EP0981632A2 - Syndrome de type 1 consecutif a une deficience glucidique de la glycoproteine - Google Patents

Syndrome de type 1 consecutif a une deficience glucidique de la glycoproteine

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Publication number
EP0981632A2
EP0981632A2 EP98925519A EP98925519A EP0981632A2 EP 0981632 A2 EP0981632 A2 EP 0981632A2 EP 98925519 A EP98925519 A EP 98925519A EP 98925519 A EP98925519 A EP 98925519A EP 0981632 A2 EP0981632 A2 EP 0981632A2
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Prior art keywords
pmm2
dna
gene
mutations
indicated
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German (de)
English (en)
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Gert University OF Leuven MATTHIJS
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Euroapi UK Ltd
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Genzyme Ltd
Genzyme UK Ltd
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Priority claimed from GBGB9708851.2A external-priority patent/GB9708851D0/en
Priority claimed from GBGB9801719.7A external-priority patent/GB9801719D0/en
Application filed by Genzyme Ltd, Genzyme UK Ltd filed Critical Genzyme Ltd
Publication of EP0981632A2 publication Critical patent/EP0981632A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to carbohydrate-deficient glycoprotein syndrome Type I (CDGl), or Jaeken disease; more particularly, it relates to the identification of the molecular defect associated therewith, to a novel gene and to diagnostic and other applications of these findings.
  • CDGl carbohydrate-deficient glycoprotein syndrome Type I
  • CDG l is the prototype of a class of genetic multi-system disorders characterised by defective glycosylation of glycoconjugates, (see Jaeken, J. et al, Pediatr. Res., 14, 179, 1980; Jaeken, J., & Carchon, H. , J. Inher. Metab. Dis. , 16, 813-820, 1993; and Jaeken, J. , et al, J. Med.
  • CDG l is inherited in an autosomal recessive manner and its locus has been mapped to chromosome 16p, (see Martinsson, T. , et al, Hum. Mol. Genet. , 3, 2037-2042,
  • the basis of the present invention is the surprising identification of a second human PMM gene, PMM2, which is located on chromosome 16pl3 and which encodes a protein having 66% identity to PMMl . Eleven different missense mutations in PMM2 were found in sixteen CDGl patients of different geographical origin and having a documented phosphomannomutase deficiency. The present results give conclusive support to the biochemical finding that phosphomannomutase deficiency is the basis for CDG 1.
  • PMM is a cytoplasmic enzyme that isomerizes mannose 6-phosphate into mannose 1-phosphate, which is then converted to GDP-mannose
  • CDG l fifty CDG l patients of different geographical origin. Moreover, intermediate activities were found in parents of CDG l patients, (see Van Schaftingen & Jaeken, 1995, ioc t), which strongly supports the hypothesis that the gene encoding phosphomannomutase is a major candidate gene for CDG 1.
  • This clone probably represents the full-length mRNA or PMM2.
  • the putative open reading frame is 738 bp long and predicts a protein of 246 amino-acids.
  • the genomic structure of the PMM2 gene has now been determined (see accompanying Fig. l-2b). Eight exons were identified, spanning approximately 20 kb of genomic DNA. The genomic structure of the PMMl and PMM2 genes is very similar, as all splice sites are conserved.
  • SSCP single-strand conformation polymo ⁇ hism
  • Table 1-1 Prevalence of the different mutations in the P M2 gene identified in CDGl patients from different geographical origins
  • Argl41-His; Asn216-Ile, Thr237-Met result in the replacement by a residue of markedly different size or hydrophobicity as compared to the normal enzyme (see accompanying Fig. 1- lb).
  • Val231-Met results in the replacement of a hydrophobic residue by a bulkier one, as do two other mutations (Alal08-Val and Val l29-Met) which concern semi-conserved positions. In the last case (Argl62-T ⁇ ), a basic residue is replaced by tryptophan. It is
  • the mutations detected in exons 4 and 5 were confirmed on genomic DNA by SSCP analysis after PCR amplification using intronic primers. This approach was used to follow the parental origin of the mutations in the informative families. For instance, in family 38, P 1 13L is of maternal origin and R 141 H is of paternal origin. The unaffected sib is a carrier of the PI 13L mutation, which fits with the linkage data (see accompanying Fig. 1-
  • CDG Type 1A CDG Type 1A
  • Figure 1-1 Sequence of the PMM2 cDNA and alignment of the predicted PMM2 protein with known phosphomannomutases.
