Am. J. Hum. Genet. 63:409–414, 1998

409

Functionally Null Mutations in Patients with the cblG-Variant Form ofMethionine Synthase DeficiencyA. Wilson,1,3 D. Leclerc,1 F. Saberi,1,3 E. Campeau,1,3 H. Y. Hwang,6 B. Shane,6 J. A. Phillips III,7D. S. Rosenblatt,2,3,4,5 and R. A. Gravel1,3,4

1Medical Research Council Group in Medical Genetics, Montreal Children’s Hospital, 2Royal Victoria Hospital, and 3Research Institutes andDepartment of Biology, Departments of 4Human Genetics and Pediatrics, and 5Department of Medicine, McGill University, Montreal;6Department of Nutritional Sciences, University of California, Berkeley; and 7Vanderbilt University School of Medicine, Nashville

Summary

Methionine synthase (MS) catalyses the methylation ofhomocysteine to methionine and requires the vitamin B12

derivative, methylcobalamin, as cofactor. We and othershave recently cloned cDNAs for MS and described mu-tations associated with the cblG complementation groupthat correspond to MS deficiency. A subset of cblG,known as “cblG variant,” shows no detectable MS ac-tivity and failure of [57Co]CN cobalamin to incorporateinto MS in patient fibroblasts. We report the mutationsresponsible for three cblG-variant patients, two of themsiblings, who presented with neonatal seizures, severedevelopmental delay, and elevated plasma homocysteine.Cell lines from all three patients were negative by north-ern blotting, though trace MS mRNA could be detectedby means of phosphorimage analysis. Reverse transcrip-tase–PCR, SSCP, and nucleotide sequence analysis re-vealed four mutations. All were functionally null, cre-ating either a frameshift with a downstream stop codonor an insert containing an internal stop codon. Of thetwo mutations found in the siblings, one of them, in-tervening sequence (IVS)-166ArG, generates a crypticdonor splice site at position 2166 of an intron beginningafter Leu113, resulting in a 165-bp insertion of intronicsequence at junction 339/340. The second is a 2-bp de-letion, 2112delTC. Mutations in the third patient in-clude a GrA substitution, well within the intron afterLys203, which results in intronic inserts of 128 or 78bp in the mRNA. The second mutation is a 1-bp inser-tion, 3378insA. We conclude that the absence of MSprotein in these cblG variants is due to mutations caus-ing premature translation termination and consequentmRNA instability.

Received March 19, 1998; accepted for publication June 1, 1998;electronically published June 29, 1998.

Address for correspondence and reprints: Dr. R. A. Gravel,MUHC–Montreal Children’s Hospital Research Institute, 4060 Ste-Catherine Street West, Montreal, Quebec H3Z 2Z3,Canada. E-mail:mc84@musica.mcgill.ca

q 1998 by The American Society of Human Genetics. All rights reserved.0002-9297/98/6302-0016$02.00

Introduction

Methionine synthase (MS) is a cobalamin-dependent en-zyme that catalyzes the methylation of homocysteine tomethionine, using 5-methyltetrahydrofolate as a methyldonor (Fenton and Rosenberg 1995; Rosenblatt 1995).The cobalamin cofactor acts as an intermediate methylcarrier, such that the methyl group of 5-methyltetrahy-drofolate is transferred first to the enzyme-boundcob(I)alamin, to form methyl cobalamin, with subse-quent transfer to homocysteine and regeneration of thecob(I)alamin cofactor (Banerjee 1997). Over time, thehighly reactive cob(I)alamin cofactor may become oxi-dized to cob(II)alamin, rendering the enzyme inactive.Regeneration of the functional enzyme takes placethrough the reductive methylation of the cob(II)alamin,occurring through a distinct enzyme and using S-aden-osyl methionine as the source of the methyl group (Lud-wig and Matthews 1997). Recently, cDNAs coding forMS (Gulati et al. 1996; Leclerc et al. 1996; Li et al.1996; Chen et al. 1997) as well as for the MS reductaseresponsible for its reactivation (Leclerc et al. 1998) werecloned, making mutation detection and characterizationof patient cell lines possible.

