Mutations in PMM2, a phosphomannomutase gene on chromosome 16p13, in carbohydrate-deficient glycoprotein type I syndrome (Jaeken syndrome).

Carbohydrate-deficient glycoprotein syndrome type 1 (CDG1 or Jaeken syndrome) is the prototype of a class of genetic multisystem disorders characterized by defective glycosylation of glycoconjugates. It is mostly a severe disorder which presents neonatally. There is a severe encephalopathy with axial hypotonia, abnormal eye movements and pronounced psychomotor retardation, as well as a peripheral neuropathy, cerebellar hypoplasia and retinitis pigmentosa. The patients show a peculiar distribution of subcutaneous fat, nipple retraction and hypogonadism. There is a 20% lethality in the first years of life due to severe infections, liver insufficiency or cardiomyopathy. CDG1 shows an autosomal recessive mode of inheritance and has been mapped to chromosome 16p. Most patients show a deficiency of phosphomannomutase (PMM)8, an enzyme necessary for the synthesis of GDP-mannose. We have cloned the PMM1 gene, which is on chromosome 22q13 (ref.9). We now report the identification of a second human PMM gene, PMM2, which is located on 16p13 and which encodes a protein with 66% identity to PMM1. We found eleven different missense mutations in PMM2 in 16 CDG1 patients from different geographical origins and with a documented phosphomannomutase deficiency. Our results give conclusive support to the biochemical finding that the phosphomannomutase deficiency is the basis for CDG1.

The normal phenotype of Pmm1-deficient mice suggests that Pmm1 is not essential for normal mouse development.

Phosphomannomutases (PMMs) are crucial for the glycosylation of glycoproteins. In humans, two highly conserved PMMs exist: PMM1 and PMM2. In vitro both enzymes are able to convert mannose-6-phosphate (mannose-6-P) into mannose-1-P, the key starting compound for glycan biosynthesis. However, only mutations causing a deficiency in PMM2 cause hypoglycosylation, leading to the most frequent type of the congenital disorders of glycosylation (CDG): CDG-Ia. PMM1 is as yet not associated with any disease, and its physiological role has remained unclear. We generated a mouse deficient in Pmm1 activity and documented the expression pattern of murine Pmm1 to unravel its biological role. The expression pattern suggested an involvement of Pmm1 in (neural) development and endocrine regulation. Surprisingly, Pmm1 knockout mice were viable, developed normally, and did not reveal any obvious phenotypic alteration up to adulthood. The macroscopic and microscopic anatomy of all major organs, as well as animal behavior, appeared to be normal. Likewise, lectin histochemistry did not demonstrate an altered glycosylation pattern in tissues. It is especially striking that Pmm1, despite an almost complete overlap of its expression with Pmm2, e.g., in the developing brain, is apparently unable to compensate for deficient Pmm2 activity in CDG-Ia patients. Together, these data point to a (developmental) function independent of mannose-1-P synthesis, whereby the normal knockout phenotype, despite the stringent conservation in phylogeny, could be explained by a critical function under as-yet-unidentified challenge conditions.

Deficiency of dolichol-phosphate-mannose synthase-1 causes congenital disorder of glycosylation type Ie.

Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndromes, lead to diseases with variable clinical pictures. We report the delineation of a novel type of CDG identified in 2 children presenting with severe developmental delay, seizures, and dysmorphic features. We detected hypoglycosylation on serum transferrin and cerebrospinal fluid beta-trace protein. Lipid-linked oligosaccharides in the endoplasmic reticulum of patient fibroblasts showed an accumulation of the dolichyl pyrophosphate Man(5)GlcNAc(2) structure, compatible with the reduced dolichol-phosphate-mannose synthase (DolP-Man synthase) activity detected in these patients. Accordingly, 2 mutant alleles of the DolP-Man synthase DPM1 gene, 1 with a 274C>G transversion, the other with a 628delC deletion, were detected in both siblings. Complementation analysis using DPM1-null murine Thy1-deficient cells confirmed the detrimental effect of both mutations on the enzymatic activity. Furthermore, mannose supplementation failed to improve the glycosylation status of DPM1-deficient fibroblast cells, thus precluding a possible therapeutic application of mannose in the patients. Because DPM1 deficiency, like other subtypes of CDG-I, impairs the assembly of N-glycans, this novel glycosylation defect was named CDG-Ie.

Unconventional intronic splice site mutation in SCN5A associates with cardiac sodium channelopathy.

