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Congenital disorder of glycosylation Ic due to a de novo deletion and an hALG-6 mutation
BBRC Biochemical and Biophysical Research Communications 339 (2006) 755–760 www.elsevier.com/locate/ybbrc
Congenital disorder of glycosylation Ic due to a de novo deletion and an hALG-6 mutation Erik A. Eklund a,1, Liangwu Sun a,1, Samuel P. Yang b, Romela M. Pasion c, Erik C. Thorland d, Hudson H. Freeze a,* a
Glycobiology and Carbohydrate Chemistry Program, The Burnham Institute, 10901 N Torrey Pines Road, La Jolla, CA 92037, USA b GenetiCare Medical Associates, 417 Mace Boulevard, Davis, CA 95616, USA c Sutter Metabolic Clinic, 5271 F Street, Sacramento, CA 95819, USA d Clinical Cytogenetics Laboratory, Mayo Clinic, 200 First St. S.W., Rochester, MN 55905, USA Received 25 October 2005 Available online 29 November 2005
Abstract We describe a new cause of congenital disorder of glycosylation-Ic (CDG-Ic) in a young girl with a rather mild CDG phenotype. Her cells accumulated lipid-linked oligosaccharides lacking three glucose residues, and sequencing of the ALG6 gene showed what initially appeared to be a homozygous novel point mutation (338G > A). However, haplotype analysis showed that the patient does not carry any paternal DNA markers extending 33 kb in the telomeric direction from the ALG6 region, and microsatellite analysis extended the abnormal region to at least 2.5 Mb. We used high-resolution karyotyping to confirm a deletion (10–12 Mb) [del(1)(p31.2p32.3)] and found no structural abnormalities in the father, suggesting a de novo event. Our findings extend the causes of CDG to larger DNA deletions and identify the first Japanese CDG-Ic mutation. 2005 Elsevier Inc. All rights reserved. Keywords: CDG-Ic; Developmental delay; Germ-line mutation; Genomic deletion; hALG6
Congenital disorders of glycosylation (CDG)2 constitute a group of metabolic syndromes where the primary defects either impair the formation of the lipid-linked oligosaccharide (LLO) precursor of N-linked glycosylation (Type I) or affect processing of the protein-bound sugar chains (Type II) [1–3]. The most common subtype, CDG-Ia, was first described in 1980 [4], and since then 12 Type I and 6 Type II subtypes have been reported. Less than a thousand patients with CDG have been diagnosed so far, but the true incidence probably is much higher (based on frequency *
Corresponding author. Fax: +1 858 713 6281. E-mail address: [email protected] (H.H. Freeze). 1 These authors contributed equally to this work. 2 Abbreviations: CDG, congenital disorders of glycosylation; GFP, green fluorescent protein; Glc, glucose; GlcNAc, N-acetyl glucosamine; hALG6, human ortholog of yeast Alg6; LLO, lipid-linked oligosaccharide; Man, mannose; PMI, phosphomannose isomerase; PMM, phosphomannomutase; UPD, uniparental disomy. 0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.11.073
analysis for certain common mutations). The spectrum of symptoms and degree of severity in CDG are unmatched by any other congenital syndrome, ranging from mentally normal children with protein-losing enteropathy and coagulopathy in CDG-Ib, to extremely severely affected children with intractable seizures, profound developmental delay, and hypotonia in e.g., CDG-Id, CDG-Ie, and CDG-If. CDG can easily be ruled out with an isoelectric focusing or mass spectrometric analysis of serum transferrin. CDG-Ic (OMIM 603147) is caused by mutations in the human ortholog of yeast Alg6, hALG6, which encodes the glucosyltransferase that adds the first glucose (Glc) residue to the growing LLO chain. It is the second most common CDG subtype with more than 30 known cases. The CDG-Ic phenotype is usually milder than CDG-Ia, and includes psychomotor retardation with delayed walking and speech, seizures, muscular hypotonia, and on occasion,
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protein-losing enteropathy [3,5,6]. LDL and clotting factor XI are generally lower in these patients than in patients affected by other CDG syndromes [15]. Sixteen different hALG6 mutations causing CDG-Ic have been described so far (see www.euroglycanet.org; the European CDG Network Database in Leuven, Belgium) where A333V accounts for the majority of the alleles [6,7]. We describe a new case of CDG-Ic, identify the first Japanese mutation, and find a large deletion of the patientÕs paternal chromosome 1, including the hALG6 locus. This is to our knowledge only the second time CDG has been connected to a large genomic deletion [8]. Methods and materials Approval of human research. Approval of human research was obtained from the Institutional Review Boards of The Burnham Institute, La Jolla, CA, and Sutter Health Central Area, Sacramento, CA. Materials. Tissue culture materials, TRIzol Reagent, SuperScript OneStep RT-PCR kit, Lipofectamine 2000, Escherichia coli Stbl3 cells, pLentiV5/TOPO plasmid, and Lentiviral Expression System were from Invitrogen (Carlsbad, CA). The oligonucleotides were ordered from Genbase (San Diego, CA). The Microsorb-MV NH2 HPLC column was purchased from Varian Instruments (Walnut Creek, CA), [2-3H]mannose (Man) (20 Ci/mmol) was from American Radiolabeled Chemicals (St. Louis, MO), and fetal bovine serum was from HyClone (Logan, UT). The pGEM-T vector and the Wizard Genomic DNA Purification Kit were from Promega (Madison, WI), the restriction enzymes and T4 DNA ligase were from New England Biolabs (Beverly, MA), and the Pfu DNA polymerase was from Stratagene (La Jolla, CA). All other chemicals used in this study were of analytical grade and purchased from Sigma Chemicals (St. Louis, MO). Cell culture. Primary fibroblast cultures were set up from a skin biopsy of the patient after a signed consent form was obtained from the parents. Control fibroblasts were from age-matched controls. All cells were grown in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin sulfate at 37 C with 5% CO2. Phosphomannose mutase and phosphomannose isomerase assays. Enzymatic assays of phosphomannose mutase (PMM) (deficient in CDG-Ia) and phosphomannose isomerase (PMI) (deficient in CDG-Ib) were performed on fibroblast extracts essentially as described previously [9,10]. LLO analysis. Metabolic labeling and preparation of LLOs from fibroblast cultures grown in 100 mm plates were performed as described earlier [11]. They were analyzed by amine adsorption hplc on a Microsorb NH2 column using an acetonitrile gradient (65–35%). 