Comprehensive mutation analysis of GLDC, AMT, and GCSH in nonketotic hyperglycinemia

Article (PDF Available)inHuman Mutation 27(4):343-52 · April 2006with134 Reads
DOI: 10.1002/humu.20293 · Source: PubMed
Nonketotic hyperglycinemia (NKH) is an inborn error of metabolism characterized by accumulation of glycine in body fluids and various neurological symptoms. NKH is caused by deficiency of the glycine cleavage multi-enzyme system with three specific components encoded by GLDC, AMT, and GCSH. We undertook the first comprehensive screening for GLDC, AMT, and GCSH mutations in 69 families (56, six, and seven families with neonatal, infantile, and late-onset type NKH, respectively). GLDC or AMT mutations were identified in 75% of neonatal and 83% of infantile families, but not in late-onset type NKH. No GCSH mutation was identified in this study. GLDC mutations were identified in 36 families, and AMT mutations were detected in 11 families. In 16 of the 36 families with GLDC mutations, mutations were identified in only one allele despite sequencing of the entire coding regions. The GLDC gene consists of 25 exons. Seven of the 32 GLDC missense mutations were clustered in exon 19, which encodes the cofactor-binding site Lys754. A large deletion involving exon 1 of the GLDC gene was found in Caucasian, Oriental, and black families. Multiple origins of the exon 1 deletion were suggested by haplotype analysis with four GLDC polymorphisms. This study provides a comprehensive picture of the genetic background of NKH as it is known to date.
HUMA N MUTATION 27(4), 343^352, 2006
Comprehensive Mutation Analysis of
in Nonketotic Hyperglycinemia
Shigeo Kure,
Kumi Kato,
Agirios Dinopoulos,
Chuck Gail,
Ton J. deGrauw,
John Christodoulou,
Vladimir Bzduch,
Rozalia Kalmanchey,
Gyorgy Fekete,
Alex Trojovsky,
Barbara Plecko,
Galen Breningstall,
Jun Tohyama,
Yoko Aoki,
and Yoichi Matsubara
Department of Medical Genetics, Tohoku University School of Medicine, Sendai, Japan;
Division of Pediatric Neurology, Cincinnati Children’s
Hospital, Cincinnati, Ohio;
Discipline of Paediatrics and Child Health, University of Sydney and Royal Alexandra Hospital for Children,
Westmead, Australia;
First Department of Pediatrics, Comenius University Children’s Hospital, Bratislava, Slovakia;
Department of Pediatrics,
Semmelweis University, Budapest, Hungary;
Institute for Medical Biology and Human Genetics, Medical University of Graz, Graz, Austria;
Department for Pediatrics, Medical University of Graz, Graz, Austria;
Department of Pediatrics (Neurology); Park Nicollet Clinic, Minneapolis,
Department of Pediatrics, Nishi-Niigata Chuo National Hospital, Niigata, Japan;
21st COE Program ‘‘Comprehensive Research
and Education Center for Planning of Drug Development and Clinical Evaluation,’’ Tohoku University, Sendai, Japan
Communicated by Jan P. Kraus
Nonketotic hyperglycinemia (NKH) is an inborn error of metabolism characterized by accumulation of glycine in
body fluids and various neurological symptoms. NKH is caused by deficiency of the glycine cleavage multi-enzyme
system with three specific components encoded by GLDC, AMT,andGCSH.Weundertookthefirst
comprehensive screening for GLDC, AMT,andGCSH mutations in 69 families (56, six, and seven families with
neonatal, infantile, and late-onset type NKH, respectively). GLDC or AMT mutations were identified in 75% of
neonatal and 83% of infantile families, but not in late-onset type NKH. No GCSH mutation was identified in this
study. GLDC mutations were identified in 36 families, and AMT mutations were detected in 11 families. In 16 of
the 36 families with GLDC mutations, mutations were identified in only one allele despite sequencing of the entire
coding regions. The GLDC gene consists of 25 exons. Seven of the 32 GLDC missense mutations were clustered
in exon 19, which encodes the cofactor-binding site Lys754. A large deletion involving exon 1 of the GLDC gene
was found in Caucasian, Oriental, and black families. Multiple origins of the exon 1 deletion were suggested by
haplotype analysis with four GLDC polymorphisms. This study provides a comprehensive picture of the genetic
background of NKH as it is known to date. Hum Mutat 27(4), 343–352, 2006.
2006 Wiley-Liss, Inc.
KEY WORDS: GLDC; AMT; GCSH; glycine encephalopathy; nonketotic hyperglycinemia; NKH; glycine cleavage
system; mutation spectrum; genotype–phenotype
Nonketotic hyperglycinemia (NKH, MIM] 605899), also
termed glycine encephalopathy, is an inborn error of amino acid
metabolism characterized by the accumulation of a large amount
of glycine in body fluids [Hamosh and Johnston, 2001]. Glycine
levels are elevated to a much greater extent in cerebrospinal fluid
(CSF) than in plasma; hence, an abnormally high value for the
CSF/plasma glycine ratio is observed. NKH is clinically classified
(by onset of symptoms) as three types: neonatal, infantile, or late-
onset. Later onset appears to be associated with a better prognosis.
The vast majority of patients fall into the neonatal category, which
involves a stereotypic presentation with severe hypotonia, apnea
requiring assisted ventilation, and intractable seizures. Approxi-
mately 30% of such patients die in the neonatal period. Survivors
often have severe psychomotor retardation, although 15–20% of
survivors achieve developmental milestones such as head control,
independent sitting, or walking [Hoover-Fong et al., 2004].