BACKGROUND: Mutations in the cardiac sodium channel, SCN5A, have been associated with one type of long-QT syndrome, with isolated cardiac conduction defects and Brugada syndrome. The sodium channelopathies exhibit marked variation in clinical phenotypes. The mechanisms underlying the phenotypical diversity, however, remain unknown. Exonic SCN5A mutations can be detected in 20% of Brugada syndrome patients. RESULTS: An intronic mutation (c.4810+3_4810+6dupGGGT) in the SCN5A gene, located outside the consensus splice site, was detected in this study in a family with a highly variable clinical phenotype of Brugada syndrome and/or conduction disease and in a patient with Brugada syndrome. The mutation was not found in a control panel of 100 (200 alleles) ethnically matched normal control subjects. We provide in vivo and in vitro evidence that the mutation can disrupt the splice donor site, activate a cryptic splice site, and create a novel splice site. Notably, our data show that normal transcripts can be also derived from the mutant allele. CONCLUSIONS: This is the first report of an unconventional intronic splice site mutation in the SCN5A gene leading to cardiac sodium channelopathy. We speculate that its phenotypical diversity might be determined by the ratio of normal/abnormal transcripts derived from the mutant allele.

CDG-Id caused by homozygosity for an ALG3 mutation due to segmental maternal isodisomy UPD3(q21.3-qter).

We report on a patient with a congenital disorder of glycosylation type Id (CDG-Id) caused by a homozygous mutation in the ALG3 gene, which results from a de novo mutation in combination with a segmental maternal uniparental isodisomy (UPD). The patient presented with severe psychomotor delay, primary microcephaly, and opticus atrophy, compatible with a severe form of CDG. Isoelectric focusing of transferrin showed a type I pattern and lipid-linked oligosaccharide analysis showed an accumulation of dol-PP-GlcNAc2Man5 in patient's fibroblasts suggesting a defect in the ALG3 gene. A homozygous ALG3 missense mutation p.R266C (c.796C > T) was identified. Further evaluation revealed that neither the mother nor the father were carrier of the p.R266C mutation. Marker analysis revealed a segmental maternal isodisomy for the chromosomal region 3q21.3-3qter. UPD for this region has not been described before. More important, the combination of UPD with a de novo mutation is an exceptional coincidence and an extraordinary observation.

PTPN11 mutations in LEOPARD syndrome.

LEOPARD syndrome is an autosomal dominant disorder with multiple lentigines, congenital cardiac abnormalities, ocular hypertelorism, and retardation of growth. Deafness and genital abnormalities are less frequently found. We report a father and daughter and a third, unrelated patient with LEOPARD syndrome. Recently, missense mutations in the PTPN11 gene located in 12q24 were found to cause Noonan syndrome. All three cases of LEOPARD syndrome reported here have a Y279C mutation in the PTPN11 gene. We hypothesise that some PTPN11 mutations are associated with the typical Noonan syndrome phenotype and that other mutations, such as the Y279C mutation reported here, are associated with both the Noonan syndrome phenotype and with skin pigmentation anomalies, such as multiple lentigines or café au lait spots.

Gross rearrangements in the MECP2 gene in three patients with Rett syndrome: implications for routine diagnosis of Rett syndrome.

Since 1999, many laboratories have confirmed that mutations in the MECP2 gene are the primary cause of Rett syndrome (RTT or RS) and identified mutations in 70 to 90% of the sporadically affected girls. Most of the screenings are PCR-based and restricted to the coding part of the gene and therefore prone to miss gross rearrangements. By Southern blot analysis we identified large deletions (>1 kb) in three female patients with classical, severe Rett syndrome. Further characterization by semi-quantitative PCR and amplification of junction fragments confirmed the presence of a 7.6-kb deletion in one patient and an 8.1-kb deletion in the other patient, both including exon 3 and the coding part of exon 4. The exact nature of the rearrangement in the third patient remained elusive. These results underline the importance of screening for major genomic rearrangements in Rett syndrome. Hum Mutat 22:116-120, 2003.

Genomic organization of the human phosphomannose isomerase (MPI) gene and mutation analysis in patients with congenital disorders of glycosylation type Ib (CDG-Ib).

CDG-Ib is the "gastro-intestinal" type of the congenital disorders of glycosylation (CDG) and a potentially treatable disorder. It has been described in patients presenting with congenital hepatic fibrosis and protein losing enteropathy. The symptoms result from hypoglycosylation of serum- and other glycoproteins. CDG-Ib is caused by a deficiency of mannose-6-phosphate isomerase (synonym: phosphomannose isomerase, EC 5.3.1.8), due to mutations in the MPI gene. We determined the genomic structure of the MPI gene in order to simplify mutation detection. The gene is composed of 8 exons and spans only 5 kb. Eight (7 novel) different mutations were found in seven patients with a confirmed phosphomannose isomerase deficiency, analyzed in the context of this study: six missense mutations, a splice mutation and one insertion. In the last, the mutation resulted in an unstable transcript, and was hardly detectable at the mRNA level. This emphasizes the importance of mutation analysis at the genomic DNA level.

