View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Am. J. Hum. Genet. 62:542–550, 1998 Lack of Homozygotes for the Most Frequent Disease Allele in Carbohydrate-Deficient Glycoprotein Syndrome Type 1A Gert Matthijs,1 Els Schollen,1 Emile Van Schaftingen,3 Jean-Jacques Cassiman,1 and Jaak Jaeken2 1Center for Human Genetics, University of Leuven, and 2Center for Metabolic Diseases, University Hospital Leuven, Leuven; and 3Laboratory of Physiological Chemistry, International Institute of Cellular and Molecular Pathology and University of Louvain, Brussels Summary Introduction Carbohydrate-deficient–glycoprotein syndrome type 1 Carbohydrate-deficient glycoprotein (CDG) syndromes (CDG1; also known as “Jaeken syndrome”) is an au- are a series of genetic disorders characterized by defec- tosomal recessive disorder characterized by defective gly- tive N-glycosylation of serum and cellular proteins (Jae- cosylation. Most patients show a deficiency of phos- ken et al. 1980, 1997b; Jaeken and Carchon 1993; Jae- phomannomutase (PMM), the enzyme that converts ken and Casaer 1997). At present, four types of CDG mannose 6-phosphate to mannose 1-phosphate in the have been described on the basis of serum transferrin synthesis of GDP-mannose. The disease is linked to chro- isoelectrofocusing (IEF). CDG type 1 (CDG1 [MIM mosome 16p13, and mutations have recently been iden- 212065]) is the most frequent type. It is a severe disorder tified in the PMM2 gene in CDG1 patients with a PMM that presents neonatally. There is a life-threatening liver deficiency (CDG1A). The availability of the genomic se- insufficiency (with an overall 20% mortality during the quences of PMM2 allowed us to screen for mutations neonatal period), combined with a severe cerebellar dys- in 56 CDG1 patients from different geographic origins. function and peripheral neuropathy, leading to severe By SSCP analysis and by sequencing, we identified 23 psychomotor retardation. These children also have skel- different missense mutations and 1 single-base-pair de- etal deformities and a characteristic deposition of adi- letion. In total, mutations were found on 99% of the pose tissue (Jaeken and Carchon 1993; Jaeken et al. disease chromosomes in CDG1A patients. The R141H 1997b). CDG type 2 (CDG2), type 3 (CDG3), and type substitution is present on 43 of the 112 disease alleles. 4 (CDG4) represent only two cases each (Stibler et al. However, this mutation was never observed in the ho- 1993, 1995; Jaeken et al. 1994). CDG2 is caused by a mozygous state, suggesting that homozygosity for these deficiency of UDP-GlcNAc:a-6-D-mannoside b-1,2-N- alterations is incompatible with life. On the other hand, acetylglucosaminyltransferase 2 (GnT 2), located in the patients were found homozygous for the D65Y and Golgi apparatus, and mutations in the GnT 2 gene F119L mutations, which must therefore be mild muta- (MGAT2) on 14q21 have been identified (Jaeken et al. tions. One particular genotype, R141H/D188G, which 1994; Tan et al. 1996). The causes of CDG3 and CDG4 is prevalent in Belgium and the Netherlands, is associ- remain unknown. ated with a severe phenotype and a high mortality. Apart CDG1 is inherited in an autosomal recessive manner, from this, there is only a limited relation between the and its locus has been mapped to chromosome 16p13 genotype and the clinical phenotype. (Martinsson et al. 1994). Linkage to the region between D16S406 and D16S500 has been confirmed in 10 of 11 informative families (Matthijs et al. 1996). In one family with two affected siblings, the disease was, however, not linked to chromosome 16p, indicating genetic hetero- geneity for CDG1 (Matthijs et al. 1996). Biochemical evidence has long suggested a basic defect in the synthesis of the dolichol-P-oligosaccharides (syn- thesis of the asparagine-N-linked oligosaccharides) in Received October 16, 1997; accepted for publication January 12, 1998; electronically published March 6, 1998. the endoplasmic reticulum (ER) (Jaeken et al. 1984; Address for correspondence and reprints: Dr. Gert Matthijs, Center Wada et al. 1992). In 1995, Van Schaftingen and Jaeken for Human Genetics, University of Leuven, Campus Gasthuisberg (1995) identified a deficiency of phosphomannomutase O&N6, Herestraat 49, B-3000 Leuven, Belgium. E-mail: (PMM) activity in patients with CDG1. This observation [email protected] ᭧ 1998 by The American Society of Human Genetics. All rights reserved. has been confirmed in more than 50 CDG1 patients from 0002-9297/98/6203-0007$02.00 different geographic origins (Jaeken et al. 1997a). We 542 Matthijs et al.: Mutations in the PMM2 Gene in CDG1A 543 have recently cloned the human PMM gene PMM2 and drome, or status epilepticus. Salient features in some have shown that it is the CDG1 gene (Matthijs et al. patients are listed in table 1. 