Deficiency of MS activity has been shown to result inthe biochemical phenotypes of hyperhomocysteinemia,homocystinuria, hypomethioninemia, and megaloblasticanemia without methylmalonic aciduria (Watkins andRosenblatt 1989; Harding et al. 1997). Patients show arange of clinical symptoms, including severe develop-mental delay, ataxia, cerebral atrophy, neonatal seizures,and blindness. Most cblG patients have reduced MS ac-tivity, with normal accumulations of labeled [57Co]CNcobalamin associated with the defective MS enzyme infibroblast-loading experiments (Sillaots et al. 1992).However, three patient cell lines, two of which are fromsiblings, show a reduced accumulation of labeled co-balamin, none of which is bound to MS. The MS activityof these cells is almost completely undetectable (Sillaotset al. 1992; Gulati et al. 1997). Because these three celllines differ from the majority of cblG lines, they havebeen classified as “cblG variants” (Sillaots et al. 1992).

410 Am. J. Hum. Genet. 63:409–414, 1998

All three patients experienced severe developmental de-lay, with neonatal seizures and elevated plasma homo-cysteine.

In this study, we report four distinct mutations in thecblG-variant patients. All four mutations would be ex-pected to result in the premature truncation of a com-plete MS protein. However, these mutations lead tomRNA instability and insufficient mRNA to produceany significant amount of MS enzyme. These resultsshow that the molecular defects in cblG variants areconsistently associated with a biochemically null phe-notype.

Material and Methods

Cell Lines

Skin fibroblasts from the cblG-variant cell linesWG1670, WG1671, and WG1655 were obtained fromthe Montreal Children’s Hospital Cell Repository forMutant Human Cell Strains. DNA from the mother ofsiblings WG1670 and WG1671 as well as from the sib-lings themselves was kindly provided by J. P. Pfotenhauer(Vanderbilt University School of Medicine, Nashville).

WG1671, the first child of unrelated parents, devel-oped generalized seizures at 3 d of age. He became pro-gressively hypotonic, and he suffered respiratory failureat 10 wk of age. Elevated plasma and urine homocy-steine (23 and 490 mmol/liter, respectively) but lowplasma methionine (5 mmol/liter) without macrocyticanemia led to the initial diagnosis of methylenetetra-hydrofolate reductase (MTHFR) deficiency. He wastreated with folinic acid, vitamin B12, vitamin B6, betaine,and methionine and was well enough to be weaned fromthe respirator by 14 wk of age. By 2 years of age, hehad severe psychomotor retardation and microcephaly.A definitive diagnosis of MS deficiency was made bymeans of complementation analysis of cultured fibro-blasts, which placed him in the cblG complementationgroup. He was subsequently treated with vitamin B12,betaine, and aspirin, and, at age 8 years, methionine wasadded. He has required femoral osteotomies and bilat-eral adductor- and heel-cord release for neuromuscularhip dislocations and contractures. At age 10 years, hehas short stature, microcephaly, rotatory nystagmus,thin fingers, and spasticity. He smiles but is not able tosit or speak.

WG1670, the younger sister of the patient describedabove, was found to have elevated plasma homocysteine(52 mmol/liter) and low methionine (8 mmol/liter) at 6 dof age. She was presumed to have MTHFR deficiencyand was started on folinic acid, vitamin B12, vitamin B6,betaine, and methionine, but these were discontinued byher mother after a few days because the child appearedto be doing well. At 3 mo of age, she had seizures and

respiratory distress, and medications were restarted. By18 mo of age, she was microcephalic and severely de-velopmentally delayed. At 2 years of age, the diagnosisof cblG was established, and she was treated with vi-tamin B12, betaine, and aspirin; at age 6 years, methio-nine supplementation was added. At 9 years, she hasshort stature, microcephaly, rotatory nystagmus, and pesplanus. She is able to walk, responds to simple com-mands, and can speak a few words.

WG1655 presented with short stature, failure tothrive, progressive weakness, hypotonia, ocular nystag-mus, jaundice, feeding difficulties, and diarrhea at 7–10wk of age (Wilden and Scott 1992). He had severe meg-aloblastic anemia and neutropenia, homocysteinemia,hypomethioninemia, and formiminoglutamic aciduriawithout methylmalonic aciduria, which led to the di-agnosis of a defect in methionine synthesis. Treatmentwith hydroxycobalamin, methionine, and folinic acid re-sulted in improved metabolite levels, improvement oftone, and reduction of nystagmus, but poor growth, de-velopmental delay, feeding difficulties requiring a gas-trostomy, persistent anemia, and immunological deficitsat age 4 years (Wilden and Scott 1992).