  • Candida albicans phosphomannomutase PMM_CANAL (cana) (sequences obtained from
  • the predicted PMM2 protein is shorter at its amino- and carboxy-terminal ends, and has an internal
  • Gly-Asp deletion of two aminoacids after codon 56, when compared to PMMl .
  • This Gly-Asp is also absent in yeast and Candida albicans PMM (see Matthijs, et al, 1997, Ioc cit). The positions of the mutations identified in this study have been indicated.
  • Hindlll (H) map of the region has been constructed by (partial) digestions of cosmids 428D1 , 408C7, 422F4 and 404H6, isolated from a chromosome 16-specif ⁇ c arrayed cosmid library (see Longmire, J.L. , et al, Genetic Analysis : Techniques and
  • the 3' end of the gene is not present in cosmids and has been characterised from the overlapping BAC clones.
  • the position of an intragenic EcoRI site ( ⁇ ) is indicated, together with the location of the eight exons that divide the coding region of the gene.
  • the approximate size of the introns was obtained from estimations of the length of restriction fragments and PCR products.
  • Intron 5 is the smallest intron at 0.5 kb, while intron 4 spans 4.5 kb.
  • the size of intron 7 could not be determined with
  • Figure 1-3 Representative SSCP patterns for mutations identified in CDG l patients.
  • the P1 13L mutation is of maternal origin in this family.
  • the R141H mutation is inherited from the father.
  • the unaffected sib also inherited the maternal disease allele, as shown with polymo ⁇ hic markers that flank the gene, and is a carrier of the P1 13L mutation.
  • a probe, specific for human PMM2 was generated by PCR from cDNA derived from a human epidermoid carcinoma cell line (BB49; mouth tumour; kindly provided by F. Brasseur, Brussels) and its identity confirmed by sequencing.
  • the primers 5'- CCCAGCGCTCTGCCTCTTCGA-SVS'-ACGTTTAACATCCCATTTCGGA (nucleotides 15 to 383) were derived from the partial cDNA sequence present in the IMAGE clone 364509, available through Genbank (Accession Nos. AA022583 and AA022584). This cDNA clone was also kindly provided by UK HGMP Resource Centre.
  • the cDNA library screened to obtain the full-length cDNA was provided by J.C. Renauld (Brussels); it is a human T-cell leukemia (Mo cells-ATCC CRL8066 (see Chen, I.S.Y. , et ai, Nature, 305, 502-505, 1983)) library inserted between the NotI and BstXl sites of pcD ⁇ A l/Amp (Invitrogen). Approximately 180,000 colonies were transferred on GeneScreen "1" filters (Amersham) and hybridized overnight at 65°C in 1 M ⁇ aCl, 1 % SDS,
  • IMAGE clone 364509 is a partial cD ⁇ A clone, derived from the 5' end of the PMM2 mR ⁇ A, down to codon 143. Most probably, the downstream adenine-rich region (nucleotide positions 491 to 515) has allowed spurious oligo-dT priming during cD ⁇ A synthesis.
  • the probe generated by PCR (see above), was used for Southern blot analysis of a
  • genomic mapping panel 5 ⁇ g of each cell-line from the Coriell Mapping panel 2 ( ⁇ IGMS, Ca den, NJ) were digested with EcoRI. Filters were hybridized overnight in 50% formamide and 5 x SSPE, 10 x Denhardt's solution, 2 % SDS, and 100 ⁇ g/ml heparin, and washed at high stringency in 0.1 x SSPE, 0. 1 % SDS at 65°C for 30 minutes. Contrarily to what is stated by NIGMS, the human chromosome 14 cell line GM10478 also contains material derived from human chromosome 16 (pl3. 1-q22.1).
  • High density human BAC colony DNA membranes (#96055, Research Genetics) were screened with the PMM2 cDNA-probe, and the positive clones were purchased from Research Genetics. Chromosome 16-specific cosmids were identified on an arrayed cosmid library, provided by N. Doggett (LANL, Los Alamos). All clones were further characterised by restriction digestion with EcoRI and Hindlll, followed by Southern
  • the genomic structure of the PMM2 gene was determined by Southern hybridizations of the cosmid clones the oligonucleotide probes derived from the cDNA sequence. These oligonucleotides were typically 20 bp in length and were 5'- end labelled using [y- 32 P]ATP and T4-PNK and hybridizations were done in 6 x SSP ⁇ , 5 x Denhardt's, 0.5% SDS and 200 ⁇ g/ml heparin. Filters were washed in 2 x SSP ⁇ , 0.1 %
  • Gene-specific primers have also been used for genomic PCR on cosmids, BACs and total human DNA.