2-Aminobenzamidelabeled Man5–9GlcNAc2 oligosaccharides (Prozyme, San Leandro, CA) were used as internal standards. Analysis of cDNA. The hALG6 cDNA was synthesized and sequenced essentially as described before [6]. The primers used for RT-PCR and sequencing were: 5 0 -AAG AAG TGA TTG ACC ACG TTT-3 0 and 5 0 AAT GGT AAT TTC ATT TAT ACA TAG C-3 0 . Other primers used for sequencing were: 5 0 -CCT AGG GTC ACT GGC-3 0 ; 5 0 -GTG TCA CTA CCA GTC-3 0 ; 5 0 -GAC TGG TAG TGA CAC-3 0 , and 5 0 -GCC AGT GAC CCT AGG-3 0 . Analysis of hALG6 genomic DNA. Genomic DNA was isolated from fibroblasts using the Wizard Genomic DNA Purification Kit. Primers used to amplify exon 4 and its boundary region of hALG6 were: 5 0 -TGC TAA AAG GGA TCA GGA ATG AAG-3 0 and 5 0 -CAA TAA TAG TAC CTT TAC CAG TTT GTC CAA-3 0 . The PCR conditions were the following: 94 C, 3 min; 30· (94 C, 30 s; 55 C, 30 s; 70 C, 45 s); 70 C, 10 min. The PCR products were purified from agarose gels and sequenced. Uniparental disomy analysis. Fifteen highly polymorphic microsatellite markers at regular intervals across chromosome 1 were selected for uniparental disomy (UPD) analysis (ABI Linkage Mapping Set, Applied
Biosystems, Foster City, CA). One primer of each set was fluorescently labeled. PCR products were run on an ABI 3100 capillary electrophoresis instrument and analyzed using Genotyper 3.7 software. Strain and plasmids. Wild type hALG6 cDNA was amplified by RTPCR using the primers: 5 0 -GAA GAT CTC AAC ATG GAG AAA TGG TAC TTG ATG-3 0 and 5 0 -AGC GTC GAC CTA GCT GAT TTT CTT CTG ATT TCT TC-3 0 . The RT-PCR product was digested with BglII and SalI, and cloned into pIRES2-eGFP plasmid between the BamHI and SalI sites. The hALG6-IRES2-eGFP fragment was amplified by Pfu DNA polymerase with two primers: 5 0 -CAC CAT GGA GAA ATG GTA CTT GAT GAC-3 0 and 5 0 -CCG CTC GAG TTA CTT GTA CAG CTC GTC CAT G-3 0 . Plasmid LV-hALG6-IRES2-eGFP was made by cloning blunt hALG6-IRES2-eGFP PCR product into pLentiV5/TOPO plasmid and transformed into E. coli Stbl3 cells. Lentiviral vectors and transduction. Lentiviral vectors, LV-hALG6IRES2-eGFP and LV-GFP, were made according to the Lentiviral Expression System protocol (Invitrogen, Carlsbad, CA). The final virus stock was titrated using the green fluorescent protein (GFP) method. The lentivirus containing the wild type hALG6 gene was then used to transduce patient fibroblasts. Seventy-two hours post-infection, the patient cells were labeled for LLO analysis as described above. A lentivirus containing only the GFP gene was used for mock transductions.
Results Patient description The patient (TM) was born to a 37-year-old G3, P1, TAB1 Japanese mother and 36-year-old Caucasian/Hispanic father at full term by vaginal delivery. Her Apgar scores were 8 at 1 min and 9 at 5 min. She had mild intrauterine growth retardation with a birth weight of 2466 g and length of 45.5 cm. There was relative macrocephaly and dolichocephaly with a head circumference of 34 cm. Other dysmorphic features included deep set eyes, synophrys, hypertelorism, flat nasal bridge, anteverted nares, indistinct philtrum, loose skin, and puffy feet that looked unusual (hallux valgus, bulbous toes, and prominent heels). Head ultrasound on day one revealed ventriculomegaly and bilateral grade 3 intraventricular hemorrhages of supposed prenatal origin. CT and MRI showed sagittal craniosynostosis, possible old ischemic lesion in the left periventricular white matter, hypoplastic corpus callosum, and absent septum pellucidum, but a normal cerebellum. Bone age X-ray, eye exam, and hearing screen done in the nursery were normal, but later a mild left-sided esotropia was confirmed. A small ASD and moderate biventricular hypertrophy were detected by echocardiogram. Routine karyotype was 46,XX. PT and PTT were normal for age. TM was a slow feeder, requiring gavage, but did go home after 3 12 weeks, fully breastfeeding. Sagittal suture repair was performed at three months of age without complications. Pre-operative MR angiography demonstrated an abnormal circle of Willis with absence of the right A-1 segment. The ASD spontaneously closed but she was found to have a bifid uvula. FISH probe for 22q11 deletion was normal. Unusual fat distribution was apparent by 10 months along with mild hepatomegaly and severe hypotonia. Developmental assessments done at 6 months, 13 months, and 26 months using the Bayley Scales of Infant
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Development showed MDI/PDI scores of 53/<50, 62/<50, and <50/<50, respectively. Testing by the school system at 33 months of age indicated scattered development with some social skills being age appropriate, receptive, and expressive language at 23–24 months, gross motor at 12 months (walking with use of a walker), and fine motor at 9 months. Liver and thyroid function tests were normal. Coagulation tests consistently showed low factor XI (42–49%) and high protein C (101–133%) activities. Protein S and antithrombin III were normal. Platelet function screen was mildly prolonged, at 166 s for collagen/epinephrine (normal 74–162 s), and 113 s for collagen/ADP (normal 58– 106 s). Recurrent epistaxis was a problem that slowly improved with age. Analysis of glycosylation With symptoms consistent with CDG, analysis of carbohydrate-deficient transferrin by electrospray-ionization mass spectrometry (ESI-MS) was performed on two occasions. It showed abnormal ratios of mono-oligo/di-oligo = 0.485 and 0.434 (normal < 0.074), and a-oligo/dioligo = 0.023 and 0.025 (normal < 0.022). Assays of PMM and PMI were normal excluding CDG-Ia and CDG-Ib (data not shown). Next the synthesis of LLO in fibroblast cultures was assessed. Cells from the patient accumulate the truncated LLO species Man9-N-acetylglucosamine2-P-P-dolichol (Man9GlcNAc2; Fig. 1A), whereas control fibroblasts mainly synthesize the full-size oligosaccharide Glc3Man9GlcNAc2 (Fig. 1B). This indicates that the addition of the first Glc residue to the growing LLO chain is impaired, and the gene encoding the enzyme responsible for this reaction (hALG6) was sequenced (see below).