All three of the patients—WG1670, WG1671, andWG1655—were classified as cblG variants, in accor-dance with findings from tests as described by Sillaotset al. (1992). Test results showed reduced accumulationsof labeled cobalamin, virtually none of which associatedwith the MS enzyme, and almost undetectable MSactivity.

Materials

Oligonucleotide primers were synthesized by R. Clar-izio (Montreal Children’s Hospital Research InstituteOligonucleotide Synthesis Facility, Sheldon Biotechnol-ogy Center, McGill University, and ACGT Corporation,Toronto). AMV reverse transcriptase (RT), Taq poly-merase, Trizol, and DNAzol reagent were purchasedfrom Gibco BRL. Restriction enzymes were purchasedfrom New England Biolabs. The a-[35S] dATP (12.5 Ci/mol) was purchased from Dupont, and the a-[32P] dATPwas from ICN Pharmaceuticals. The random-primedDNA-labeling kit was from Boehringer. The GenecleanIII kit was purchased from Bio 101, the T/A cloning kitfrom Invitrogen, and the Wizard Mini-preps from Pro-mega. Sequenase kits were obtained from United StatesBiochemicals.

Northern Blot Analysis

Total RNA from human fibroblast cell lines WG1670,WG1671, and WG1655 and control 8074 was isolatedby use of the Trizol reagent. Cells were lysed in culturedishes as recommended by the manufacturer. RNA con-

Wilson et al.: Null Mutations in cblG Variants 411

Table 1

cDNA Sequence Segments Used in SSCP Analysis

PCRSegmentNumber

NucleotidePositiona

AnnealingTemperature

(7C) Primer Pairsb

Uncut PCRProduct

(bp)c EnzymeDigestion Products

(bp)d

1 256–635 66 3107/1209A 690 PstI 136, 286, 2682 532–1657 62 309A/1827 1,125 AluI 261, 216, 363,

105, 1803 1468–2057 60 1907A/1907B 590 HinfI 485, 1054 1963–2698 60 1406E/1773 735 AvaII 165, 176, 3945 2610–3856 60 1783/1107A 1,246 MseI 184, 196, 203,

414, 249

a Counted from the A of the ATG initiation codon.b Sense/antisense oligonucleotides used to generate each segment.c Expected size.d Size of PCR fragments after digestion, listed in order from 5′ end of segment.

centrations were measured spectrophotometrically at260 nm. A total of 15 mg of RNA from each patientwas denatured and subjected to electrophoresis in 1.5%agarose formaldehyde gels and was then transferred toZetabind nylon membranes. The membranes were pre-hybridized at 657C in 0.5 M phosphate buffer (pH 7.0),1 mM EDTA, 7% SDS, 1% BSA, and 50 mg single-stranded salmon-sperm DNA for 2 h. Hybridization wasperformed overnight in fresh prehybridization bufferwith a 32P-labeled human MS cDNA probe. The cDNAprobes, generated through PCR with oligonucleotidesD1730 and D1733 (Leclerc et al. 1996), were labeledwith a-[32P] dATP by use of the random-primed DNA-labeling kit. A phosphorimage analyzer (Fuji BAS 2000Bio-Imaging Analyser; Fuji Medical Systems) was usedto detect radioactive signals.

Mutation Analysis

Total cellular RNA was isolated from fibroblast pelletsby use of the method of Chirgwin et al. (1979). GenomicDNA was isolated with the use of DNAzol reagent ac-cording to the manufacturer’s specifications. Reversetranscription was performed with 25 mg total RNA inreactions containing 2.5 U of AMV RT and 500 ng ofMS-specific terminal oligonucleotide primer (1107A inthe report by Leclerc et al. [1996]) in a total reactionvolume of 54 ml. Resultant cDNA was used as templatefor PCR. PCR for five overlapping cDNA segments (ta-ble 1) was performed in reactions containing 5 ml oftemplate; 1 ml each of dTTP, dGTP, and dCTP (10 mM);0.5 ml of dATP (10 mM); and 1 ml of a-[35S] dATP (12.5Ci/mol). SSCP analysis was performed as described else-where (Leclerc et al. 1996). Fragments displaying shiftswere either subcloned and sequenced or sequenced di-rectly. Sequencing was performed with use of the primersof the initial PCR amplification, on both genomic DNAand cDNA.

