Characterization of two unusual truncating PMM2 mutations in two CDG-Ia patients

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Abstract

Congenital disorders of glycosylation type Ia (CDG-Ia) is a recessive metabolic disorder caused by mutations in the PMM2 gene and characterized by a defect in the synthesis of N-glycans. The clinical presentation ranges from very severe multi-organ failure to mild neurological problems. A plethora of PMM2 mutations has been described and the vast majority are missense mutations. This selection reflects the requirement of a minimal phosphomannomutase activity to be compatible with life.

We describe the characterization of two unusual truncating mutations in two CDG-Ia patients. The first patient is compound heterozygous for the PMM2 mutation p.V231M (c.691G>A) and a deep intronic point mutation (c.639-15.479C>T). The latter variant activates a cryptic splice site which results in an in-frame insertion of a pseudoexon of 123 bp between exon 7 and 8.

The second patient is compound heterozygous for the mutation p.V44A (c.131T>C) and an Alu retrotransposition mediated complex deletion of approximately 28 kb encompassing exon 8.

These types of mutations have not been described before in CDG-Ia patients. Their detection stresses the importance to combine PMM2 mutation screening on genomic DNA with analysis of the transcripts and/or with the enzymatic analysis of the phosphomannomutase activity. Next to the exonic deletions, which already receive more attention than before, it is likely that deep intronic mutations represent an increasingly important category of mutations.

Introduction

CDG-Ia is, with an estimated frequency of 1/20.000–1/50.000, the most frequent type of the CDG type I syndromes. These syndromes are all characterized by a defect in the synthesis of N-glycan precursors in the endoplasmic reticulum [1], [2]. More than 500 patients have been diagnosed world wide.

The clinical presentations of these patients range from very severe to very mild with only slight neurological problems. The ‘classical’ presentation, as first described in 1984 by Prof. Jaeken, represents the more severe side of the spectrum. These patients present with multi-organ failure, often resulting in neonatal death. Recently, a number of patients have been described with only slight neurological problems including hypotonia, mild ataxia and slight developmental problems [3], [4], [5], [6].

CDG-Ia is caused by mutations in the PMM2 gene, one of the two mammalian paralogous genes encoding phosphomannomutase (PMM) [7], [8]. PMM converts mannose-6-P into mannose-1-P which is further processed to become dolichol-P-mannose, the mannose donor in N-glycosylation. The spectrum of PMM2 mutations is steadily growing, still, the majority of the mutations are of the missense type (96.5% in CDG mutation database http://www.euroglycanet.org/home.html) [9]. The truncating mutations include nonsense mutations, small deletions or insertions and splice mutations. Recently, two mutations in the branch site of intron 7 have been added to this list [10]. The preference for missense mutations is the consequence of the requirement for a minimal PMM activity to be compatible with life. This hypothesis has been confirmed in humans by the lack of homozygotes for the frequent mutation R141H which encodes an inactive enzyme [1], [11]. Also, compound heterozygotes for R141H and a truncating mutation or two truncating mutations have never been described. Recently, it has been shown that knock out mice are not viable, whereby the homozygous embryos disappear before day 2.5 [12].

We describe two new unusual truncating mutations in this paper. The first is a deep intronic point mutation which introduces a pseudoexon. The second mutation is a large deletion including exon 8. These types of mutations have not been described before in PMM2. They are in general missing from the mutation spectrum of disease genes, because simple PCR-based mutation analysis at the genomic level does not easily identify them.

Section snippets

Phosphomannomutase and phosphomannose isomerase activities

Phosphomannomutase and phosphomannose isomerase activities in fibroblasts or fresh leukocytes were determined essentially as described in [8].

Mutation analysis of PMM2

Genomic DNA, extracted from fibroblasts, was screened for mutations in the PMM2 gene by direct sequencing, essentially as described in [13]. For cDNA analysis, RNA was extracted from cultured fibroblasts of the patients using the QIAamp RNA blood mini kit as described in the manufacturer’s protocol (Qiagen, Venlo, Netherlands). Subsequently, 5 μg of RNA

Clinical description of the patients

Patient 1, son of unrelated parents, was born at term after a complicated pregnancy (endangered abortion, trombopenia, weak fetus movements before delivery) with weight 4190 g, Apgar score 8 (at 5 min). After the first ten minutes of life his clinical status deteriorated with respiratory failure, arrhythmia, petechiae, generalized edema (a second Apgar score is not available). Hypoglycaemia, decreased cholesterol, albumin, and antithrombin III levels were found. Ultrasound revealed pericardial

Discussion

Until now, two PMM2 mutations have been found in all CDG-Ia patients with a marked phosphomannomutase deficiency in fibroblasts and/or leukocytes (more than 230 patients in our laboratory alone). Most of the mutations are point mutations within the coding region of the gene, resulting in missense or nonsense mutations, or splice mutations affecting the conserved bases of the splice donor or splice acceptor. All of these can easily be identified by direct sequencing on genomic DNA. Two

Acknowledgments

This work was performed in collaboration with several members of EUROGLYCANET, a network for CDG research, funded by the Sixth Framework Program of the European Commission. This work was further supported by a grant from the Fund for Scientific Research (FWO, Flanders, Belgium) and from the Spanish Ministerio de Sanidad y Consumo (FIS PI041319, Spain).

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