Patients with the infantile type of NKH are often asymptomatic
in the neonatal period and the phenotype is characterized by mild
to moderate psychomotor retardation, behavioral problems,
seizures, and chorea. The clinical presentations of late-onset
NKH are heterogeneous. In previous studies, two families did not
have seizures or mental retardation, but exhibited progressive
paraplegia and optic atrophy [Bank and Morrow, 1972; Steiman
et al., 1979]. Another family was reported to present with mental
retardation and choreoathetosis [Singer et al., 1989].
The fundamental defect lies in the glycine cleavage system
(GCS; EC2.1.2.10) [Tada et al., 1969]. The GCS is a
mitochondrial complex enzyme system that consists of four
Published online 31 January 2006 in Wi ley InterScience (www.
in terscience.w i
DOI 10.1002/humu.20293
Th e Su pp leme n t ary Material re ferre d to in t h is artic l e ca n b e ac-
cessed at http:/ /www. interscience.wi /jpages/1059-7794/
Recei ved 11 Jul y 2005; ac c epted revis ed m a n us cript 9 Novemb er
Grant sponsor: Ministry of Education, Culture, Sports, Science, and
Technology; Ministry of Health, Labor, an d Public Welfare inJapan.
Correspon den ce to: Sh igeo Kure, M.D. , Departmen t of Me di ca l
Genetics, Tohoku University School of Medicine, 1-1 Seiryomachi,
Aobaku, Sendai 980- 8574, Japan.
individual proteins [Kikuchi, 1973]: glycine decarboxylase (also
called P-protein), aminomethyltransferase (T-protein), hydrogen
carrier protein (H-protein), and dihydrolipoamide dehydrogenase
(L-protein). The enzymatic analysis of NKH patients revealed that
approximately 80% of patients with NKH have a GLDC deficiency
and the rest have an AMT deficiency [Tada and Hayasaka, 1987].
Mitochondrial precursors of human P, T, H, and L-proteins consist of
1,020, 403, 173, and 509 amino acids, respectively. Dihydrolipoa-
mide dehydrogenase is a housekeeping enzyme that serves as an E3
component of other a-keto acid dehydrogenase complexes, such as
pyruvate dehydrogenase. Deficiency of dihydrolipoamide dehydro-
genase causes progressive neurological deterioration with lactic
acidosis but not hyperglycinemia [Hong et al., 1996]. The three
GCS-specific components (P, T, and H-proteins) are encoded by
distinct genes: GLDC (MIM] 238300) on chromosome 9p24 [Isobe
et al., 1994], AMTon 3p21.1-21.2 [Nanao et al., 1994], and GCSH
(MIM] 238330) on 16p24 [Kure et al., 2001], respectively.
To date, a limited number of NKH mutations have been
reported (Human Gene Mutation Database, Cardiff; http:// The GLDC mutations reported to date
include the S564I mutation that is prevalent in Finnish patients
[Kure et al., 1992], the R515S mutation found in 5% of Caucasian
patients [Toone et al., 2000], microdeletions [Kure et al., 1991],
large deletions [Takayanagi et al., 2000; Sellner et al., 2005], one
abnormal splicing [Flusser et al., 2005], one nonsense mutation
[Sellner et al., 2005], and 10 missense mutations [Toone et al.,
2002; Korman et al., 2004; Kure et al., 2004; Boneh et al., 2005;
Dinopoulos et al., 2005]. The AMT gene (MIM] 238310)
mutations identified to date include nine missense mutations
[Nanao et al., 1994, 1994a; Kure et al., 1998; Toone et al., 2000,
2001, 2003], one microdeletion [Kure et al., 1998, 1998b], and
one splicing mutation [Toone et al., 2000]. In GCSH we have
found one abnormal splicing in a patient with a transient form of
NKH [Kure et al., 2002]. Since multiple genes are responsible for
NKH, previous studies screened only a small number of GLDC
and AMT exons and/or a few patients, which hampered
elucidation of the genetic background of NKH.
The purpose of the present study was to establish the mutation
spectrum of NKH by performing a comprehensive screening for
mutations in GLDC, AMT, and GCSH in 69 families with three
different types of NKH. The structure of AMT has been
determined [Nanao et al., 1994, 1994b]. Also, we previously
reported the exon-intron organizations of GLDC [Takayanagi et al.,
2000] and GCSH [Kure et al., 2001], which provided us with basic
information to amplify the entire coding regions for the three genes.
To increase the sensitivity of mutational screening, we directly
sequenced all amplicons without employing prescreening scanning
methods such as single-strand conformation polymorphism.
Patients were examined in the metabolic disease clinics of a
number of referring hospitals. NKH was clinically suspected based
on the presentation of symptoms characteristic of each disease
type and electroencephalograms (EEG) recordings, and were
subsequently confirmed by amino acid analysis. The CSF/serum
glycine ratio at diagnosis was 40.04 in all patients, whereas it was
o0.03 in normal neonates. Patients were classified into three
clinical subtypes (neonatal, infantile, and late-onset) based on the
onset of clinical symptoms.
Neonatal type. We studied 56 families with neonatal onset.
Initial symptoms, including hypotonia, apnea, and coma, devel-
oped within 7 days after birth–in most cases within 3 days. Almost
all of the patients showed a burst suppression pattern on EEG
within 2 weeks after birth, and hypsarrhythmia thereafter.
Infantile type. Six families were enrolled. Symptoms started
at 3–12 months of age. Five of the six patients had no symptoms in
the neonatal period. Very mild hypotonia and apnea were observed
in patient P107 [Dinopoulos et al., 2005]. Mild mental retardation
and abnormal behaviors, such as aggressiveness, developed in
Late-onset type. We studied seven patients with late-onset
NKH. All seven patients had elevated glycine concentrations in
repeated amino acid analysis of CSF and/or plasma. Spastic
paraplegia without mental retardation developed in three of the
seven patients. These patients resembled those in previously
reported families [Bank and Morrow, 1972; Steiman et al., 1979].
