Characterization of two unusual truncating PMM2 mutations in two CDG-Ia patients
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).
References (22)
- et al.
High residual activity of PMM2 in patients’ fibroblasts: possible pitfall in the diagnosis of CDG-Ia (phosphomannomutase deficiency)
Am. J. Hum. Genet.
(2001) - et al.
Underdiagnosis of mild congenital disorders of glycosylation type Ia
Pediatr. Neurol.
(2005) - et al.
Phosphomannomutase deficiency is a cause of carbohydrate-deficient glycoprotein syndrome type I
FEBS Lett.
(1995) - et al.
PMM2 intronic branch-site mutations in CDG-Ia
Mol. Genet. Metab.
(2006) - et al.
Effect of mutations found in carbohydrate-deficient glycoprotein syndrome type IA on the activity of phosphomannomutase 2
FEBS Lett.
(1999) - et al.
Lack of homozygotes for the most frequent disease allele in carbohydrate-deficient glycoprotein syndrome type 1A
Am. J. Hum. Genet.
(1998) - et al.
Lack of Hardy–Weinberg equilibrium for the most prevalent PMM2 mutation in CDG-Ia (congenital disorders of glycosylation type Ia)
Eur. J. Hum. Genet.
(2000) Genetic defects in the human glycome
Nat. Rev. Genet.
(2006)- et al.
Congenital disorder of glycosylation type Ia in a 6-year-old girl with a mild intellectual phenotype: two novel PMM2 mutations
J. Inherit. Metab. Dis.
(2005) - et al.
Congenital disorder of glycosylation type Ia revealed by hypertransaminasemia and failure to thrive in a young boy with normal neurodevelopment
J. Pediatr. Gastroenterol. Nutr.
(2005)
Mutations in PMM2, a phosphomannomutase gene on chromosome 16p13, in carbohydrate-deficient glycoprotein type I syndrome (Jaeken syndrome)
Nat. Genet.
Cited by (42)
Non-functional alternative splicing caused by a Latino pathogenic variant in a case of PMM2-CDG
2021, Molecular Genetics and Metabolism ReportsCitation Excerpt :All predictions were made with the DYSF transcript ENST00000268261. The cDNA-based polymerase chain reaction (PCR) product corresponding to the coding sequence of PMM2 was obtained using forward primer PMM2s 5′-TGCCAACGTGTCTTGTAAGG-3′ and reverse primer PMM2as 5′-GGAAGTTTCTGGCACTGGAG-3′ [32]. Sequencing of PCR products was performed by an ABI Prism 3130xl autoanalyzer (Applied Biosystems, Foster City, CA) and results were visualized using SnapGene Viewer 2.2.2 (GSL Biotech LLC, Chicago, IL, USA).
Identification through exome sequencing of the first PMM2-CDG individual of Mexican mestizo origin
2020, Molecular Genetics and Metabolism ReportsCitation Excerpt :A bioinformatic filter was constructed including all the genes previously reported to be related with CDG. The cDNA-based polymerase chain reaction (PCR) product corresponding to the coding sequence of PMM2 was obtained using forward primer PMM2s 5′-TGCCAACGTGTCTTGTAAGG-3′ and reverse primer PMM2as 5′-GGAAGTTTCTGGCACTGGAG-3′ [3]. The PCR product corresponding to exon 5 of PMM2 was amplified from gDNA using forward primer PMM2-E5F 5′-GAAACATTGACCACACTAGCC-3′ and reverse primer PMM2-E5R 5′-GTGTTGGGATTACAGGCATG-3′ [4].
Renal involvement in PMM2-CDG, a mini-review
2018, Molecular Genetics and MetabolismHuman genomic variants and inherited disease: Molecular mechanisms and clinical consequences
2018, Emery and Rimoin's Principles and Practice of Medical Genetics and Genomics: FoundationsThrombotic complications in patients with PMM2-CDG
2013, Molecular Genetics and MetabolismCitation Excerpt :Out of the 100 patients evaluated we found 14 cases of thrombosis (11 cases in the variable observation period prior to the patient was reported in the literature, and 3 in our own patient group). In this group of 14 patients with thrombosis, 7 developed an arterial and 7 a venous thrombosis [3,6,7,10,17,18,22–26]. Two patients also developed disseminated intravascular coagulation (DIC) secondary to arterial thrombosis [3,10].
Human Gene Mutation in Inherited Disease: Molecular Mechanisms and Clinical Consequences
2013, Emery and Rimoin's Principles and Practice of Medical Genetics