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Analysis of hALG6 The patientÕs hALG6 cDNA, encoding the a1,3-glucosyltransferase, was sequenced and a novel point mutation that appeared homozygous was detected (338G > A; data not shown). This causes the amino acid exchange R113H. No other mutations were detected. The mutation was confirmed on gDNA, exon 4 (Fig. 2A). Analysis of the parentsÕ gDNA showed that the mother was heterozygous (Fig. 2B) for the mutation whereas the father was a non-carrier (Fig. 2C). This prompted haplotype analysis, which demonstrated lack of any paternal DNA markers extending 33 kb in the telomeric direction from the hALG6 region (data not shown), raising the possibility of paternal deletion or maternal UPD. UPD studies were performed on the proband and her parents with 15 highly polymorphic microsatellite markers distributed across chromosome 1. Results for 14 markers were consistent with biparental inheritance (Fig. 3A). However, marker D1S2737, 2.5 Mb telomeric to the hALG6 gene, originated only from a maternal allele (Fig. 3B). These data were consistent with either a paternal deletion of this locus or maternal segmental UPD for a small region of chromosome 1. In order to distinguish between these two possibilities, semi-quantitative PCR was performed with D1S2737 and amplicons from the b-globin and dystrophin loci as controls. The results supported the presence of a deletion (data not shown). High resolution karyotyping finally confirmed TMÕs deletion [del(1)(p31.2p32.3)] and found no structural abnormalities in her father, suggesting a de novo event.
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Time (min) Fig. 1. LLO analysis. 3H-Man labeled LLOs from TM (A) and control cells (B) were separated using an acetonitrile gradient by amine adsorption hplc. The elution position of Man9GlcNAc2 (M9N2) and Glc3Man9GlcNAc2 (G3M9N2) oligosaccharide standards are indicated with arrows.
Fig. 2. Mutational analysis. Sequencing of exon 4 of the ALG6 gene in (A) the patient, (B) the mother, and (C) the father reveals an apparent ‘‘homozygous’’ 338G > A transition in the patient, heterozygosity in the mother, and normal sequence in the father.
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Fig. 3. Uniparental disomy studies. (A) Pedigree demonstrating inheritance of 15 different microsatellite markers. All markers demonstrated biparental inheritance, except D1S2737 (bold). (B) Microsatellite analysis of marker D1S2737 indicating inheritance of only a maternal allele(s) at this locus. Allele sizes are indicated below the peaks.
Lentiviral correction of the LLO synthesis In order to prove that the mutations are disease-causing, we transduced the fibroblasts with a lentivirus expressing the wild type hALG6 gene and analyzed the LLO production (Fig. 4A). A lentivirus expressing only GFP was used for mock transductions (Fig. 4B). The hALG6 transduced cells produced considerably more full-size LLO, proving that the mutations are causing the LLO phenotype, but the correction was not complete indicating that not all cells were transduced. Earlier experiments with these constructs have shown 65–75% transduction efficiency, consistent with these data. Discussion The causes of most congenital genetic syndromes are small genetic changes such as point mutations, single or few nucleotide deletions or insertions. CDG is no exception and all but one patient [8] reported so far stem from heterozygous parents with one or more of these changes. In the course of diagnosing this CDG-Ic patient, we first thought the patient was a homozygote for the novel mutation
338G > A, since sequencing of both cDNA and gDNA showed a homozygous pattern. Upon analysis of the father, however, no mutation was found at the gDNA level, eventually raising the question of a paternal deletion. Using haplotype analysis, microsatellite analysis, and high resolution karyotyping a deletion was defined as [del(1)(p31.2p32.3)]. Two other cases with a deletion in this area have been described, but they all encompass larger deletions [12]. Semi-quantitative PCR comparing a microsatellite marker in the deleted region, D1S2737, and the b-globin and dystrophin loci, supported the presence of a deletion. The father was shown to have no deletion, arguing for either a spontaneous germ-line mutation or a new somatic mutation, where the latter would lead to mosaicism. Both are rare, but known, causes of recessive disorders [13,14]. Since the patientÕs syndrome affects many different organs of different embryonic origins, a germ-line mutation would be the most likely explanation in this case. Common risk factors for germ-line mutations are that they are most often paternal and that they increase with age [15]. With a deletion encompassing such a large span of gDNA, the patientÕs phenotype could potentially be con-
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includes the hALG6 locus. The patientÕs phenotype is typical of CDG-Ic and no additional ‘‘non-CDG typical’’ symptoms that could result from loss of other genes are noted. The case illustrates the importance, prior to genetic counseling for recurrence risks, of performing molecular analysis of each parent, especially when the patient ‘‘appears’’ to be homozygous. There are instances when the ‘‘classic definition’’ of recessive trait—that both parents of an affected individual must be carriers—does not apply. This information can dramatically change the odds of having a future normal pregnancy. Acknowledgments
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Time(min) Fig. 4. Lentiviral correction of LLO synthesis. Fibroblasts from the patient were either transduced with lentivirus expressing wild type hALG6 or GFP. Seventy-two hours post-transduction LLOs were labeled, extracted, and analyzed by HPLC. Cells transduced with hALG6 (A) have a significantly higher proportion full size LLO, Glc3Man9GlcNac2 (G3M9N2), compared to the mock transduced counterpart (B). The elution position of Man7GlcNac2 (M7N2), Man9GlcNac2 (M9N2), and Glc3Man9GlcNac2 (G3M9N2) oligosaccharide standards are indicated with arrows. The transduction efficiency of 65–75% [17] is in accordance with these data.