The rest of the patients presented with mental retardation
after they entered school or during adolescence. There is some
confusion in terms of phenotypic classification of the mild form of
the infantile type and the late-onset type, since there are some
reports of patients in whom developmental delay or mild
hypotonia started in the middle or late infantile periods [Flannery
et al., 1983; Singer et al., 1989]. We classified such patients as
having the infantile type—not the late-onset type. In the present
study we classified patients as having the late-onset type when
they were free of any symptoms during infancy.
Mutational Screening
Exons and flanking intron sequences of GLDC (GenBank
NM_000170, NT_008413.16), AMT (GenBank NM_00481,
NT_086638.1), and GCSH (GenBank NM_004483,
NC_00016.8) were amplified by PCR from genomic DNA,
followed by direct sequencing analysis using the dye-primer
sequencing method as previously described [Kure et al., 2001].
The 18-mer oligonucleotides of the M13 and reverse sequencing
primers were added to the 5
end of the forward and reverse PCR
primers, respectively. We initially screened for a large deletion
involving GLDC exon 1 by semiquantitative PCR using the
pseudogene of GLDC (GLDCP) as a gene dose control
[Takayanagi et al., 2000]. PCR primer sequences for amplifying
GLDC exons 1–6 were previously reported [Takayanagi et al.,
2000], and those for other exons are described in Supplementary
Table S1 (available online at
jpages/1059-7794/suppmat). Each PCR cycle consisted of denatur-
ing at 981C for 10 sec, annealing at 551C for 30 sec, and extension
at 721C for 30 sec, with repetition for 35 cycles. For PCR
amplification of GLDC exon 1, 10% dimethylformamide was
added to the reaction mixture. Fifty control subjects were
subsequently screened for any detected base changes to exclude
noncausative polymorphisms. If no mutations were detected in the
sequencing analysis of GLDC , we screened for mutations in AMT
by amplifying all of the nine exons. When no mutations were
identified in either GLDC or AMT, we sequenced all of the five
exons in GCSH. For characterization of GLDC and AMT missense
mutations, each mutated amino acid residue was compared with
the corresponding amino acid in rat [Sakata et al., 2001], chicken
[Kume et al., 1991], pea [Turner et al., 1992], and E. coli
[Okamura-Ikeda et al., 1993], as shown in Tables 2 and 3.
Characterization of the GLDC Deletion
We identified the minimum deleted region of both alleles of
each homozygous patient of the GLDC exon 1 deletion. Fifteen
sequence tagged sites (STSs) were used for this deletion mapping,
344 HUMAN MUT ATION 27(4), 343 ^352, 2006
Human Mutation DOI 10.1002/humu
as illustrated in Fig. 1. PCR primer sequences and amplification
conditions for D9S281 and RH92434 were obtained from the
website of the UC SC Genome Browser (
The PCR primer sequences and amplicon sizes of STSs 1–8 are
presented in Supplementary Table S1. STSs 1–7 were located 5
upstream of the GLDC gene, and STS 8 was located in intron 2.
Amplification primers for GLDC exons 1–5 were also used in the
deletion mapping. We amplified these 15 STSs by using genomic
DNA of Patients P5 and P36 as a template in order to test whether
each STS was involved in the homozygously deleted region.
Structural information about the 5
upstream region of GLDC and
the location of Alu motifs was obtained from the UCSC Genome
Browser (Fig. 1).
Mutation Screenin g
We performed mutational screening in 69 NKH families (56
neonatal type, six infantile type, and seven late-onset type). First,
the GLDC exon 1 deletion was screened by semiquantitative PCR
amplification using the GLDCP as a control of the gene copy
number. This deletion was found in six families (Table 1).
Subsequent extensive sequencing of GLDC, AMT, and GCSH
coding exons revealed that 42 of the 56 neonatal-type families
(75%) had GLDC or AMT mutations in at least one of two alleles.
GLDC mutations were found in 31 (74%) and AMT mutations
were detected in 11 (26%) of the 42 families. No differences were
FIGURE 1. Mapping of the GLDC deletions. Genomic regions that were homozygously deleted in patients P5 and P36 were de¢ned by
ampli¢cation of 13 STS markers.TheJMJD2 C gene and the gene-like structure, LOC158352, are shown based on the information of
the UCSC Genome Browser. E1^5 indicate amplicons including the GLDC exons 1^5, respectively. Amplicons (1) indicate that the
STS was successfully ampli¢ed, while (^) means that the STS failed to be ampli¢ed.
FIGURE 2. NKH mutations identi¢ed in this and previous studies.The GLDC (A) and AMT (B) exons are indicated by open boxes, and
noncoding regions are shaded. Missense and nonsense mutations are shown above the exon boxes, and deletions/insertions
and mutations of splicing errors are indicated below the exon boxes.
