMT1 or POMT2 in six previously genetically uncharacterised patients who had very low enzyme activity. In conclusion, the lymphoblast-based enzymatic assay is a sensitive and useful method (i) to select patients harbouring POMGNT1, POMT1 or POMT2 mutations; (ii) to assess the pathogenicity of new or already described mutations.

PMM2 intronic branch-site mutations in CDG-Ia.

Congenital Disorders of Glycosylation (CDG, OMIM#212065)-Ia is an autosomal recessive disorder, characterized by central nervous system dysfunction and multiorgan failure associated with mutations in the PMM2 gene. We report two patients who are compound heterozygotes with respect to two new intronic mutations that affect a highly conserved adenosine in a consensus branch-site sequence. The mutations, one in intron 7: c.340 -23A > G (IVS7 -23A > G) and the other in intron 2: c.179 -25A > G (IVS2 -25A > G), are associated with the c.422G > A (R141H) and c.193 G > T (D65Y) mutations, respectively. The c.179 -25A > G and the c.340 -23A > G changes cause exon 3 and exon 8 to be lost at the RNA level, respectively. This kind of mutation can cause a problem in molecular diagnosis of CDG-Ia if intronic primers are not correctly chosen, and if molecular diagnosis is not performed at both the DNA and mRNA levels.

Involvement of renin-angiotensin system in pressure-flow relationship: role of angiotensin-converting enzyme gene polymorphism.

BACKGROUND: The renin-angiotensin system is involved in blood pressure regulation. The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene is known to be associated with variation of plasma and cellular ACE concentrations. Furthermore, changes in arterial function have been suggested to be associated to the DD genotype. The aim of the study was to investigate the arterial vascular response to a physiologic stimulus (i.e., flow) according to the I/D ACE gene polymorphism. METHODS: Sixty patients scheduled for coronary artery bypass grafting (n = 24) or valve surgery (n = 36) under normothermic cardiopulmonary bypass were genotyped in a blind manner by polymerase chain reaction. Mean arterial pressure was measured at pump flows ranging from 1 to 3 l x min(-1) x m (-2) by 0.25 l x min(-1) x m(-2) step each 15 s, to obtain a pressure-flow relation. Independent factors associated with the variation of the slope of the pressure-flow relation curve were assessed by multivariate analysis. RESULTS: We found a D allelic frequency of 0.54. Patients were separated in two groups (DD, n = 16; ID/II, n = 44). There were no significant difference with regard to preoperative and intraoperative data between the two groups. DD patients had their pressure-flow relation curves shifted upward (with higher pressures as flow increased), indicating a lesser decrease in vascular resistance. Furthermore, DD genotype was the only independent predictor of the slope of the curves (21.5 +/- 4.2 vs. 18.1 +/- 5 mmHg/[l x min(-1) x m(-2)] for DD and ID/II, respectively; P = 0.02; values are mean +/-SD). CONCLUSION: These results show that vasomotor properties are influenced by the I/D polymorphism of the ACE gene.