  • Standard PCR conditions were (50 ⁇ l) 50 ng DNA, 200 ⁇ M dNTP, 25 pmol each primer, 10 mM Tris-HCl, pH 8.3, 50 niM KC1, 1.5 mM MgCl 2 , 0.01 % gelatin and 1 U AmpliTaq polymerase (Perkin Elmer).
  • the Long Expand PCR kit was used, as prescribed by the manufacturer (Boehringer Mannheim).
  • Fluorescently-labelled primers (FITC, fluorescein-isothiocyanate) were used for cycle- sequencing on cosmid DNA, using Amersham's Thermo-sequenase kit and 2 to 6 ⁇ g of DNA.
  • FISH Fluorescence in situ hybridizations
  • DNA from the different BAC clones purified after alkaline lysis of the bacterial hosts, was labelled with biotin by nick-labelling, and used for FISH on human metaphase
  • PCR-fragments were purified using Qiaquick-PCR purification kit (Qiagen) and typically 50 to 100 ng was used with 1 pmol of fluorescently-labelled primer, for 15 to 22 cycles.
  • the first may be given the sub-heading "Prenatal diagnosis in CDG l families: beware of heterogeneity" and may be outlined as follows:
  • Carbohydrate-deficient glycoprotein syndrome type I (CDG l) 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 16pl3. 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.
  • CDG l Carbohydrate-deficient glycoprotein syndrome type I
  • OMIM Jaeken disease
  • CDGl glycosylation of glycoconjugates
  • the diagnosis of CDGl has an essential role which is observed after iso-electric focusing (IEF) of serum transferrins [3,4].
  • IEF iso-electric focusing
  • the transferrin assay does not reveal an aberrant pattern in amniotic fluid or in foetal blood [7,8].
  • CDG l locus has been mapped to chromosome I6p [9]
  • prenatal diagnosis has become possible by linkage analysis.
  • a major concern has arisen with the identification of genetic heterogeneity in CDG l . It was initially shown that the disease did not link to chromosome 16pl3 in a family with two affected siblings [10], and we describe another case in the present report. Obviously, a prenatal test with linked genetic markers on
  • chromosome 16p is of no value in such cases.
  • the diagnosis has not been confirmed by phosphomannomutase measurements or mutation analysis in the proband or in the parents.
  • the polymo ⁇ hic markers are ordered from telomeric to centromeric, top to bottom.
  • the dark chromosome is associated with the disease.
  • the disease is not linked to chromosome 16.
  • Enzymatic activities are expressed as a % mean values in controls.
  • NA no phosphomannomutase data.
  • the proband in family 15 ( Figure 2- la) has a phosphomannomutase deficiency with a value below 0.1 mU/mg protein in fibroblasts (HS in Table 1 in Van Schaftingen and Jaeken [11].
  • DNA analysis with linked genetic markers on cultured amniocytes revealed that the foetus was not affected but was a heterozygotus carrier. Phosphomannomutase measurements on these amniocytes showed intermediate values, compatible with a carrier status. A healthy baby was born, as expected.
  • the proband had the /l/OSV and the common R141H mutations, as shown by SSCP analysis and confirmed by sequencing [12].
  • SSCP analysis of DNA isolated from cultured trophoblasts revealed that the foetus had inherited the paternal R141H mutation but not the maternal A108V mutation ( Figure 2-2).
  • a phosphomannomutase value of 1.2 mU/mg protein was compatible with the carrier status.
  • family 45 no material was available from the
  • phosphomannomutase activity should first determine the phosphomannomutase activity in fibroblasts, leukocytes or lymphocytes of the proband, or if not possible, in leukocytes of the parents, and look for mutations in the PMM2 gene. If no phosphomannomutase deficiency is found, no prenatal diagnosis should be offered at this stage. If a phosphomannomutase deficiency is found and the mutations are identified, the prenatal diagnosis should primarily be based on the detection of mutations. If the mutations are not found, phosphomannomutase measurements should be combined with linkage analysis.
  • Wada Y. et al Structure of serum transferrin in carbohydrate-deficient glycoprotein
  • Van Schaftingen E. Jaeken J. : Phosphomannomutase deficiency is a cause of carbohydrate-deficient glycoprotein syndrome type I. FEBS Lett 1995; 311: 318-320.