founded by the loss of other genes besides the one involved in the particular syndrome observed. In our case, the presentation of the patient was typical for a relatively mild CDG case, typical of CDG-Ic patients [3]. Most of the symptoms—facial dysmorphisms, coagulopathy, failure to thrive, developmental delay, and subcutaneous fat pads— are well known in CDG and there is no obvious reason to believe they are connected to loss of other genes in a dominant fashion. The only finding in the patient that is somewhat unusual is the absence of the A-1 segment in the Circle of Willis, however unilateral hypoplasia of arteria cerebri anterior is a well-described normal variant [16]. CDG-Ic is the second most prevalent CDG subtype and 16 point mutations in hALG6 have been shown to be disease causing (according to the Leuven mutation database, www.euroglycanet.org, and [17]). Using a lentiviral approach we show that transduction of the patientÕs cells with wild type hALG6 partially restores the synthesis of full size LLOs, proving that these two genetic changes are pathogenic. The maternal point mutation, 338G > A, is the first hALG6 mutation described in a Japanese genetic background. All but three (who were Indian [6]) of the reported CDG-Ic patients were of European ancestry, and by far the most common amino acid exchange is the A333V. This mutation was also found in the Indian patients raising the question of its origin [6]. In conclusion, we describe a case of CDG-Ic where the patientÕs paternal chromosome 1 has a deletion that
The authors are grateful to Dr. Terrance Wardinsky for referring TM initially because he suspected a diagnosis of CDG. Drs. Sherri Bale and John Compton at GeneDx (Gaithersburg, MD) are acknowledged for help with mutational and haplotype analysis and Dr. Paul Saxon at Sutter Medical Center for help with the cytogenetic studies. The research was supported by NIH Grant R01DK65615 (to H.H.F.) and a postdoctoral fellowship from STINT/VR (Sweden; K2004-99PK-14887-02B) to E.A.E. References [1] J. Jaeken, Komrower Lecture. Congenital disorders of glycosylation (CDG): itÕs all in it! J. Inherit. Metab. Dis. 26 (2003) 99–118. [2] H.H. Freeze, Human disorders in N-glycosylation and animal models, Biochim. Biophys. Acta 1573 (2002) 388–393. [3] T. Marquardt, J. Denecke, Congenital disorders of glycosylation: review of their molecular bases, clinical presentations and specific therapies, Eur. J. Pediatr. 162 (2003) 359–379. [4] J. Jaeken, M. Vanderschueren-Lodeweyckx, P. Casaer, L. Snoeck, L. Corbeel, E. Eggermont, R. Eeckels, Familiar psychomotor retardation with markedly fluctuating serum prolactin, FSH and GH levels, partial TBG deficiency, increased serum arylsulphatase A and increased CSF protein: a new syndrome? Pediatr. Res. 14 (1980) 179. [5] S. Gru¨newald, T. Imbach, K. Huijben, M.E. Rubio-Gozalbo, A. Verrips, J.B. de Klerk, H. Stroink, J.F. de Rijk-van Andel, J.L. Van Hove, U. Wendel, G. Matthijs, T. Hennet, J. Jaeken, R.A. Wevers, Clinical and biochemical characteristics of congenital disorder of glycosylation type Ic, the first recognized endoplasmic reticulum defect in N-glycan synthesis, Ann. Neurol. 47 (2000) 776–781. [6] J.W. Newell, N.S. Seo, G.M. Enns, M. McCraken, J.F. Mantovani, H.H. Freeze, Congenital disorder of glycosylation Ic in patients of Indian origin, Mol. Genet. Metab. 79 (2003) 221–228. [7] T. Imbach, P. Burda, P. Kuhnert, R.A. Wevers, M. Aebi, E.G. Berger, T. Hennet, A mutation in the human ortholog of the Saccharomyces cerevisiae ALG6 gene causes carbohydrate-deficient glycoprotein syndrome type-Ic, Proc. Natl. Acad. Sci. USA 96 (1999) 6982–6987. [8] E. Schollen, S. Grunewald, L. Keldermans, B. Albrecht, C. Korner, G. Matthijs, CDG-Id caused by homozygosity for an ALG3 mutation due to segmental maternal isodisomy UPD3(q21.3-qter), Eur. J. Med. Genet. 48 (2005) 153–158. [9] E. Van Schaftingen, J. Jaeken, Phosphomannomutase deficiency is a cause of carbohydrate-deficient glycoprotein syndrome type I, FEBS Lett. 377 (1995) 318–320. [10] R. Niehues, M. Hasilik, G. Alton, C. Korner, M. Schiebe-Sukumar, H.G. Koch, K.P. Zimmer, R. Wu, E. Harms, K. Reiter, K. von Figura, H.H. Freeze, H.K. Harms, T. Marquardt, Carbohydratedeficient glycoprotein syndrome type Ib. Phosphomannose isomerase
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deficiency and mannose therapy, J. Clin. Invest. 101 (1998) 1414– 1420. [11] E.A. Eklund, J.W. Newell, L. Sun, N.S. Seo, G. Alper, J. Willert, H.H. Freeze, Molecular and clinical description of the first US patients with congenital disorder of glycosylation Ig, Mol. Genet. Metab. 84 (2005) 25–31. [12] M.M. Lai, M.F. Robards, A.C. Berry, C.N. Fear, C. Hart, Two cases of interstitial deletion 1p, J. Med. Genet. 28 (1991) 128– 130. [13] C. Badens, M.G. Mattei, A.M. Imbert, C. Lapoumeroulie, N. Martini, G. Michel, D. Lena-Russo, A novel mechanism for thalassaemia intermedia, Lancet 359 (2002) 132–133.
[14] J. Zlotogora, Parents of children with autosomal recessive diseases are not always carriers of the respective mutant alleles, Hum. Genet. 114 (2004) 521–526. [15] J.F. Crow, The origins, patterns and implications of human spontaneous mutation, Nat. Rev. Genet. 1 (2000) 40–47. [16] H.E. Riggs, C. Rupp, Variation in form of circle of Willis. The relation of the variations to collateral circulation: anatomic analysis, Arch. Neurol. 8 (1963) 8–14. [17] L. Sun, E.A. Eklund, J.L.K. Van Hove, H.H. Freeze, J.A. Thomas, Clinical and molecular characterization of the first adult congenital disorder of glycosylation (CDG) type Ic patient, Am. J. Med. Genet. 137 (2005) 22–26.