HUMAN MUTATION 27(4), 343^ 352 , 2006 345
Human Mutation DOI 10.1002/humu
TA B LE 1. PatientsWith GLDC orAMT Mutations
Family number
Ethnicity Age of onset Consanguinity CSF Gly (mM) CSF/serum Gly ratio Gene Mutation 1 Mutation 2 Reference
Neonatal type
P14 Caucasian Day 1 No 296 0.25 GLDC Exon 1 deletion c.2714T4G This study
P21 Caucasian Day 1 Yes 71 0.24 AMT c.230C4Tc.230C4T This study
P26 Caucasian Day 1 Yes 86 0.27 AMT c. 1 25A4Gc.125A4GKureetal.[1998a]
P29 Caucasian Day 1 No 43 0.04 GLDC c.793delC ND This study
P31 Caucasian Day 1 No 196 0.26 AMT c.4 7112T4Cc.887G4A This study
P32 Caucasian Day 1 No 32 0.20 GLDC c.1786C4Tc.1786C4T This study
P36 Caucasian Day 1 No 220 0.18 GLDC Exon 1 deletion Exon 1 deletion This study
P39 Caucasian Day 1 No 187 0.13 GLDC c.1 5 95C4Gc.1832T4G This study
P48 Caucasian Day 1 No 198 0.20 GLDC c.266511G 4C ND This study
P49 Caucasian Day 1 No 213 0.17 GLDC c.2203^2A4G ND This study
P59 Caucasian Day 1 Yes 202 0.23 GLDC c. 1 996C4T c.1996C4T This study
P5 Oriental Day 1 No 240 0.18 GLDC Exon 1 deletion Exon 1 deletion Ohya et al. [1991]
P6 Oriental Day 1 No 160 0.09 GLDC c. 245T4Gc.1821_18311del Kure et a l. [2004]
P19 Oriental Day 1 No 264 0.30 GLDC c. 449A4Cc.2368C4TKureetal.[2004]
P25 Oriental Day 1 No 74 0.34 AMT c. 54delC c.8 26G4CKureetal.[1998b]
P74 Oriental Day 1 No 117 0.12 GLDC c.23 11G4A ND This study
P86 Oriental Day 1 No 177 0.20 GLDC c. 22 13_2214delGT ND This study
P93 Oriental Day 1 No 130 0.13 GLDC c.2105C 4T ND This study
P115 Oriental Day 1 No 145 0.11 AMT c. 14 7delG c. 9 70_972delATG This study
P124 Oriental Day 1 No 300 0.11 GLDC c.25511G4Ac.806C4T This study
P30 Caucasian Day 2 No 121 0.19 AMT c.60delG c.47 112T4C This study
P44 Caucasian Day 2 No 98 0.11 GLDC c.806C4Tc.25511G4A This study
P46 Caucasian Day 2 No 155 0.13 GLDC c.2 293C4T c.1 705 G 4A This study
P47 Caucasian Day 2 No 78 0.17 GLDC c.2 5 19T4A ND This study
P61 Caucasian Day 2 No 186 0.17 AMT c. 982_972GC4Tc.452_466del This study
P75 Caucasian Day 2 No 167 0.16 AMT c.212 A 4Cc.217C4T This study
P3 Oriental Day 2 No 151 0.10 GLDC c. 2306 C4T c. 2846C4T This study
P4 Oriental Day 2 No 387 0.28 GLDC c.2258 A 4C c.2839^1G4C This study
P7 Oriental Day 2 No 164 0.10 GLDC c.2266_2268delTTC ND Kure et al. [1991]
P8 Oriental Day 2 No 209 0.31 GLDC c. 2 080G4C ND This study
P10 Oriental Day 2 No 517 0.20 GLDC c.887T 4G ND This study
P77 Oriental Day 2 No 148 0.12 GLDC c. 449A4Cc.192611G4A This study
P91 Oriental Day 2 No 132 0.08 AMT c.6 1delC c.53 5delC This study
P69 Oriental Day 3 No 220 0.12 GLDC c.23 11G4A ND This study
P70 Oriental Day 3 No 68 0.19 GLDC Exon 1 deletion ND This study
P120 Oriental Day 3 No 324 0.19 GLDC c.25 74T4G ND This study
P23 Oriental Day 4 No 83 0.14 GLDC c.2 1 8 2G4C ND This study
P104 Caucasian Day 4 No 240 0.26 GLDC c.1166C4T c .144 3insG This study
P15 Black Day 5 No 333 0.08 GLDC Exon 1 deletion c.2891insA This study
P34 Caucasian Day 6 No 174 0.08 AMT c. 139G4T ND This study
P76 Caucasian Day 6 No 215 0.18 AMT c.1 36G4Ac.230C4T This study
P72 Black Day 7 No 117 0.20 GLDC c.266511G4Cc.176G4C This study
Infantile type
P50 Caucasian 3 months No 200 0.25 GLDC Exon 1 deletion c.2311G4A This study
P78 Caucasian 6 months No 46 0.06 GLDC c.2 2 16G4Ac.2216G4ADinopoulosetal.[2005]
P12 Caucasian 12 months No 41 0.04 GLDC c.221 6G4A ND Christodoulou et al. [1993]
P107 Caucasian Unclear
No 150 0 .09 GLDC c.1166C4Tc.1166C4TDinopoulosetal.[2005]
P108 Caucasian Unclear
No 55 0. 05 GLDC c.1166C4Tc.1166C4TDinopoulosetal.[2005]
DNA mutation numbering is based on cDNA sequence: 11 corresponds to theA of the ATG translation initiation codon. GLDC:NM_000 17 0; AMT:NM_00481 .
Developmental delay in infantile period.
ND, not detected.
346 HUMAN MUTATION 27(4), 343^352, 2006
Human Mutation DOI 10.1002/humu
observed in CSF glycine levels between 42 mutation-positive and
14 mutation-negative individuals. GLDC mutations were detected
in five (83%) of the six patients with infantile NKH. No mutations
were found in GLDC, AMT,orGCSH in any of seven patients
with late-onset NKH. NKH mutations were highly heterogeneous.
Only nine of the 47 mutation-positive individuals were homo-
zygous for a single mutation (three individuals from consangui-
neous families (P21, P26, and P59), and five homozygotes of
recurrent mutations (P36, P5, P78, P107, and P108)). Patient P32
was homozygous for a private mutation (p.R596X), even though
there was no evidence that he was the product of a consangui-
neous marriage. No GCSH mutations were identified in this study.