  • Jaeken J. et ak Phosphomannomutase deficiency is the main cause of carbohydrate- deficient glycoprotein syndrome with type I isoelectrofocusing pattern of serum sialotransferrins. J Inher Metab Dis 1997; 20: 447-449.
  • CDGl carbohydrate-deficient glycoprotein syndrome type I
  • Carbohydrate-deficient glycoprotein syndrome type I (CDG l) is the paradigm of a group of genetic multi-system disorders characterized by a deficiency in the glycosylation pathway (see (1) for a recent review).
  • CDG l is an autosomal recessive disorder, with a major disease locus located on chromosome band 16pl3. Van Schaftingen and Jaeken (2) have
  • PMMl The first human phosphomannomutase or PMM gene, PMMl is localized on chromosome band 22ql3 and could therefore not harbor the primary defect in CDG l patients (5). More recently, PMM2 was mapped to the CDG l candidate region on chromosome 16p 13 (6). Mutations in PMM2 have been identified in CDG l patients with a documented phosphomannomutase deficiency,
  • PMM2 carries a number of mutations at positions corresponding to those where mutations
  • the CDG l gene had been localized to chromosome band 16pl3 by linkage analysis (7,8). Critical cross-overs in patients and in carriers have allowed us to reduce the candidate
  • Figure 3-2 presents the final mapping data on the PMM2 gene and our contribution to the physical map of chromosome 16 (10).
  • a YAC contig covering this minimal region was constructed, starting from the physical map which had been constructed for chromosome 16 at the Center for Human Genome Studies (Los Alamos National Laboratory, Los Alamos; see ( 1 1)).
  • the YAC contig spans the region of interest, be it that only a single YAC, 802G4, spans the gap between markers D 16S406 and
  • D16S3087 and is positive for marker s54A6.
  • the order of the markers on the Los Alamos map was accepted, but markers D16S513 and D 16S406 might be switched. There is conflicting data in the literature about the order of these markers and their position relative to D 16S495 and D 16S502 ((9) and data from the Whitehead Institute and from Los
  • YAC 802G4 was unstable in our hands, and a new screening of the CEPH MEGA YAC library with marker D16S3020 did not identify any other YAC clones to bridge the region. Therefore, a BAC library was screened with markers s52C5, D 16S3087, s54A6, D 16S3020, D 16S406 and D 16S502, and several BACs could be positioned on the map and linked to the YAC contig. Overlaps between different BACs (as indicated in figure 3-2) were identified by hybridization of Southern blots of the BACs with end fragments, generated by vectorette PCR. No BACs were identified for D 16S502. As soon as the
  • PMM2 cDNA was available (6), it was used as a probe for hybridization of Southern blots of the YACs: positive signals were obtained for YAC clones 802G4 and 909F5. These results were confirmed by PCR on YAC DNA with primers specific for exon 4 of PMMl. This result localizes the gene between D16S3020 and D16S502/D16S406. Marker D16S3020 is close to (within 20 kb) and upstream (5') of the PMM2 gene, because positive signals were obtained by PCR with the BAC clones and several of the cosmids that contain the 5' end of the gene (see below); the chromosomal orientation of the gene has not been determined.
  • PMMl has been unambiguously localized to 22ql3 (5). Hybridizations at low stringency with a PMMl cDNA probe on a chromosomal mapping panel did not reveal any other signals. PMM2 was identified after a cDNA sequence, similar to but different from PMMl was identified among the IMAGE data in Genbank (6). Several BAC clones were isolated
  • the samd cDNA probe identified two EcoRI fragments of )12 kb and 0.8 kb (not shown). The larger band was also observed with DNA isolated from the cell-lines GM 10567, which contains chromosome 16 as the sole human chromosome in a mouse background, and GM 10487, known to contain chromosome 14 and material derived from chromosome 16p.
  • the 0.8 kb fragment originates from chromosome 18, because it was only observed with DNA from GM1 1010, a cell-line with human chromosome 18 in a hamster background (not shown).
  • FISH analysis revealed that BAC clones 27D21 , 41M16 and 342N15, which contain the ) 12 kb EcoRI fragment, mapped to the candidate region on the short arm of chromosome 16 (16pl3), and contained (part of) the PMM2 gene, while BAC clones 147L11 , 1 10G4, 322C 1 and 31 1N20, containing the
  • the region upstream of PMMl ⁇ has no apparent characteristics of a promoter region, but contains two
  • cosmids were isolated by hybridization of arrayed chromosome-specific cosmid libraries from chromosome 22 (reference 15) and chromosome 16 (reference 16) with the respective full length cDNA probes.