Congenital disorder of glycosylation Ic due to a de novo deletion and an hALG-6 mutation Erik A. Eklund a,1, Liangwu Sun a,1, Samuel P. Yang b, Romela M. Pasion c, Erik C. Thorland d, Hudson H. Freeze a,* a
Glycobiology and Carbohydrate Chemistry Program, The Burnham Institute, 10901 N Torrey Pines Road, La Jolla, CA 92037, USA b GenetiCare Medical Associates, 417 Mace Boulevard, Davis, CA 95616, USA c Sutter Metabolic Clinic, 5271 F Street, Sacramento, CA 95819, USA d Clinical Cytogenetics Laboratory, Mayo Clinic, 200 First St. S.W., Rochester, MN 55905, USA Received 25 October 2005 Available online 29 November 2005
Abstract We describe a new cause of congenital disorder of glycosylation-Ic (CDG-Ic) in a young girl with a rather mild CDG phenotype. Her cells accumulated lipid-linked oligosaccharides lacking three glucose residues, and sequencing of the ALG6 gene showed what initially appeared to be a homozygous novel point mutation (338G > A). However, haplotype analysis showed that the patient does not carry any paternal DNA markers extending 33 kb in the telomeric direction from the ALG6 region, and microsatellite analysis extended the abnormal region to at least 2.5 Mb. We used high-resolution karyotyping to confirm a deletion (10–12 Mb) [del(1)(p31.2p32.3)] and found no structural abnormalities in the father, suggesting a de novo event. Our findings extend the causes of CDG to larger DNA deletions and identify the first Japanese CDG-Ic mutation. 2005 Elsevier Inc. All rights reserved. Keywords: CDG-Ic; Developmental delay; Germ-line mutation; Genomic deletion; hALG6
Congenital disorders of glycosylation (CDG)2 constitute a group of metabolic syndromes where the primary defects either impair the formation of the lipid-linked oligosaccharide (LLO) precursor of N-linked glycosylation (Type I) or affect processing of the protein-bound sugar chains (Type II) [1–3]. The most common subtype, CDG-Ia, was first described in 1980 [4], and since then 12 Type I and 6 Type II subtypes have been reported. Less than a thousand patients with CDG have been diagnosed so far, but the true incidence probably is much higher (based on frequency *
Corresponding author. Fax: +1 858 713 6281. E-mail address: [email protected] (H.H. Freeze). 1 These authors contributed equally to this work. 2 Abbreviations: CDG, congenital disorders of glycosylation; GFP, green fluorescent protein; Glc, glucose; GlcNAc, N-acetyl glucosamine; hALG6, human ortholog of yeast Alg6; LLO, lipid-linked oligosaccharide; Man, mannose; PMI, phosphomannose isomerase; PMM, phosphomannomutase; UPD, uniparental disomy. 0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.11.073
analysis for certain common mutations). The spectrum of symptoms and degree of severity in CDG are unmatched by any other congenital syndrome, ranging from mentally normal children with protein-losing enteropathy and coagulopathy in CDG-Ib, to extremely severely affected children with intractable seizures, profound developmental delay, and hypotonia in e.g., CDG-Id, CDG-Ie, and CDG-If. CDG can easily be ruled out with an isoelectric focusing or mass spectrometric analysis of serum transferrin. CDG-Ic (OMIM 603147) is caused by mutations in the human ortholog of yeast Alg6, hALG6, which encodes the glucosyltransferase that adds the first glucose (Glc) residue to the growing LLO chain. It is the second most common CDG subtype with more than 30 known cases. The CDG-Ic phenotype is usually milder than CDG-Ia, and includes psychomotor retardation with delayed walking and speech, seizures, muscular hypotonia, and on occasion,
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protein-losing enteropathy [3,5,6]. LDL and clotting factor XI are generally lower in these patients than in patients affected by other CDG syndromes [15]. Sixteen different hALG6 mutations causing CDG-Ic have been described so far (see www.euroglycanet.org; the European CDG Network Database in Leuven, Belgium) where A333V accounts for the majority of the alleles [6,7]. We describe a new case of CDG-Ic, identify the first Japanese mutation, and find a large deletion of the patientÕs paternal chromosome 1, including the hALG6 locus. This is to our knowledge only the second time CDG has been connected to a large genomic deletion [8]. Methods and materials Approval of human research. Approval of human research was obtained from the Institutional Review Boards of The Burnham Institute, La Jolla, CA, and Sutter Health Central Area, Sacramento, CA. Materials. Tissue culture materials, TRIzol Reagent, SuperScript OneStep RT-PCR kit, Lipofectamine 2000, Escherichia coli Stbl3 cells, pLentiV5/TOPO plasmid, and Lentiviral Expression System were from Invitrogen (Carlsbad, CA). The oligonucleotides were ordered from Genbase (San Diego, CA). The Microsorb-MV NH2 HPLC column was purchased from Varian Instruments (Walnut Creek, CA), [2-3H]mannose (Man) (20 Ci/mmol) was from American Radiolabeled Chemicals (St. Louis, MO), and fetal bovine serum was from HyClone (Logan, UT). The pGEM-T vector and the Wizard Genomic DNA Purification Kit were from Promega (Madison, WI), the restriction enzymes and T4 DNA ligase were from New England Biolabs (Beverly, MA), and the Pfu DNA polymerase was from Stratagene (La Jolla, CA). All other chemicals used in this study were of analytical grade and purchased from Sigma Chemicals (St. Louis, MO). Cell culture. Primary fibroblast cultures were set up from a skin biopsy of the patient after a signed consent form was obtained from the parents. Control fibroblasts were from age-matched controls. All cells were grown in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin sulfate at 37 C with 5% CO2. Phosphomannose mutase and phosphomannose isomerase assays. Enzymatic assays of phosphomannose mutase (PMM) (deficient in CDG-Ia) and phosphomannose isomerase (PMI) (deficient in CDG-Ib) were performed on fibroblast extracts essentially as described previously [9,10]. LLO analysis. Metabolic labeling and preparation of LLOs from fibroblast cultures grown in 100 mm plates were performed as described earlier [11]. They were analyzed by amine adsorption hplc on a Microsorb NH2 column using an acetonitrile gradient (65–35%). 