TABLE 2. GLDC Mutations
Mutation Location
of mutation
No . of
mutation Comments
Missense mutations Evolutionary conservation
Human Rat Chicken Pea E.coli
c. 17 6G4C Exon 1 p.R59T 1 Yes Arg Arg Arg Ser Ser
c.2 45T4G Exon 1 p.L82W 1 No Lue Lue Val Val Val
c. 449A4C Exon 3 p.N150T 2 No Asn Asn Asn Asn Asn
c.806C4T Exon 6 p.T269M 2 Yes Thr Thr Thr Thr Thr
c.88 7T4G Exon 7 p.L296R 1 Yes Leu Leu Leu Leu Ile
c. 11 66C4T Exon 9 p. A389V 3 No Ala Ala Ala Ala Ala
c. 1595C4G Exon 13 p.T532R 1 Yes Thr Thr Thr His Thr
c. 170 5G4A Exon14 p.A569T 1 Yes Ala Ala Ala Met Ile
c. 1832T4G Exon 15 p.V611G 1 Yes Val Val Ile Phe Val
c. 2080G4C Exon18 p.A694P 1 Yes Ala Ala Ala Ala Cys
c.2 105C4T Exon18 p.S701F 1 Yes Ser Ser Ser Ser Ser
c.2 182G4C Exon 18 p.G728R 1 Yes Gly Gly Gly Gly Gly
c.2 2 1 6G4A Exon19 p.R739H 2 No Arg Arg Arg Ser Ser
c.2258A4C Exon 19 p.H753P 1 Yes His His His His His
c.2 293C4T Exon 19 p.P765S 1 Yes Pro Pro Pro Pro Pro
c.2306C4T Exon19 p.P769L 1 Yes Pro Pro Pro Pro Pro
c.2311G4A Exon19 p.G771R 3 Yes G ly Gly Gly Gly Gly
c.2368C4T Exon 20 p.R790W 1 No Arg Arg ^ ^ ^
c.2 5 1 9T4A Exon 21 p.M840K 1 Yes Met Met Met Met Ile
c.2 714T4G Exon 23 p.V905G 1 Yes Val Val Ile Ile Val
c. 2846C4T Exon 24 p.P949L 1 Yes Pro Pro Pro Pro Pro
Nonsense mutations
c. 1786C4T Exon15 p.R596X 1 Yes
c.1996 C 4T Exon 17 p.Q666X 1 Yes
c.2 574T4GExon22p.Y858X 1Yes
Deletions and insertions
Exon1 deletion Exon 1 Deletion including exon 1 6 No A recurrent
c.793delC Exon 6 Deletion of ¢rst letter
of 265Gln codon
1 Yes FS after codon
at codon 296
c. 182 1_1831del Exon 15 11-bp deletion started at
third letter of 607Gly
1 No FS after codon
at codon 669
c.1443insG Exon 11 Insertion of G after third
letter of 481Leu codon
1 Yes FS at codon
at codon 491
c.2 2 1 3_2214delGT Exon 19 Deletion of GT in
738Cys codon
1 Yes FS after codon
at codon 745
c.2 266_2268delTTC Exon19 Deletion ofTTC in
756Phe codon
1 No No FS/TRM
at codon 1019
c.2891insA Exon 24 Insertion of A after second
letter of 964Tyr codon
1 Yes No FS/TRM
at codon 964
Aberrant splicing
c.2 5511G4A Intron 2 Disruption of splicing
donor site, gt4at
c.1926 11G 4A Intron 17 Disruption of splicing
donor site, gt4at
c.2203^ 2A4G Intron18 Disruption of splicing
acceptor site, ag4gg
c.266511G4C Intron 22 Disruption of splicing
donor site, gt4ct
c.2839^1G4C Intron 23 Disruption of splicing
acceptor site, ag4cg
DNA mutation numbering is based on cDNA sequence: 11 corresponds to theA of the ATG translation initiation codon. GDLC:NM_000 17 0 .
FS, frameshift; TRM, termination codon.
HUMAN MUTA TION 27( 4), 343^352, 2006 34 7
Human Mutation DOI 10.1002/humu
GLDC Mutations
Deletion of exon 1 was detected in six patients (four
Caucasian, one oriental, and one black). In this study we
identified 36 GLDC mutations, 28 of which were novel (Table 2).
None of the identified mutations were detected in 100 control
alleles. There were 21 missense mutations, three nonsense
mutations, seven deletion/insertion mutations, and five splicing
mutations Table 3. For all of the 21 missense mutations the
substituted amino acids are conserved in rats, and 13 of the 21
amino acids are conserved from humans to E. coli. Such a high
degree of evolutionary conservation presumably reflects the
functional importance of each of these amino acids. Seven
mutations were found in multiple individuals with no apparent
relationship. A missense mutation p.N150 T was identified in
oriental families, while p.T269 M, p.A389 V, p.R739 H, and
c.266511G4C were found in Caucasian families. Sequencing
analysis of the GLDC gene revealed the presence of several
polymorphisms, which were also found in control subjects
(Table 4). Five of the polymorphisms were found in at least
10% of the control alleles tested. Three of them were found
within exons (exon 1: c.249G4A; exon 4, c.501G4A; and exon
25, c.3070C4G(3
noncoding region)). Two of them were
located in introns (intron 9, c.1262136A4G; and intron 19,
c.2203-6insA). The c.2203-6insA polymorphism was located at
the intron 19/exon 20 boundary, and substitutes the ttaaaaaaaa-
tacag/GAAGAAA (A8 allele) to the ttaaaaaaaaatacag/GAA
GAAA (A9 allele). Other polymorphisms that were less
frequently observed were c.438G4A (p.T146T) in exon 3,
c.1261136A4G (intron 9), and c.1261152G4A (intron 9).
AMT Mutations
A total of 17 mutations (including 13 novel mutations) were
identified, including eight missense mutations, eight deletion/
insertion mutations, and one splicing mutation. The evolutionary
conservation of each mutated amino acid is shown in Table 3.