The confirmation of mutations in genomic DNA was

made by PCR-dependent diagnostic tests. Genomic PCRwas performed with 3 ml of template, 500 ng of specificprimers, 1 ml each of dNTPs (10 mM), and 3 U of Taqpolymerase in a 46-ml volume. Mutation 2112delTC wasidentified through genomic sequencing and was con-firmed by heteroduplex analysis by use of oligonucleo-tides AA111 (5′-GTG ATA GGA AGC CAG ATT GAG)and 511B (5′-GAA AAT TTC CCC ATA TTT TGC TAAC) as primers for the PCR reaction. The product washeated to 957C for 3 min, cooled to room temperature,and run on 8% polyacrylamide gels. Mutation IVS-166ArG was identified through genomic sequencing us-ing primer pairs 1939 (5′-CTA CCT CAT GGT TTGGGA GGA GAA) and 1938 (5′-GAT TCA TGA CCATCT AAT ACT CAG). The mutation was detected bya test for the loss of an artificially created MseI restrictionsite. Primer pairs used for this diagnostic test were2802A (5′-CTT GTC TTT CCT TGC GCC TTT TA)and 2808C (5′-AGA GCA CAT GTT CAT CCG TAGGC), where the underlined T in 2802A replaced the Cin the normal sequence. To test for the mutation, 15 mlof PCR product was digested in 2.2 ml of New EnglandBiolab’s Buffer 2, 2.2 ml of 10 # BSA, and 2.5 ml ofMseI. The GrA substitution found in the intron begin-ning after Lys203 was identified through genomic se-quencing by use of primer pairs 309A (5′-GAG CAGGCC AAA GGA CTT CTG GAT) and 2802B (5′-CTGCTC TTC AAC TGA GGA GTG GCT). The mutationwas confirmed by a test for the presence of an artificiallycreated BfaI site. The PCR primers used were AE358(5′-TTT ACA GAT TCT ATT TTT TTG TTC T) andAE359 (5′-TCT GAT TCC CAC AAC AAT GAA A),where the underlined C in AE358 replaced the G in thenormal sequence. To test for the mutation, 15 ml of PCRproduct was digested in 1.9 ml of NE Buffer 4 and 2 mlof BfaI. Mutation 3378insA was first identified throughgenomic sequencing with primer pairs 1774 (5′-TGCCTC TCA GAC TTC ATC GCT CCC) and AE373 (5′-

412 Am. J. Hum. Genet. 63:409–414, 1998

Figure 1 Results of northern analysis, showing levels of mRNAin variant cell lines WG1670, WG1671, and WG1655. The controlpattern is on the right, with bands at 10 and 7.5 kb compared withmolecular-weight standards. At the bottom is the pattern obtained for18S RNA as a control for differences in sample loading.

Figure 2 mRNA abnormalities in mutant fibroblast lines. A,Agarose gel of RT-PCR products showing extra bands indicative ofsplice defects in fibroblast lines WG1671 and WG1655. C1 is a controlcell line; B, the water blank. The bands on the left are molecular-weight standards. B, Mechanism of splicing defects. In cell linesWG1670 and WG1671, the ArG substitution at position 2166 ofthe intron after exon 3, beginning at Leu113, creates a 3′ acceptor site165 bp upstream of the normal acceptor site. This new 3′ site replacesthe original downstream site, which has not been altered. In cell line1655, the GrA substitution near the middle of the intron after exon6, beginning at Lys203, creates a 3′ acceptor site and consequent gen-eration of cryptic donor sites 78 and 128 bp farther downstream. Allinserts contain stop codons.