2-Aminobenzamidelabeled Man5–9GlcNAc2 oligosaccharides (Prozyme, San Leandro, CA) were used as internal standards. Analysis of cDNA. The hALG6 cDNA was synthesized and sequenced essentially as described before [6]. The primers used for RT-PCR and sequencing were: 5 0 -AAG AAG TGA TTG ACC ACG TTT-3 0 and 5 0 AAT GGT AAT TTC ATT TAT ACA TAG C-3 0 . Other primers used for sequencing were: 5 0 -CCT AGG GTC ACT GGC-3 0 ; 5 0 -GTG TCA CTA CCA GTC-3 0 ; 5 0 -GAC TGG TAG TGA CAC-3 0 , and 5 0 -GCC AGT GAC CCT AGG-3 0 . Analysis of hALG6 genomic DNA. Genomic DNA was isolated from fibroblasts using the Wizard Genomic DNA Purification Kit. Primers used to amplify exon 4 and its boundary region of hALG6 were: 5 0 -TGC TAA AAG GGA TCA GGA ATG AAG-3 0 and 5 0 -CAA TAA TAG TAC CTT TAC CAG TTT GTC CAA-3 0 . The PCR conditions were the following: 94 C, 3 min; 30· (94 C, 30 s; 55 C, 30 s; 70 C, 45 s); 70 C, 10 min. The PCR products were purified from agarose gels and sequenced. Uniparental disomy analysis. Fifteen highly polymorphic microsatellite markers at regular intervals across chromosome 1 were selected for uniparental disomy (UPD) analysis (ABI Linkage Mapping Set, Applied
Biosystems, Foster City, CA). One primer of each set was fluorescently labeled. PCR products were run on an ABI 3100 capillary electrophoresis instrument and analyzed using Genotyper 3.7 software. Strain and plasmids. Wild type hALG6 cDNA was amplified by RTPCR using the primers: 5 0 -GAA GAT CTC AAC ATG GAG AAA TGG TAC TTG ATG-3 0 and 5 0 -AGC GTC GAC CTA GCT GAT TTT CTT CTG ATT TCT TC-3 0 . The RT-PCR product was digested with BglII and SalI, and cloned into pIRES2-eGFP plasmid between the BamHI and SalI sites. The hALG6-IRES2-eGFP fragment was amplified by Pfu DNA polymerase with two primers: 5 0 -CAC CAT GGA GAA ATG GTA CTT GAT GAC-3 0 and 5 0 -CCG CTC GAG TTA CTT GTA CAG CTC GTC CAT G-3 0 . Plasmid LV-hALG6-IRES2-eGFP was made by cloning blunt hALG6-IRES2-eGFP PCR product into pLentiV5/TOPO plasmid and transformed into E. coli Stbl3 cells. Lentiviral vectors and transduction. Lentiviral vectors, LV-hALG6IRES2-eGFP and LV-GFP, were made according to the Lentiviral Expression System protocol (Invitrogen, Carlsbad, CA). The final virus stock was titrated using the green fluorescent protein (GFP) method. The lentivirus containing the wild type hALG6 gene was then used to transduce patient fibroblasts. Seventy-two hours post-infection, the patient cells were labeled for LLO analysis as described above. A lentivirus containing only the GFP gene was used for mock transductions.
Results Patient description The patient (TM) was born to a 37-year-old G3, P1, TAB1 Japanese mother and 36-year-old Caucasian/Hispanic father at full term by vaginal delivery. Her Apgar scores were 8 at 1 min and 9 at 5 min. She had mild intrauterine growth retardation with a birth weight of 2466 g and length of 45.5 cm. There was relative macrocephaly and dolichocephaly with a head circumference of 34 cm. Other dysmorphic features included deep set eyes, synophrys, hypertelorism, flat nasal bridge, anteverted nares, indistinct philtrum, loose skin, and puffy feet that looked unusual (hallux valgus, bulbous toes, and prominent heels). Head ultrasound on day one revealed ventriculomegaly and bilateral grade 3 intraventricular hemorrhages of supposed prenatal origin. CT and MRI showed sagittal craniosynostosis, possible old ischemic lesion in the left periventricular white matter, hypoplastic corpus callosum, and absent septum pellucidum, but a normal cerebellum. Bone age X-ray, eye exam, and hearing screen done in the nursery were normal, but later a mild left-sided esotropia was confirmed. A small ASD and moderate biventricular hypertrophy were detected by echocardiogram. Routine karyotype was 46,XX. PT and PTT were normal for age. TM was a slow feeder, requiring gavage, but did go home after 3 12 weeks, fully breastfeeding. Sagittal suture repair was performed at three months of age without complications. Pre-operative MR angiography demonstrated an abnormal circle of Willis with absence of the right A-1 segment. The ASD spontaneously closed but she was found to have a bifid uvula. FISH probe for 22q11 deletion was normal. Unusual fat distribution was apparent by 10 months along with mild hepatomegaly and severe hypotonia. Developmental assessments done at 6 months, 13 months, and 26 months using the Bayley Scales of Infant
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Development showed MDI/PDI scores of 53/<50, 62/<50, and <50/<50, respectively. Testing by the school system at 33 months of age indicated scattered development with some social skills being age appropriate, receptive, and expressive language at 23–24 months, gross motor at 12 months (walking with use of a walker), and fine motor at 9 months. Liver and thyroid function tests were normal. Coagulation tests consistently showed low factor XI (42–49%) and high protein C (101–133%) activities. Protein S and antithrombin III were normal. Platelet function screen was mildly prolonged, at 166 s for collagen/epinephrine (normal 74–162 s), and 113 s for collagen/ADP (normal 58– 106 s). Recurrent epistaxis was a problem that slowly improved with age. Analysis of glycosylation With symptoms consistent with CDG, analysis of carbohydrate-deficient transferrin by electrospray-ionization mass spectrometry (ESI-MS) was performed on two occasions. It showed abnormal ratios of mono-oligo/di-oligo = 0.485 and 0.434 (normal < 0.074), and a-oligo/dioligo = 0.023 and 0.025 (normal < 0.022). Assays of PMM and PMI were normal excluding CDG-Ia and CDG-Ib (data not shown). Next the synthesis of LLO in fibroblast cultures was assessed. Cells from the patient accumulate the truncated LLO species Man9-N-acetylglucosamine2-P-P-dolichol (Man9GlcNAc2; Fig. 1A), whereas control fibroblasts mainly synthesize the full-size oligosaccharide Glc3Man9GlcNAc2 (Fig. 1B). This indicates that the addition of the first Glc residue to the growing LLO chain is impaired, and the gene encoding the enzyme responsible for this reaction (hALG6) was sequenced (see below).