All of these amino acids are conserved among humans, rats, and
chickens, and six of eight amino acids are also conserved
in peas, which suggests that they are functionally important.
All of the deletion/insertion mutations generated a profoundly
truncated AMT polypeptide. Two polymorphic sites were
observed in two AMT exons: exon 2, c.327T4C; and exon 8,
Characterization of the GLDC Exon 1 Deletion
To define the boundaries of the exon 1 deletions, eight STSs were
designed: seven STSs at the region 150 kb upstream of GLDC,and
one STS in intron 2 of GLDC (Fig. 1; Supplementary Table S1).
Two additional published STSs (D9S281 and RH92434) and GLDC
exons 1–5 were also used for the deletion mapping. We amplified a
total of 15 STSs using genomic DNA as the templates obtained
from Patients P5 and P36, who were homozygous for the GLDC
exon 1 deletion. In Patient P5, STSs 2–8 and exons 1–3 were not
amplified, indicating that the 80–100 kb was homozygously deleted.
In Patient P36, STSs 6–8, and exons 1–2 failed to be amplified,
demonstrating a 35–45 kb homozygous deletion. Both deletions did
not extend to a known gene 5
adjacent to GLDC: jumonji domain
containing protein2C (JMJD2C). Alu repeats in this region were
identified using the UCSD Genome Browser (Fig. 1).
TABLE 3. AMT Mutations
Mutation Location
No . of
mutation Comments
Missense mutations Evolutionary Conservation
Human Rat Chicken Pea E.coli
c. 1 2 5A4G Exon 2 p.H42R 1 No His His His His His
c.136 G 4A Exon 2 p.G47R 1 Yes Gly Gly Gly Gly Ala
c.139 G 4T Exon 2 p.G47W 1 Yes Gly Gly Gly Gly Ala
c.2 1 2A4C Exon 2 p.H71P 1 Yes His His His Asn Ala
c.2 17C4T Exon 2 p. R 73C 1 Yes Arg Arg Arg Arg Arg
c.230C4T Exon 2 p.S77L 1 Yes Ser Ser Ser Ser Gly
c.826G 4C Exon 7 p.D276H 1 No Asp Asp Asp Asp Glu
c.88 7G4A Exon 8 p.R296H 1 No Arg Arg Lys Arg Lys
Deletions and insertion
c.54delC Exon 1 Deletion of third letter
of 18Phe codon
1 No FS at codon 20/TRM
at codon 95
c.60delG Exon 1 Deletion of third letter
of 20Pro codon
1 Yes FS at codon 21/TRM
at codon 95
c.61delC Exon1 Deletion of ¢rst letter
of 21Ala codon
1 Yes FS at codon 21/TRM
at codon 95
c.147delG Exon 2 Deletion of third letter
of 49Met codon
1 Yes FS at codon 50/TRM
at codon 95
c. 452_466del Exon 4 Deletion start at second
letter of 151Lys codon
1 Yes No FS/TRM
at codon 398
c.535delC Exon 5 Deletion of ¢rst letter
of 179Leu codon
1 Yes FS at codon 179/ TRM
at codon 180
c. 97 0_972delATG Exon 8 Deleion of 320Met
1 Yes No FS/TRM
at codon 482
c. 982_972GC4T Exon 8 GC in 328Ala codon
was substitued by T
1 Yes FS at codon 328/TRM
at codon 337
Aberrant splicing
c. 4 7 112T4C Intron 4 Disruption of splicing
donor site , gt4gc
DNA mutation numbering is based on cDNA sequence: 11 corresponds to theA of the ATG translation initiation codon. AMT:NM_00481 .
FS, frameshift; TRM, termination codon.
348 HUMAN MUTATION 27(4), 343^352, 2006
Human Mutation DOI 10.1002/humu
Hap lot yp e An alysi s of t he GLDC Mutations
The identification of five polymorphisms within GLDC allowed
us to determine the haplotypes of the mutant alleles with the exon
1 deletion. Haplotypes of the deletion allele were identified in four
families (P5, P14, P36, and P50), but could not be determined in
two families (P15 and P86; Table 4). Four mutant alleles shared
the same haplotype (G, A, A8, C), with the genotypes of four
polymorphic sites being written as follows: (c.501G4A, c.12321
36A4G, c.2203-6ins A, c3070C4G). Two additional haplotypes,
(G, A, A9, C) and (A, G, A9, C) were also found, suggesting
multiple origins of the exon 1 deletion.
Mutation Spectra of the GL DC and AMT Genes
The GLDC mutations detected in this study, as well as those
from previous reports, are illustrated in Fig. 2A. The most striking
feature of the mutation distribution is the clustering of the
missense mutations in exon 19. Seven of the 32 missense
mutations (22%) were identified in exon 19. The distribution of
the AMT mutations is shown in Fig. 2B. No obvious clustering of
the mutations was found in this gene.
We undertook an extensive screening for GLDC, AMT, and
GCSH mutations in a cohort of patients with NKH, and the
results reveal a comprehensive picture of the mutation spectrum of
NKH to date. In this study, 36 GLDC mutations and 17 AMT
mutations were detected, including 28 novel GLDC and 13 novel
AMT mutations. In total, 75% of neonatal and 83% of infantile
patients were positive for GLDC or AMT mutations. Mutations in
NKH patients were highly heterogeneous. Patients were found to
be compound heterozygotes in 38 of 47 mutation-positive cases
(81%). This is in sharp contrast with findings in countries with a
high incidence of NKH, such as Finland [von Wendt et al., 1979]
and Israel [Korman and Gutman, 2002]. In those countries there
are common mutations and many homozygotes for each common
mutation. A GLDC missense mutation (p.564I) has been
identified in Finland [Kure et al., 1992], and a GLDC missense
mutation (p.M1 T) and AMT missense mutation (p.H42R) have
been found in Israel [Boneh et al., 2005; Flusser et al., 2005].