  • FIG. 3-5 shows the genomic structure of PMMl and PMM2 with a detailed EcoRI and
  • Hindlll map The position of the different cosmids is indicated.
  • PMM2 the overlapping BAC clones have been included.
  • the intron lengths have been determined by PCR. They vary from 160 bp (intron 3) to 4.9 kb (intron 5) in PMMl and from 0.5 kb (intron 5) and ) 4 kb (intron 7) in PMMl.
  • the PMMl gene thus spans approximately 13 kb of genomic DNA, whereas the size of
  • PMMl is at least 17 kb.
  • PMMl is the CDGl gene, as mutations in this gene have been identified in patients with the syndrome (6). There is currently no disease associated with defects in PMMl .
  • a YAC contig and partial BAC contig were constructed across the CDG l minimal region, which had been reduced to an interval on the genomic map of less than 1 cM between markers D 16S406 and D 16S404.
  • the positional cloning approach and the physical mapping effort were suspended due to the availability of a candidate gene, i.e. a cDNA for PMMl (6).
  • PMMl has now been precisely mapped to the candidate region for CDGl, within 20 kb of D 16S3020.
  • PMMl has previously been isolated by a similar approach and mapped to bin 16.2 on chromosome 22 (reference 5).
  • both proteins display phosphomannomutase activity, be it that the
  • Candida albicans PMM and the difference is mainly due to a deletion of 7 aminoacids in the N-terminal part of the protein.
  • PGM phosphoglucomutase
  • 13 and 22ql l-ql3 may also be paralogous genes and related MYH genes are clustered on 14q 12 and 17pl3, whereas members of the somatostatin receptor family have been mapped to 22q l3 (SSTR3), 16p l3 (SSTR5), 14ql3 (SSTR1), 17q24 (SSTR2) and 2Oql 1.2 (SSTR4).
  • SSTR3 22q l3
  • SSTR5 16p l3
  • 14ql3 SSTR1
  • 17q24 SSTR2
  • 2Oql 1.2 SSTR4
  • the gene on chromosome 18 is a processed pseudogene of PMMl. It is closely related to the PMMl cDNA sequence and the absence of homology in the 5' upstream region suggests that it has arisen by retrotransposition. The 3' tail of the processed pseudogene has not been fully sequenced, so it is not known whether the poly A stretch, characteristic for processed pseudogenes. is present. The presence of a stopcodon at position 143 implies that
  • this gene cannot be translated into a functional enzyme. Most likely, this processed pseudogene has never been actively transcribed. The fact that the pseudogene has
  • YAC analysis YAC clones were obtained from "mega" YAC libraries constructed at CEPH. The selected
  • YAC clones were grown on ura " t ⁇ " plates and individual colonies were picked, grown in selective AHC medium, and analyzed by pulsed-field gel electrophoresis (PFGE) to check the size of the insert.
  • PFGE pulsed-field gel electrophoresis
  • Yeast DNA was prepared in agarose plugs (200 ⁇ l) according to Ragoussis (30). The STS content of the YACs was checked by PCR. For PCR analysis, a quarter of a plug was dissolved in 1 ml H,0 and 5 ⁇ l of this solution was used in 25 ⁇ l reaction volume, under standard PCR conditions.
  • 951F4, 925B10 and 936B6 was blotted and the filter was hybridized with the PMM2 cDNA probe under standard conditions (31).
  • the cDNA probe contained the entire coding, and part of the 3' untranslated region (to nucleotide 1077).
  • Cosmids for PMM2 were identified by hybridization of a chromosome 16 specific arrayed cosmid library (16).
  • BAC clones for markers s52C5, D16S3087 and D16S406 and PMM2 were obtained by hybridization of "high density human BAC colony DNA membranes" (Research Genetics).
  • the probe for s52C5 was generated by PCR from the amplicon of s52C5 and random primed labeling.
  • the specific PCR primers were used as probes for oligonucleotide hybridization.
  • the primer sequences were obtained from GDB.
  • PMM2 the insert of the PMM2 cDNA clone was purified and labeled.
  • the BAC clones for marker s54A6 were isolated by PCR analysis of BAC DNA pools (Research Genetics).
  • BAC ends were rescued by vectorette PCR.
  • Vectorette libraries were constructed as described by Riley et al. (32) with minor modifications.
  • 200 ng DNA was digested with 5 U Rsal and ligated to 6 pmol Rsal vectorette cassette (top strand: 5'- CAAGGAGAGGACGCTGTCTGTCGAAGG-3'; bottom strand: 5'- CTCTCCCTTCTCGAATCGTAACCGTTC-3').