CTC CCT ACT GCC TGG CCT CTG TC). The diag-nostic test for the 3378insA mutation was performed byassaying for the loss of an artificially created AflII site.The oligonucleotide primers used for PCR were AF345(5′-GAC TAC AGC AGC ATC ATG CTT AA) andAH450 (5′-GAC ACT GGT TCT AAG GGC TGA T),where the underlined C and T in AF345 replaced thenormal nucleotides G and C, respectively. To test for themutation, 15 ml of PCR product was digested in 2 ml ofNew England Biolab’s Buffer 2, 2 ml of 10 # BSA, and1 ml of AflII. All digestions were performed at 377Covernight before the samples were subjected to electro-phoresis on 8% polyacrylamide gels.

Results

Fibroblast cell lines WG1670, WG1671, andWG1655 were analyzed by means of northern blot, witha 1-kb PCR-generated fragment from the human MScDNA used as a probe. Normal cells showed bands at7.5 and 10 kb, as expected (Chen et al. 1997). In con-trast, none of the patient samples gave a detectable signalunder the same exposure conditions, although trace sig-nals could be detected, after long exposure, by phos-phorimage analysis (fig. 1). It was possible to amplifythese trace-level transcripts through RT-PCR, which al-lowed an analysis of the resultant cDNA. The cDNAwas divided into five overlapping segments, as shown intable 1. These fragments were amplified and evaluatedfor possible mutations.

When RT-PCR products were run with use of agarosegel electrophoresis, slower-running bands were found as-

sociated with fragment 1 of both WG1671 and WG1655(fig. 2A). Subcloning and sequencing of the larger bandassociated with WG1671 revealed a 165-bp insertion ofintronic DNA at junction 339/340 after exon 3 (num-bered from the A of the initiation codon; fig. 2B). Se-quencing of genomic DNA showed the insertion to bethe result of a substitution, IVS-166ArG, which gen-erates a cryptic 3′ acceptor splice site at position 2166of the intron beginning after Leu113. The insertion con-tains an in-frame stop codon 9 bp into the sequence, anindication that translation is terminated. The mutationwas confirmed by diagnostic PCR with use of an arti-ficially created MseI restriction site (fig. 3A). Alterationof the sense oligonucleotide created an MseI cut site incontrol DNA. The presence of the ArG mutation elim-inated the restriction site. The mutation was confirmedas heterozygous in genomic DNA from both siblings(WG1670 and WG1671), since both cut and uncutbands were present on the diagnostic gel. DNA from themother was also made available, and testing for the mu-tation gave negative results.

The second mutation in WG1670 and WG1671 wasdetected as a heteroduplex during SSCP analysis of frag-

Wilson et al.: Null Mutations in cblG Variants 413

Figure 3 DNA-based diagnostic tests for the identified muta-tions. DNA was amplified by PCR and either digested or not digestedwith the designated restriction enzyme, and the fragments were re-solved by PAGE. A, Test for IVS-166ArG. An artificially created MseIsite was introduced through alteration of the sense oligonucleotide2802A, so that MseI digestion of control DNA cleaves the 214-bpPCR product into fragments of 139, 53, and 23 bp (the last not shown).The mutant sequence gives 139- and 76-bp fragments as cleavageproducts. B, Test for 2112delTC. Electrophoresis of the PCR productsleaves trailing heteroduplexes in samples where the mutation is het-erozygous. C, Test for GrA mutation in intron after Lys203. An ar-tificially created BfaI site was introduced through alteration of thesense oligonucleotide AE358, so that BfaI digestion results in cleavageof the 150-bp PCR product to 126 and 24 bp (latter not shown) ifthe mutation is present. D, Test for 3378insA. An artificially createdAflII was introduced through alteration of the sense oligonucleotideAF345, so that digestion with AflII cleaves the 151-bp PCR productto 132 and 19 bp (latter not shown) if the wild-type sequence is present.For all panels, C, C1 and C2 are control DNA samples, and the num-bered samples correspond to the WG designation of patient cell lines.

ment 4. It was sequenced directly from cDNA and ge-nomic DNA PCR products and was shown to be due toa 2-bp deletion, 2112delTC, resulting in a frameshiftand a downstream stop codon. A diagnostic test for themutation was made by heteroduplex analysis of PCRproducts amplified from genomic DNA (fig. 3B). It wasconfirmed in both siblings. DNA from the mother wasnegative for the IVS-166ArG mutation and positive for2112delTC, which confirmed that the mutations are onseparate chromosomes (fig. 3A and B).