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Analysis of hALG6 The patientÕs hALG6 cDNA, encoding the a1,3-glucosyltransferase, was sequenced and a novel point mutation that appeared homozygous was detected (338G > A; data not shown). This causes the amino acid exchange R113H. No other mutations were detected. The mutation was confirmed on gDNA, exon 4 (Fig. 2A). Analysis of the parentsÕ gDNA showed that the mother was heterozygous (Fig. 2B) for the mutation whereas the father was a non-carrier (Fig. 2C). This prompted haplotype analysis, which demonstrated lack of any paternal DNA markers extending 33 kb in the telomeric direction from the hALG6 region (data not shown), raising the possibility of paternal deletion or maternal UPD. UPD studies were performed on the proband and her parents with 15 highly polymorphic microsatellite markers distributed across chromosome 1. Results for 14 markers were consistent with biparental inheritance (Fig. 3A). However, marker D1S2737, 2.5 Mb telomeric to the hALG6 gene, originated only from a maternal allele (Fig. 3B). These data were consistent with either a paternal deletion of this locus or maternal segmental UPD for a small region of chromosome 1. In order to distinguish between these two possibilities, semi-quantitative PCR was performed with D1S2737 and amplicons from the b-globin and dystrophin loci as controls. The results supported the presence of a deletion (data not shown). High resolution karyotyping finally confirmed TMÕs deletion [del(1)(p31.2p32.3)] and found no structural abnormalities in her father, suggesting a de novo event.
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Time (min) Fig. 1. LLO analysis. 3H-Man labeled LLOs from TM (A) and control cells (B) were separated using an acetonitrile gradient by amine adsorption hplc. The elution position of Man9GlcNAc2 (M9N2) and Glc3Man9GlcNAc2 (G3M9N2) oligosaccharide standards are indicated with arrows.
Fig. 2. Mutational analysis. Sequencing of exon 4 of the ALG6 gene in (A) the patient, (B) the mother, and (C) the father reveals an apparent ‘‘homozygous’’ 338G > A transition in the patient, heterozygosity in the mother, and normal sequence in the father.
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Fig. 3. Uniparental disomy studies. (A) Pedigree demonstrating inheritance of 15 different microsatellite markers. All markers demonstrated biparental inheritance, except D1S2737 (bold). (B) Microsatellite analysis of marker D1S2737 indicating inheritance of only a maternal allele(s) at this locus. Allele sizes are indicated below the peaks.
Lentiviral correction of the LLO synthesis In order to prove that the mutations are disease-causing, we transduced the fibroblasts with a lentivirus expressing the wild type hALG6 gene and analyzed the LLO production (Fig. 4A). A lentivirus expressing only GFP was used for mock transductions (Fig. 4B). The hALG6 transduced cells produced considerably more full-size LLO, proving that the mutations are causing the LLO phenotype, but the correction was not complete indicating that not all cells were transduced. Earlier experiments with these constructs have shown 65–75% transduction efficiency, consistent with these data. Discussion The causes of most congenital genetic syndromes are small genetic changes such as point mutations, single or few nucleotide deletions or insertions. CDG is no exception and all but one patient [8] reported so far stem from heterozygous parents with one or more of these changes. In the course of diagnosing this CDG-Ic patient, we first thought the patient was a homozygote for the novel mutation
338G > A, since sequencing of both cDNA and gDNA showed a homozygous pattern. Upon analysis of the father, however, no mutation was found at the gDNA level, eventually raising the question of a paternal deletion. Using haplotype analysis, microsatellite analysis, and high resolution karyotyping a deletion was defined as [del(1)(p31.2p32.3)]. Two other cases with a deletion in this area have been described, but they all encompass larger deletions [12]. Semi-quantitative PCR comparing a microsatellite marker in the deleted region, D1S2737, and the b-globin and dystrophin loci, supported the presence of a deletion. The father was shown to have no deletion, arguing for either a spontaneous germ-line mutation or a new somatic mutation, where the latter would lead to mosaicism. Both are rare, but known, causes of recessive disorders [13,14]. Since the patientÕs syndrome affects many different organs of different embryonic origins, a germ-line mutation would be the most likely explanation in this case. Common risk factors for germ-line mutations are that they are most often paternal and that they increase with age [15]. With a deletion encompassing such a large span of gDNA, the patientÕs phenotype could potentially be con-
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includes the hALG6 locus. The patientÕs phenotype is typical of CDG-Ic and no additional ‘‘non-CDG typical’’ symptoms that could result from loss of other genes are noted. The case illustrates the importance, prior to genetic counseling for recurrence risks, of performing molecular analysis of each parent, especially when the patient ‘‘appears’’ to be homozygous. There are instances when the ‘‘classic definition’’ of recessive trait—that both parents of an affected individual must be carriers—does not apply. This information can dramatically change the odds of having a future normal pregnancy. Acknowledgments
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Time(min) Fig. 4. Lentiviral correction of LLO synthesis. Fibroblasts from the patient were either transduced with lentivirus expressing wild type hALG6 or GFP. Seventy-two hours post-transduction LLOs were labeled, extracted, and analyzed by HPLC. Cells transduced with hALG6 (A) have a significantly higher proportion full size LLO, Glc3Man9GlcNac2 (G3M9N2), compared to the mock transduced counterpart (B). The elution position of Man7GlcNac2 (M7N2), Man9GlcNac2 (M9N2), and Glc3Man9GlcNac2 (G3M9N2) oligosaccharide standards are indicated with arrows. The transduction efficiency of 65–75% [17] is in accordance with these data.