Mutations in PMM2 that cause congenital disorders of glycosylation, type Ia (CDG-Ia).

The PMM2 gene, which is defective in CDG-Ia, was cloned three years ago [Matthijs et al., 1997b]. Several publications list PMM2 mutations [Matthijs et al., 1997b, 1998; Kjaergaard et al., 1998, 1999; Bjursell et al., 1998, 2000; Imtiaz et al., 2000] and a few mutations have appeared in case reports or abstracts [Crosby et al., 1999; Kondo et al., 1999; Krasnewich et al., 1999; Mizugishi et al., 1999; Vuillaumier-Barrot et al., 1999, 2000b]. However, the number of molecularly characterized cases is steadily increasing and many new mutations may never make it to the literature. Therefore, we decided to collate data from six research and diagnostic laboratories that have committed themselves to a systematic search for PMM2 mutations. In total we list 58 different mutations found in 249 patients from 23 countries. We have also collected demographic data and registered the number of deceased patients. The documentation of the genotype-phenotype correlation is certainly valuable, but is out of the scope of this molecular update. The list of mutations will also be available online (URL: http://www.kuleuven. ac.be/med/cdg) and investigators are invited to submit new data to this PMM2 mutation database.

Fine mapping of Noonan/cardio-facio cutaneous syndrome in a large family.

Noonan syndrome (NS) is an autosomal dominant condition with facial dysmorphy, congenital cardiac defects and short stature. A gene for NS has previously been linked to a 14 cM region in 12q24. We performed linkage analysis in a four generation Belgian family with NS in some individuals and cardio-facio-cutaneous (CFC) syndrome in others. Clinical data and linkage data in this family indicate that NS and CFC syndrome result from a variable expression of the same genetic defect. We report a maximum lod score of 4.43 at zero recombination for marker D12S84 in 12q24. A crossover in this pedigree narrows the candidate gene region for NS to a 5 cM interval between markers D12S84 and D12S1341.

Lack of Hardy-Weinberg equilibrium for the most prevalent PMM2 mutation in CDG-Ia (congenital disorders of glycosylation type Ia).

The R141H mutation in the PMM2 gene is the most frequent mutation in type Ia of the congenital disorders of glycosylation (formerly carbohydrate-deficient glycoprotein syndromes)(CDG-Ia). However, it has never been observed in the homozygous state. Homozygosity for this mutation is probably incompatible with life. In this study, we determined the frequency of R141H in two normal populations: in neonates of Dutch origin 1/79 were carriers, whilst in the Danish population, a carrier frequency of 1/60 was found. These figures are clearly in disequilibrium with the frequency of CDG-Ia that has been estimated at 1/80,000 to 1/40,000 in these populations. Haplotype analysis of 43 patients with the R141H mutation of different geographic origins indicated that the R141H is an old mutation in the Caucasian population. Based on the new data, the disease frequency has been calculated at 1/20,000 in these populations. It is concluded that the disease is probably underdiagnosed.

Prenatal diagnosis in CDG1 families: beware of heterogeneity.

Carbohydrate-deficient glycoprotein syndrome type 1 (CDG1) is an autosomal recessive, metabolic disorder with severe psychomotor retardation and a high mortality rate in early childhood. Most patients have a deficiency of phosphomannomutase, due to mutations in PMM2, a gene located on chromosome 16p13. Over a period of 18 months we offered prenatal diagnosis to eight families. In six cases and prior to the identification of the gene, the diagnosis was based on linkage analysis and phosphomannomutase measurements. Subsequently direct mutation analysis has been used in two families. It is shown here that phosphomannomutase activities are strongly reduced in cultured amniocytes and trophoblasts of affected foetuses. We refrained from offering prenatal testing in two other families, because either the disease did not link to chromosome 16 and/or normal phosphomannomutase activities were measured in fibroblasts from the proband. This confirms earlier suggestions of heterogeneity for CDG1.

Identification and localization of two mouse phosphomannomutase genes, Pmm1 and Pmm2.