In 16 of 36 families (44%) with GLDC mutations, we were able
to identify a mutation in only one of two mutant alleles despite
extensive mutational screening of GLDC, AMT, and GCSH.
Mutations may present in promoter regions or introns of the
GLDC gene, or large deletions or duplications may exist. Deletion
of GLDC exon 1 was detected in eight of the 36 families (22%)
with GLDC mutations. The deletion was found in Caucasian,
oriental, and black patients. Subsequent haplotype analysis
suggested multiple origins of the deletion. There is a high-density
repeat region of Alu motifs in the 5
upstream region and the
introns of GLDC, which are reported to trigger a homologous
recombination between two Alu motifs and cause a large deletion
(e.g., the C1-inhibitor gene, C1-INH [Stoppa-Lyonnet et al.,
1991]; lysyl hydroxylase gene, PLOD1 [Pousi et al., 1994]; and
a MutL mismatch repair gene, hMLH1 [Mauillon et al., 1996]).
We therefore speculate that the deletions of GLDC exon 1 are
caused by homologous recombination between Alu repeats.
Recently, Sellner et al. [2005] reported a deletion of GLDC
exons 2–15 that was flanked by Alu motifs. Alu-mediated
homologous deletion may occur not only in the 5
region of
GLDC, but also in other regions of GLDC.
No mutations were detected in seven patients with late-onset
NKH, despite intensive screening of the entire coding regions of
GLDC, AMT, and GCSH, which suggests that gene(s) other than
GCS genes may be responsible for late-onset NKH. Several reports
have described hyperglycinemic patients with no evidence of
neurological symptoms until 1 year of age [Bank and Morrow,
1972; Steiman et al., 1979; Lane et al., 1991, 1998; Wiltshire
et al., 2000; Ellaway et al., 2001]. The diagnosis of NKH was not
confirmed by mutational analysis in any of those patients. Patients
with atypical GE tend to have a relatively modest elevation of
serum and CSF glycine concentrations, which may be also caused
by other genetic disorders or therapeutic agents [Korman and
Gutman, 2002]. Vanishing white matter disease is a type of
leukoencephalopathy with characteristic MRI findings that is
commonly associated with mild elevation of CSF glycine
concentration [van der Knaap et al., 1999]. This disorder might
have been classified as a late-onset NKH if the responsible genes
(EIF2B5 and EIF2B2) had not been identified [Leegwater et al.,
2001]. For a more accurate diagnosis and better understanding of
late-onset NKH patients, genetic characterization is of paramount
importance. There are several other proteins that are functionally
related to the GCS reaction. Lipoylation of H-protein is catalyzed
by lipolyltransferase. The gene encoding the enzyme would be a
good candidate for NKH [Fujiwara et al., 1999]. Two types of
glycine transporters, GlyT1 and GlyT2, have been identified [Zafra
et al., 1997]. GlyT2 is located only in the brain stem and spinal
cord, while GlyT1 is distributed in the brain, kidney, and liver, and
the latter transporter may have a role in maintaining the glycine
level in both CSF and plasma. Thus, the GlyT1 gene is another
good candidate gene for NKH.
A mutation in at least one of two alleles was identified in 47
of 62 patients with neonatal or infantile type NKH, 36 of whom
(77%) had GLDC mutations. The dominance of the GLDC
mutations over AMT mutations is in agreement with previous
TABLE 4. Haplotype Analysis of the GLDC AlleleWith the Exon 1 Deletion
Patients Ethnicity Allele
Polymorphic site
Exon 4
c. 50 1G4A
Intron 9
c. 1 26 2136A4G
Intron 19
Exon 25
noncoding region)
c.30 7 0C4G
P 5 Oriental Allele 1 G A A8 C
Allele 2 G A A8 C
P14 Caucasian Allele1 G A A8 C
P 36 Caucasian Allele 1 G A A8 C
Allele 2 G A A9 C
P 50 Caucasian Allele 1 A G A9 C
DNA mutation numbering is based on cDNA sequence: 11 corresponds to theA of the ATG translation initiation codon. GDLC:NM_000 17 0 .
HUMAN MUTA TION 27( 4), 343^352, 2006 349
Human Mutation DOI 10.1002/humu
enzymatic studies [Tada and Hayasaka, 1987; Toone et al.,
2000]. No GCSH mutations were identified in any of the three
clinical subtypes of NKH, suggesting that GCSH mutations are
extremely rare in NKH. We recently identified a heterozygous
splicing mutation in GCSH in a Japanese family with a peculiar
type of NKH–transient type NKH [Kure et al., 2002]. Transient
NKH is indistinguishable from neonatal NKH in terms of the
onset of disease, but serum and CSF glycine are normalized
within 2–8 weeks [Luder et al., 1989; Schiffmann et al., 1989].
A girl with atypical NKH was reported to have low enzymatic
activity of the H-protein [Hiraga et al., 1981]. Unlike other
patients with NKH, this patient showed progressive deterioration
and extensive spongy degeneration of white matter with marked
gliosis on postmortem examination [Trauner et al., 1981]. It may
be that GCSH mutations will be found in patients with atypical
NKH rather than the more readily recognized clinical form
of NKH.
Seven of 32 GLDC missense mutations were clustered in exon
19 (Fig. 2). Glycine decarboxylase consists of 1,020 amino acids,
and exon 19 encodes 37 of 1,020 amino acids (3.6%), that is, 22%
of the GLDC missense mutations were clustered in only 3.6% of
the protein-coding region. Two octapeptides encoded in exon
19–His749 to Phe756 (HLNLHKTF) and Pro759 to Gly766
(PHGGGGPG)–are perfectly conserved in humans, chickens,
peas, and E. coli. Crystal structure analysis revealed that His749,
Asn751, His753, Lys754, and His760 (underlined) formed an
active-site pocket of the GLDC enzyme [Nakai et al., 2005], and
the cofactor of GLDC, pyridoxal phosphate, was covalently bound
to Lys754 [Fujiwara et al., 1987]. Amino acid changes in this
conserved region frequently abolish GLDC enzyme activity, which
may be a possible explanation for the high incidence of GLDC
missense mutations in this region.
