Fatal outcome after heart surgery in PMM2-CDG due to a rare homozygous gene variant with double effects

Abstract

Variants in Phosphomannomutase 2 (PMM2) lead to PMM2-CDG, the most frequent congenital disorder of glycosylation (CDG). We here describe the disease course of a ten-month old patient who presented with the classical PMM2-CDG symptoms as cerebellar hypoplasia, retinitis pigmentosa, seizures, short stature, hepato- and splenomegaly, anaemia, recurrent vomiting and inverted mamillae. A severe form of tetralogy of Fallot was diagnosed and corrective surgery was performed at the age of 10 months. At the end of the cardiopulmonary bypass, a sudden oedematous reaction of the myocardium accompanied by biventricular pump failure was observed immediately after heparin antagonization with protamine sulfate. The patient died seven days after surgery, since myocardial function did not recover on ECMO support. We here describe the first patient carrying the homozygous variant g.18313A > T in the PMM2 gene (NG_009209.1) that either can lead to c.394A > T (p.I132F) or even loss of 100 bp due to exon 5 skipping (c.348_447del; p.G117Rfs*4) which is comparable to a null allele. Proliferation and doubling time of the patient's fibroblasts were affected. In addition, we show that the induction of cellular stress by elevating the cell culture temperature to 40 °C led to a decrease of the patients' PMM2 transcript as well as PMM2 protein levels and subsequently to a significant loss of residual activity. We assume that metabolic stressful processes occurring after cardiac surgery led to the drop of the patient's PMM activity below a life-sustaining niveau which paved the way for the fatal outcome.

Keywords: Congenital disorder(s) of glycosylation; Exon skipping; N-glycosylation; PMM2; Phosphomannomutase 2; Splicing variant.

Fig. 1

Intra-operative transesophageal echocardiography showed significant hypertrophy with increased septum thickness. Numbers indicate the penetration depth of echo in cm.

Fig. 2

A Isoelectric focussing of serum transferrin revealed elevated amounts of disialo- and asialotransferrin in case of the patient (lane 4), indicating a CDG-I disease. As internal standards also a healthy control (lane 1) and CDG-I (lane 2) as well as CDG-II patients' samples (lane 3) are included. B Sanger Sequencing of PMM2 exon 5 showed homozygosity for variant c.394A > T (p.I132F) in the patient. The parents are heterozygous carriers. C Due to the loss of exon 5, amplification of the PMM2 cDNA by RT-PCR revealed a 100 bp shorter PCR product in case of the patient than in the control. D PMM2 protein alignment demonstrated Ile¹³² as a highly conserved amino acid in a highly conserved stretch through different species. E Schematic intron-exon structure of PMM2 from exon 3 to exon 7 (gDNA) and the spliced mRNA fragment (mRNA) of a healthy control and the patient with deletion of exon 5 leading to a shift in the open reading frame and a mutated C-terminus ending in Lys-Lys-Arg-Lys-Lys-Ile-* (short: KKRKKI*). F PMM2 activity analysis of a control and the patient. At 37 °C the patient's activity was reduced to 7%, whereas at 40 °C, the activity was only 3% as compared to the value measured at 37 °C. G Western blot analysis for PMM2 of control and patient fibroblasts. In comparison to the control, the patient's PMM2 expression was clearly reduced to 19% at 37 °C and further declined to 10% at 40 °C. H PMM2 qRT-PCR studies of control and patient fibroblasts. At 37 °C the PMM2 expression in the patient cells was diminished to 47% compared to the control, whereas only 4% residual PMM2 transcript level could be detected at 40 °C in case of the patient. I HPLC analysis of LLOs derived from control (in blue) and patient's fibroblasts (in orange) after metabolic labelling with [2-³H]mannose displayed accumulations of shortened oligosaccharide structures Man_2-5 (Man_2-5GlcNAc₂-PP-Dol) accompanied by depletion of the full-length oligosaccharide Glc₃. (Glc₃Man₉GlcNAc₂-PP-Dol). J – L Analysis of cell growth. (J) XCelligence device analysis of cell proliferation of a control (blue) and the patient (orange) measured over 5 days. (K) PMM2-deficient cells needed about 1,25 times longer for doubling compared to control fibroblasts and showed a significantly reduced cell proliferation (L). Used symbols: = dolichol, = galactose, = N-Acetylglucosamine, = mannose, = glucose, = sialic acid

Fig. S1

In silico structure prediction of wildtype and mutated PMM2 proteins. (A) Predicted impact of p.I132F on PMM2 protein conformation. Blue represents a rigidification of the structure. (B and C) Interactomic interactions in wildtype and mutated PMM2 proteins. Isoleucine 132 (B, wildtype) and phenylalanine (C, mutated) are colored in light-blue and are shown together with the interacting amino acid residues. (D) PDBePISA prediction of the PMM2 (PDB 2AMY) homodimer. Red circles indicate the site of mutation.