1997a). Another PMM gene, PMM1, could be assigned The blood samples and/or fibroblast or lymphoblast to chromosome 22q13 (Matthijs et al. 1997b). Both cultures from patients were provided to us after a request PMM1 and PMM2 have been expressed in Escherichia for enzymatic assays and molecular diagnosis, and the coli and have been found to be active proteins (Pirard referring physicians and the families have been informed et al. 1997; E. Van Schaftingen and M. Pirard, unpub- about the results. Amniocytes were analyzed in the con- lished data). We have previously reported 11 missense text of prenatal diagnosis. mutations in 16 CDG1 patients with a documented PMM deficiency (CDG1A) (Matthijs et al. 1997a). These SSCP Analysis and Sequencing mutations have been identified at the cDNA level, after reverse transcriptase–PCR amplification, followed by DNA was isolated either from fresh blood or from SSCP analysis and sequencing. To search for mutations fibroblast or lymphoblast cultures from patients by use in genomic DNA, the PMM2 intron/exon structure has of a high-salt-extraction procedure. On the basis of the been determined, whereby eight exons have been iden- available sequence, primers were designed for the PCR tified (Schollen et al. 1998), and primers flanking each amplification of nine DNA fragments suitable for SSCP translated exon have been designed. We here describe analysis. One primer in each pair was labeled with FITC. the results of an exhaustive mutation analysis of the The primer sequences are given in table 2. PCR reactions m PMM2 gene in patients with a documented PMM were typically done in 25 l, and the cycling conditions Њ Њ Њ Њ deficiency. were 30 s at 95 C, 30 s at 50 –60 C, and 30 s at 72 C, for 32 cycles. A 10–15-ml portion of each PCR product was mixed with an equal volume of formamide and then Patients and Methods was denatured for 5 min at 95ЊC, loaded onto a non- denaturing polyacrylamide gel (0.5 # Hydrolink MDE Patients [J. T. Baker] in 0.6 # TBE [10 # TBE ϭ 1 M Tris, Fifty-six patients from 12 countries were included in 0.82 M boric acid, and 10 mM EDTA]), and electro- Њ the study; all except 2 were of Caucasian origin. A di- phoresed for 10 h at 4 C at 400 V. The gels were directly agnosis of CDG1 was made in all these patients, on the scanned on a Fluorimager (Vistra), and the signals were basis of clinical manifestations, and was substantiated analyzed with the ImagequaNT software (Molecular by the typical IEF pattern of serum transferrins: there is Dynamics). both a strong reduction in the intensity of the normal The PCR fragments were sequenced by cycle sequenc- tetrasialotransferrin band and a concomitant increase in ing or solid-phase sequencing. Prior to cycle sequencing the disialo- and asialotransferrin concentration. PMM with the Thermosequenase kit (Amersham), the PCR deficiency was documented in most cases (see table 1 fragments were purified by use of the Qiaquick-PCR and the Results section). The clinical features of CDG1 purification kit (Qiagen), and, typically, 50–100 ng was patients have recently been reviewed by Jaeken and Ca- used with 1 pmol of fluorescently labeled primer, for saer (1997) and Jaeken et al. (1997b). In brief, the neu- 15–22 cycles. Solid-phase sequencing using a biotiny- rological picture includes abnormal eye movements, lated template and streptavidin-coated DynaBeads (Dy- combined with slow head movements in the neonatal nal) was done according to established procedures. period, and axial hypotonia with hyporeflexia. Most children present with an alternating strabismus. There Results is a severe psychomotor retardation and failure to thrive, with ataxia and, sometimes, deafness. Additional fea- Fifty-six patients and their affected siblings were in- tures, presenting after infancy, are hypogonadism, reti- cluded in this series. Included are three pairs of patients nitis pigmentosa, joint contractures, and strokelike ep- in which each pair has at least one common ancestor isodes. Most patients never attain the ability to walk (table 1). The diagnosis of CDG1 with PMM deficiency without support, but there is no regression. Other symp- was confirmed biochemically in 46 patients for whom toms include mild facial dysmorphism (with large, some- fibroblasts, lymphoblasts, or fresh leukocytes were avail- what dysplastic ears), skeletal deformities, and a typical able; in 2 of these patients, a partial deficiency of PMM subcutaneous deposition of adipose tissue (“fat pads”). was found in fibroblasts (patients 47 [SAO] and 48 There is a mild to moderate hepatomegaly, and some [GSS]), and, in 1 family (family 4, patients FG and FP, infants develop pericardial effusion and/or cardiomy- both affected), intermediate values were measured in opathy. Approximately 20% of the patients die before lymphoblasts. From the remaining 10 patients, whose the age of 5 years, as a consequence of liver failure, diagnosis had been made previously on clinical grounds, severe infection, cardiac insufficiency, nephrotic syn- no cells were available for the enzymatic assay; in 7 of 544 Am.