Sequencing of the slower-running bands associatedwith the RT-PCR of fragment 1 from cell line WG1655yielded two insertions in the mRNA, one of 78 bp andanother of 128 bp, beginning after Lys203. The 78-bpinsertion was found to be a truncated version of the 128-bp insertion, missing the last 50 bp. By means of genomicsequencing, the insertions were discovered to be the re-sult of a GrA substitution, near the center of the intronafter exon 6, that created a single cryptic 3′ acceptorsplice site (fig. 2B). For each insert, a cryptic 5′ donorsplice site was recruited to complete the new exon, oneat 78 bp and the other at 128 bp downstream of themutation. Neither of the newly created 5′ donor sites is

preferred, since the two transcripts appear to occur inequal amounts. An in-frame stop codon occurs 9 bp intothe insertions. The mutation was confirmed by use ofdiagnostic PCR on genomic DNA, with an artificiallycreated BfaI restriction site (fig. 3C). Alteration of thesense oligonucleotide created a BfaI restriction site if theGrA mutation was present. Because the mutation washeterozygous, both cut and uncut bands were presenton the diagnostic gel.

In RT-PCR and SSCP analysis of segment 5, the secondmutation in WG1655 was detected as a band shift, rel-ative to the controls. This segment was subcloned andsequenced from both cDNA and genomic DNA. A 1-bpinsertion, 3378insA, was detected, which results in aframeshift and a downstream stop codon. The mutationwas confirmed by diagnostic PCR on genomic DNA,with use of an artificially created AflII restriction site.Alteration of the sense oligonucleotide created an AflIIsite that was eliminated if the inserted A was present(fig. 3D). Because the mutation was heterozygous, bothcut and uncut bands were present in the diagnostic gel.Screening for the four mutations in the other cblG celllines gave negative results, showing that the mutationsare unique to these individuals.

Discussion

All four mutations described here—the two splice mu-tations and two frameshifts—are compatible with pre-mature termination of translation and consequent insta-bility of MS mRNA, as confirmed by the near-completeabsence of mRNA detected by northern blotting. Thesemolecular defects provide an explanation for the absenceof cobalamin-associated MS and for the profound de-ficiency of enzyme activity in cultured fibroblasts thatdefines the cblG-variant group (Sillaots et al. 1992). Incontrast, other cblG patients incorporate [57Co]CN co-balamin into their MS protein, although they too havedeficient MS activity.

The three cblG-variant patients showed onset of dis-ease in the first few days or weeks of life, with profoundneurological findings, hyperhomocysteinemia, and hy-pomethioninemia. Poor growth, hypotonia, nystagmus,and developmental delay were common features. Oneof the three patients (WG1655) had severe megaloblasticanemia. These findings are similar to those for patientswith severe cblG disease, as described by Watkins andRosenblatt (1989). Although the onset of disease mayalso be early in non–cblG-variant patients, it may notoccur until either later in childhood or even adulthood(Carmel et al. 1988). Patients with cblE disease, due toa deficiency in MS reductase (Leclerc et al. 1998), alsoshow a broad spectrum of clinical findings that are sim-ilar to those seen in the cblG patients.

It is significant that all four alleles in the two families

414 Am. J. Hum. Genet. 63:409–414, 1998

we studied had null-type mutations. Thus, the relativelyrare cblG variants do not share a common genetic ab-normality but instead share a biochemical characteristic,the absence of MS protein. Although this leads to asevere early-onset cblG phenotype, our findings confirmthat complete MS deficiency is not lethal.

Acknowledgments

We thank G. Dunbar and P. Zhao for growing the cell cul-tures. We are grateful to J. P. Pfotenhauer for providing DNAfrom siblings WG1670 and WG1671 and from their mother.We also thank N. Matiaszuk and H. Lue-Shing for technicalassistance. These studies were supported by grant GR-13297from the Medical Research Council of Canada Group in Med-ical Genetics and by grant HL58955-01 from the NationalHeart, Lung, and Blood Institute.

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