Phosphomannomutases catalyze the reversible conversion of mannose 6-phosphate to mannose 1-phosphate. In humans, two different isozymes have recently been identified, PMM1 and PMM2. We have previously shown that mutations in the PMM2 gene cause the most frequent type of the congenital disorders of glycosylation, CDG-Ia. Here, we present data on the two mouse orthologous genes, Pmm1 and Pmm2. The chromosomal localization of the two mouse genes has been determined. We also present the gene structure and the exon-intron organization of Pmm1 and Pmm2. Pmm1 maps to mouse chromosome 15, Pmm2 to chromosome 16. These chromosomal regions are syntenic with regions on human chromosomes 22 and 16, respectively. The Pmm1 gene is composed of eight exons and spans approximately 9.5 kb. The genomic structure is extremely well conserved between the human and mouse gene. The Pmm2 gene consists of eight exons and spans a larger genomic region ( approximately 20 kb). An alignment of the human and mouse protein sequences confirms the conservation among this family of phosphomannomutases. The two mouse genes are expressed in many tissues, but the expression pattern is slightly different between Pmm1 and Pmm2. The most striking difference is the high expression of Pmm1 in brain tissue, whereas Pmm2 is only weakly expressed in this tissue.

Stable expression of VLA-4 and increased maturation of the beta 1-integrin precursor after transfection of CHO cells with alpha 4m cDNA.

A full-length cDNA coding for the murine alpha 4 integrin subunit (alpha 4m) was transfected into CHO-K1 cells and cell lines that expressed VLA-4 at their surface as a result of the association of transfected alpha 4m with endogenous hamster beta 1 were selected. Functionality of the expressed alpha 4m beta 1 was shown by adhesion assays on VCAM-1 and antibody (anti-VCAM-1) inhibition. Pulse chase experiments indicated that transfection of the murine alpha 4 cDNA into CHO cells led to an increase in maturation and a decrease in degradation of the beta 1 precursor subunit compared to control CHO-K1 cells. This was supported by FACS analysis, using an anti-hamster beta 1 monoclonal antibody, which showed that more beta 1 subunit was expressed at the surface of these stably transfected alpha 4m expressing cells. These results support the hypothesis that degradation of precursor beta 1 is at least partly determined by the quantity of alpha subunits available intracellulary for heterodimer formation.

Effect of mutations found in carbohydrate-deficient glycoprotein syndrome type IA on the activity of phosphomannomutase 2.

Seven mutant forms of human phosphomannomutase 2 were produced in Escherichia coli and purified. These mutants had a Vmax of 0.2-50% of the wild enzyme and were unstable. The least active protein (R141H) bears a very frequent mutation, which has never been found in the homozygous state whereas the second least active protein (D188G) corresponds to a mutation associated with a particularly severe phenotype. We conclude that total lack of phosphomannomutase 2 is incompatible with life. Another conclusion is that the elevated residual phosphomannomutase activity found in fibroblasts of some patients is contributed by their mutated phosphomannomutase 2.

The genomic structure of the murine alpha 4 integrin gene.

The VLA-4 (alpha 4 beta 1) integrin is a leukocyte glycoprotein involved in both cell-extracellular matrix and cell-cell interactions. We report here the cloning of the murine alpha 4 gene whose protein product is antigenically related to the human VLA-4 alpha chain. The alpha 4 m gene is about 75 kb long and consists of 28 exons, ranging in size from 46 bp (exon 13) to 437 bp (exon 1). The introns varied from 79 bp (intron 8) to more than 17 kb (intron 2). Three mRNA transcripts from this alpha 4 m gene can be visualized on Northern blot. After cloning the 3' untranslated region (3' UTR), four polyadenylation sites could be identified, presumably responsible for the presence of three to four transcripts of the alpha 4 gene, differing substantially in length.

Cloning and characterization of the promoter region of the murine alpha-4 integrin subunit.

To study the differential expression of the murine VLA-4 (alpha 4 beta 1) integrin, the 5'-flanking region of the gene for the alpha subunit (alpha 4m) was isolated and a cDNA for alpha 4m was obtained with reverse transcriptase polymerase chain reaction (RT-PCR). The cDNA sequence contained a difference in the signal peptide region compared to the previously described cDNA (Neuhaus et al., 1991). As a consequence, another start codon is predicted, resulting in a decrease in size of the signal peptide. This was confirmed by genomic sequencing. The promoter region was delimited by ribonuclease protection assay (RPA) and transfection experiments fusing 5'-upstream fragments to the luciferase gene. A fragment extending from -936 to +221 was capable of controlling the expected cell-type-specific expression. Sequence comparison of the mouse alpha 4m promoter region with the human alpha 4h promoter revealed little homology. Like most integrin subunits, alpha 4m lacks TATA anc CCAAT boxes. Putative recognition sites for DNA-binding nuclear factors (AP1, AP2, Sp1, and PU1) were identified. The characterization of the promoter region and further identification of the transcription regulatory elements should provide insight in the regulation of alpha 4m integrin gene expression.