  • the end fragments were then amplified by PCR with the universal vectorette primer (5'-
  • BAC clones for PMM2 and cosmids for PMMl and PMM2 were analyzed by hybridization of Southern blots of EcoRI and Hindlll digestions. cDNA or genomic probes were labeled with ⁇ AP-dCTP by random primed labeling and used for hybridization overnight in 50% formamide, 5X SSPE, 10X Denhardt's solution, 2% SDS and lOO ⁇ g/ml heparin. Filters
  • Oligonucleotide probes derived from the cDNA sequence and used for the determination of the genomic structure, were typically 20 bp long and 5 '-labeled using y- 32 P-ATP and T4 PNK according to established protocols (31). Hybridizations with oligonucleotide probes were done in 6X SSPE, 5X Denhardt's solution, 0.5 % SDS and 200 ⁇ g/ml heparin. Filters were washed in 2X SSPE, 0. 1 % SDS at 45 ° C for 5 min. A detailed Hindlll map of cosmids 428D1,
  • 408C7, 422F4 and 404H6 was generated by partial digestion.
  • Four ⁇ g of purified cosmid DNA was digested to completion with NotI (20U) in 20 ⁇ l, and further digested with Hindlll (5U) in lOO ⁇ l. Aliquots of 25 ⁇ l were taken at 2, 5, 10 and 60 minutes. The reaction was stopped by adding l ⁇ l 0. 1 M EDTA. Samples were analyzed by field-inversion gel electrophoresis (FIGE), blotted and hybridized with T3 and T7 oligonucleotides.
  • FIGE field-inversion gel electrophoresis
  • Gene-specific primers have been used for genomic PCR on cosmids, BACs and total human DNA under standard conditions.
  • the Long Expand PCR kit was used as prescribed by the manufacturer (Boehringer Mannheim).
  • Fluorescently labeled primers FITC, fluorescein-isothiocyanate
  • FITC fluorescein-isothiocyanate
  • CDG Carbohydrate-deficient glycoprotein
  • Phosphomannomutase deficiency is the main cause of carbohydrate-deficient glycoprotein syndrome with type I isoelectrofocusing pattern of serum sialotransferrins. J. Inher. Metab.
  • PMM the human homologue of SEC53 or yeast phosphomannomutase
  • HMOX1 heme oxygenase- 1
  • HMOX2 heme oxygenase-2
  • FIGURE LEGENDS (Third Section)
  • Figure 3-1 Delineation of the candidate region for CDGl by genetic analysis of families with a documented PMM deficiency in the proband.
  • the genetic map of the CDG l region is shown, and the polymo ⁇ hic markers were ordered according to the available maps (10, 1 1), with the telomere to the left, centromere to the ⁇ ght.
  • the previously published candidate region extends from D 16S406 to D 16S405 and spans 13 cM (7).
  • the regions represented by thin lines were excluded by cross-overs between the marker alleles and the disease patients or carriers in the different families ( : not informative).
  • the minimal region is defined by markers D 16S406 and D 16S404, that are less than 1 cM apart on the genetic map.
  • Figure 3-2 Physical map of the CDG l candidate region on chromosome band 16pl3.2.
  • a YAC contig is shown for the region from D 16S502 to D 16S404 (not to scale).
  • Polymo ⁇ hic markers and STSs are shown, whereby the vertical lines indicate positive hits by PCR.
  • the BAC clones were isolated by screening a BAC library with
  • Figure 3-3 Evidence that sequences related to PMMl are present on chromosome 18p.
  • BACs 1 10G4, 322C1 and 311N20 Similar results were obtained with BACs 1 10G4, 322C1 and 311N20.
  • the BAC clones containing the ) 12 kb fragment are derived from chromosome 16p (reference 6).
  • Figure 3-4 Alignment of the PMMl cDNA sequence with the genomic sequence of the processed pseudogene PMMl ⁇ on chromosome 18.
  • the translation of the reading frame for PMMl is given above the DNA sequence (single letter codes).
  • the PMM2 sequence is numbered from the ATG codon, the sequence of the pseudogene starts at the upstream EcoRI site, which was used for cloning (not shown). Due to insertions and deletions in the PMMl ⁇ sequence, the reading frame is interrupted or altered several times. The open reading frame of the pseudogene ends at the position corresponding to codon 143 in PMM2. The homology was calculated from the alignment shown in the figure. When the sequence difference would lead to a different codon in the putative reading frame of the pseudogene, the corresponding amino acid letter code is represented in bold. The codons in PMM2 that were found mutated in CDGl patients, and
  • arrowhead indicates the nucleotide of interest.