founded by the loss of other genes besides the one involved in the particular syndrome observed. In our case, the presentation of the patient was typical for a relatively mild CDG case, typical of CDG-Ic patients [3]. Most of the symptoms—facial dysmorphisms, coagulopathy, failure to thrive, developmental delay, and subcutaneous fat pads— are well known in CDG and there is no obvious reason to believe they are connected to loss of other genes in a dominant fashion. The only finding in the patient that is somewhat unusual is the absence of the A-1 segment in the Circle of Willis, however unilateral hypoplasia of arteria cerebri anterior is a well-described normal variant [16]. CDG-Ic is the second most prevalent CDG subtype and 16 point mutations in hALG6 have been shown to be disease causing (according to the Leuven mutation database, www.euroglycanet.org, and [17]). Using a lentiviral approach we show that transduction of the patientÕs cells with wild type hALG6 partially restores the synthesis of full size LLOs, proving that these two genetic changes are pathogenic. The maternal point mutation, 338G > A, is the first hALG6 mutation described in a Japanese genetic background. All but three (who were Indian [6]) of the reported CDG-Ic patients were of European ancestry, and by far the most common amino acid exchange is the A333V. This mutation was also found in the Indian patients raising the question of its origin [6]. In conclusion, we describe a case of CDG-Ic where the patientÕs paternal chromosome 1 has a deletion that
The authors are grateful to Dr. Terrance Wardinsky for referring TM initially because he suspected a diagnosis of CDG. Drs. Sherri Bale and John Compton at GeneDx (Gaithersburg, MD) are acknowledged for help with mutational and haplotype analysis and Dr. Paul Saxon at Sutter Medical Center for help with the cytogenetic studies. The research was supported by NIH Grant R01DK65615 (to H.H.F.) and a postdoctoral fellowship from STINT/VR (Sweden; K2004-99PK-14887-02B) to E.A.E. References [1] J. Jaeken, Komrower Lecture. Congenital disorders of glycosylation (CDG): itÕs all in it! J. Inherit. Metab. Dis. 26 (2003) 99–118. [2] H.H. Freeze, Human disorders in N-glycosylation and animal models, Biochim. Biophys. Acta 1573 (2002) 388–393. [3] T. Marquardt, J. Denecke, Congenital disorders of glycosylation: review of their molecular bases, clinical presentations and specific therapies, Eur. J. Pediatr. 162 (2003) 359–379. [4] J. Jaeken, M. Vanderschueren-Lodeweyckx, P. Casaer, L. Snoeck, L. Corbeel, E. Eggermont, R. Eeckels, Familiar psychomotor retardation with markedly fluctuating serum prolactin, FSH and GH levels, partial TBG deficiency, increased serum arylsulphatase A and increased CSF protein: a new syndrome? Pediatr. Res. 14 (1980) 179. [5] S. Gru¨newald, T. Imbach, K. Huijben, M.E. Rubio-Gozalbo, A. Verrips, J.B. de Klerk, H. Stroink, J.F. de Rijk-van Andel, J.L. Van Hove, U. Wendel, G. Matthijs, T. Hennet, J. Jaeken, R.A. Wevers, Clinical and biochemical characteristics of congenital disorder of glycosylation type Ic, the first recognized endoplasmic reticulum defect in N-glycan synthesis, Ann. Neurol. 47 (2000) 776–781. [6] J.W. Newell, N.S. Seo, G.M. Enns, M. McCraken, J.F. Mantovani, H.H. Freeze, Congenital disorder of glycosylation Ic in patients of Indian origin, Mol. Genet. Metab. 79 (2003) 221–228. [7] T. Imbach, P. Burda, P. Kuhnert, R.A. Wevers, M. Aebi, E.G. Berger, T. Hennet, A mutation in the human ortholog of the Saccharomyces cerevisiae ALG6 gene causes carbohydrate-deficient glycoprotein syndrome type-Ic, Proc. Natl. Acad. Sci. USA 96 (1999) 6982–6987. [8] E. Schollen, S. Grunewald, L. Keldermans, B. Albrecht, C. Korner, G. Matthijs, CDG-Id caused by homozygosity for an ALG3 mutation due to segmental maternal isodisomy UPD3(q21.3-qter), Eur. J. Med. Genet. 48 (2005) 153–158. [9] E. Van Schaftingen, J. Jaeken, Phosphomannomutase deficiency is a cause of carbohydrate-deficient glycoprotein syndrome type I, FEBS Lett. 377 (1995) 318–320. [10] R. Niehues, M. Hasilik, G. Alton, C. Korner, M. Schiebe-Sukumar, H.G. Koch, K.P. Zimmer, R. Wu, E. Harms, K. Reiter, K. von Figura, H.H. Freeze, H.K. Harms, T. Marquardt, Carbohydratedeficient glycoprotein syndrome type Ib. Phosphomannose isomerase
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deficiency and mannose therapy, J. Clin. Invest. 101 (1998) 1414– 1420. [11] E.A. Eklund, J.W. Newell, L. Sun, N.S. Seo, G. Alper, J. Willert, H.H. Freeze, Molecular and clinical description of the first US patients with congenital disorder of glycosylation Ig, Mol. Genet. Metab. 84 (2005) 25–31. [12] M.M. Lai, M.F. Robards, A.C. Berry, C.N. Fear, C. Hart, Two cases of interstitial deletion 1p, J. Med. Genet. 28 (1991) 128– 130. [13] C. Badens, M.G. Mattei, A.M. Imbert, C. Lapoumeroulie, N. Martini, G. Michel, D. Lena-Russo, A novel mechanism for thalassaemia intermedia, Lancet 359 (2002) 132–133.
[14] J. Zlotogora, Parents of children with autosomal recessive diseases are not always carriers of the respective mutant alleles, Hum. Genet. 114 (2004) 521–526. [15] J.F. Crow, The origins, patterns and implications of human spontaneous mutation, Nat. Rev. Genet. 1 (2000) 40–47. [16] H.E. Riggs, C. Rupp, Variation in form of circle of Willis. The relation of the variations to collateral circulation: anatomic analysis, Arch. Neurol. 8 (1963) 8–14. [17] L. Sun, E.A. Eklund, J.L.K. Van Hove, H.H. Freeze, J.A. Thomas, Clinical and molecular characterization of the first adult congenital disorder of glycosylation (CDG) type Ic patient, Am. J. Med. Genet. 137 (2005) 22–26.