  • An EcoRI site present in the coding region
  • Figure 3-6 Alignment of the exon/intron boundaries for PMMl and PMMl.
  • the coding sequence is represented in uppercase. Values between square brackets represent additional nucleotides present in one gene as compared to the other.
  • the third may be given the sub-heading "Lack of homozygotes for the most frequent disease allele in carbohydrate-deficient glycoprotein syndrome type IA" and may be outlined as follows:
  • Carbohydrate-deficient glycoprotein syndrome type I (CDG l or Jaeken syndrome, OMIM
  • R141H/D188G which is prevalent in Belgium and the Netherlands, is associated with a severe phenotype and a high mortality. Apart from this, there is only a limited relation between the genotype and the clinical phenotype.
  • Carbohydrate-deficient glycoprotein (CDG) syndromes are a series of genetic disorders characterized by defective N-glycosylation of serum and cellular proteins (Jaeken et al. 1980; Jaeken and Carchon 1993; Jaeken et al. 1997a; Jaeken and Casaer, 1997).
  • CDG type I CDG l
  • CDG type I CDG l
  • CDG l is the most frequent type. It is a severe disorder which presents neonatally. There is a life-threatening liver insufficiency (with an
  • CDG type II CDG2
  • CDG3 type III
  • CDG4 type IV
  • CDG2 is caused by a deficiency of UDP-GlcNAc: ⁇ -6-D-mannoside ⁇ 8- l ,2-N-acetylglucosaminyltransferase II (GnT II), located in the Golgi apparatus, and mutations in the GnT II gene (MGAT2) on 14q21 have been identified (Jaeken et al. 1994; Tan et al. 1996). The causes of CDG3 and CDG4 remain unknown. CDGl is inherited in an autosomal recessive manner and its locus was mapped to chromosome 16pl3 (Martinsson et al. 1994).
  • CDG type IA phosphomannomutase deficiency
  • CDG l Fifty-six patients from 12 countries were included in the study. All except 2 were of Caucasian origin. A diagnosis of CDG l was made in all these patients, based on the clinical manifestations, substantiated by the typical IEF pattern of serum transferrins: there is a strong reduction in the intensity of the normal tetrasialotransferrin band, and a concomitant increase in the disialo- and asialotransferrin concentration. The phosphomannomutase deficiency was documented in most cases (see table 4-2 and Results). The clinical features of CDG l patients have recently been reviewed in Jaeken et al. (1997a) and Jaeken and Casaer (1997).
  • the neurological picture includes abnormal eye movements, combined with slow head movements in the neonatal period and axial hypotonia with hyporeflexia. Most children present with an alternating strabismus. There is a severe psychomotor retardation and failure to thrive, with ataxia and sometimes deafness. Additional features, presenting after infancy, are hypogonadism, retinitis pigmentosa. joint contractures and stroke-like episodes. Most patients never achieve walking without support, but there is no regression. Other symptoms include: mild facial dysmo ⁇ hism (with large, somewhat dysplastic ears) and skeletal deformities, and a typical subcutaneous deposition of adipose tissue ("fat pads").
  • the blood samples and/or fibroblast or lymphoblast cultures from patients were provided to us with a request for enzymatic assays and molecular diagnosis, and the referring physicians and the families have been informed about the results. Amniocytes were analyzed in the context of prenatal diagnosis.
  • DNA was isolated from fresh blood, or fibroblast or lymphoblast cultures from patients using a high-salt extraction procedure. Based on the available sequence, primers were designed for the PCR amplification of 9
  • DNA fragments suitable for SSCP analysis.
  • One primer in each pair was FITC (fluorescein-isothiocyanate) labeled.
  • the primers sequences are given in table 4-1.
  • PCR reactions were typically done in 25 ⁇ l, and the cycling conditions were 30" at 95°C, 30" at 50 to 60°C, 30" at 72°C, for 32 cycles.
  • Ten to 15 ⁇ l of the PCR products were mixed with an equal volume of formamide, denatured for 5' at 95°C, loaded onto a non- denaturing polyacrylamide gel (0.5X Hydrolink MDE, J.T.
  • the mutation screening was done by a non-radioactive SSCP analysis of fragments amplified from genomic DNA.
  • the PCR fragments encompassed the individual exons, and the flanking sequences. Whenever a fragment revealed an aberrant SSCP pattern, the
  • Phosphomannomutase measurements are also useful for the identification of carriers. If cells from an affected child are not available, indirect evidence can be obtained from the phosphomannomutase activities in leukocytes from the parents. This has proven worthwhile in 2 cases with an urgent request for prenatal diagnosis, in which we derived information on the phosphomannomutase deficiency from the parents, before initiating the molecular analysis. In view of the genetic heterogeneity, prenatal testing should only be offered in
  • the R141H mutation is by far the most frequent (Caucasian) one.
  • the D188G mutation seems to be restricted to Belgian (Flemish) and Dutch patients.
  • the V44A mutation is probably of Spanish origin, while the D65Y mutation, homozygous in a Portuguese patient, is also found in a French patient with Portuguese ancestors.
  • the V129M mutation might well be of Italian origin. We have not tried to link the most frequently observed mutations to a common haplotype of the flanking polymo ⁇ hic markers, but it seems that some mutations are old mutations that have been present in the different populations for centuries.
  • the R141H mutation is caused by a CGC to CAC transition, but the equally likely CGC to TGC transition (R141C, which is found in a processed pseudogene, derived from PMMl and located on chromosome 18p; see SchoUen et al, 1998) has not been observed in patients.
  • the R 141H mutation may be an old mutation, like the frequent ⁇ F508 in cystic fibrosis (Morral et al. 1993).
  • some mutations must have occurred independently on different chromosomes in the different populations.
  • the R123G mutation shared by 2 Spanish and 2 Dutch patients, is syntenic with a polymo ⁇ hism at nucleotide 324 in the coding region, and was also identified in a Japanese and a French patient, but without the polymo ⁇ hism.
  • the T237R and T237M mutations are caused by C to T transitions on opposite strands in the same CpG- dinucleotide. It is known that CpG-dinucleotides are hot spots for mutations. An interesting observation is that the corresponding CpGs have also been mutated in the processed
  • a complex genotype has been observed in 2 families. DNA from the parents has been used to establish the phase of these mutations.
  • the A233T mutation is respectively syntenic with the T237R and T237M mutations in patients SN/SJ and LS, whereas the T237R is present on the other allele in the latter family.
  • the alternative combination of the T237R and T237M mutations with the A233T mutation is remarkable, and the occurrence of 2 different mutations in the same codon on the 2 chromosomes in patient LS is intriguing.
  • the D65Y is rare, but the F1 19L is a frequent mutation, and the occurrence of one F 1 19L/F 1 19L patient in a series of 56 reflects Hardy-Weinberg equilibrium. The observation suggests that these are mild mutations. This might be reflected by the relatively high value of the residual phosphomannomutase activity in fibroblasts of the D65Y/D65Y patient. However, the same D65Y mutation, in combination with the R141H mutation was found in a patient with a severe phenotype, who died at a very young age (patient 2). The FI 19L/R141H is a particularly frequent genotype, thus the combination of the two most frequent disease mutations is not lethal.
  • Eeckels R Familial psychomotor retardation with markedly fluctuating serum proteins, FSH and GH levels, partial TBG deficiency, increased serumarylsulphatase A and increased CSF protein: a new syndrome ?
  • CDG Carbohydrate-deficient glycoprotein
  • yeast phosphomannomutase SEC53
  • Candida albicans PMM Cons. : conserved
  • A233T is observed in combination with either the T237R or the T237M mutation (patients 54 and 55).
  • Sequence analysis revealed the occurrence of the F I 19L mutation in the homozygous state.
  • the results of the direct sequencing are shown for A) a normal control (homozygous for the normal sequence); B) the father of patient SN (heterozygous) and C) patient PG (homozygous for the mutation). Codon 119 is boxed.

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Abstract

L'invention concerne, entre autres, une molécule d'ADN purifiée et isolée, caractérisée en ce qu'elle comprend une séquence nucléotidique codant une protéine humaine phosphomannomutase 2 (PMM2) ou une partie de celle, comme on peut le voir sur les figures jointes.
EP98925519A 1997-04-30 1998-04-30 Syndrome de type 1 consecutif a une deficience glucidique de la glycoproteine Withdrawn EP0981632A2 (fr)

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GBGB9708851.2A GB9708851D0 (en) 1997-04-30 1997-04-30 Carbohydrate-deficient glycoprotein syndrome type I
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GBGB9801719.7A GB9801719D0 (en) 1998-01-27 1998-01-27 Carbohydrate-deficient
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