Patients P107 and P108 were homozygous for the p.A389 V
mutation, while patient P104 was a compound heterozygote of the
p.A389 V mutation and a null mutation, c.1443insG (Table 1).
The p.A389 V mutation had approximately 8% residual activity in
the COS7 expression analysis [Dinopoulos et al., 2005]. Patients
P107 and P108 did not present with comas or seizures in the
neonatal period, but exhibited developmental delay and abnormal
behaviors that developed with age. In contrast, Patient P104 had a
typical presentation of the neonatal form of NKH, but subse-
quently the course of the disease was less severe. The presence of
the pA389 V mutant allele, which allows some residual enzyme
activity, may explain this milder phenotype of classic NKH. The
clinical course of P104 resembled those of P6 and P19, who were
expected to have 5–8% residual activity by the in vitro expression
analysis [Kure et al., 2004]. These results suggest that only a few-
percent difference in GCS residual activity dramatically alters the
clinical picture, such as age of onset and prognosis. A previous
enzymatic study of patients with neonatal and infantile NKH
supports this suggestion [Hayasaka et al., 1987]. GCS activities
ranged from 0 to 0.7 nmoles of CO
formed/mg protein/hr in the
neonatal type, while it ranged from 0.7 to 1.4 in the infantile type
(for which the control range was 3.9–5.2). However, an exception
was observed. Both P69 and P50 were compound heterozygotes of
GLDC exon 1 deletion and p.G771R. Patient P69 manifested
typical symptoms on the second day of life, whereas Patient P50
did not develop symptoms until 3 months of age. Thus far, residual
activity is a major determinant of the clinical course, but it is
probably modified by environmental factors and/or genotypes of
other than the GCS genes.
Elucidation of the responsible gene(s) for late-onset NKH, and
screening for deletions in all GLDC exons are needed to establish
a more complete picture of the genetic background and develop
the genotype–phenotype relationships in NKH.
We are grateful to the families who participated in this work.
We thank Dr. Avihu Boneh (Royal Children’s Hospital,
Melbourne), Dr. Helena Haekansson (Lalmar Central Hospital,
Sweden), Dr. Shiro Tono-Oka (Kagoshima City Hospital, Japan),
Dr. Toshimitsu Takayanagi (National Hospital Organization Saga
National Hospital, Japan), Dr. Mitsuru Kubota (Hokkaido
University Hospital, Japan), and Dr. Masaki Takayanagi (Chiba
Children’s Hospital, Japan) for referring the NKH patients.
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352 HUMAN MUT ATION 27(4), 343 ^ 352, 2006
Human Mutation DOI 10.1002/humu
    • "Children with prenatal mild ventriculomegaly had significantly larger cortical grey matter than controls and a large ratio of grey matter to white matter, both of which are features of autism [309]. Whole-genome sequencing applied to ASD families revealed links between autism and defective versions of the aminomethyl transferase gene (AMT) [310], another gene involved in glycine cleavage and linked to nonketotic hyperglycinaemia [311]. A case study concerned a boy with transient neonatal nonketotic hyperglycinaemia and autism [312]. "
    [Show abstract] [Hide abstract] ABSTRACT: Glyphosate, a synthetic amino acid and analogue of glycine, is the most widely used biocide on the planet. Its presence in food for human consumption and animal feed is ubiquitous. Epidemiological studies have revealed a strong correlation between the increasing incidence in the United States of a large number of chronic diseases and the increased use of glyphosate herbicide on corn, soy and wheat crops. Glyphosate, acting as a glycine analogue, may be mistakenly incorporated into peptides during protein synthesis. A deep search of the research literature has revealed a number of protein classes that depend on conserved glycine residues for proper function. Glycine, the smallest amino acid, has unique properties that support flexibility and the ability to anchor to the plasma membrane or the cytoskeleton. Glyphosate substitution for conserved glycines can easily explain a link with diabetes, obesity, asthma, chronic obstructive pulmonary disease (COPD), pulmonary edema, adrenal insufficiency, hypothyroidism, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, prion diseases, lupus, mitochondrial disease, non- Hodgkin’s lymphoma, neural tube defects, infertility, hypertension, glaucoma, osteoporosis, fatty liver disease and kidney failure. The correlation data together with the direct biological evidence make a compelling case for glyphosate action as a glycine analogue to account for much of glyphosate’s toxicity. Glufosinate, an analogue of glutamate, likely exhibits an analogous toxicity mechanism. There is an urgent need to find an effective and economical way to grow crops without the use of glyphosate and glufosinate as herbicides.
    Full-text · Article · Jun 2016
    • "Glycine cleavage H protein (GCSH) deficiency So far, no patient with clear mutations in the GCSH gene has been identified (Van Hove et al 1993; Kure et al 2006). A complex GCSH gene rearrangement has been reported (Koyata and Hiraga 1991), and there was biochemical evidence for GCSH deficiency in an early publication (Hiraga et al 1981). "
    [Show abstract] [Hide abstract] ABSTRACT: Lipoate is a covalently bound cofactor essential for five redox reactions in humans: in four 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). Two enzymes are from the energy metabolism, α-ketoglutarate dehydrogenase and pyruvate dehydrogenase; and three are from the amino acid metabolism, branched-chain ketoacid dehydrogenase, 2-oxoadipate dehydrogenase, and the GCS. All these enzymes consist of multiple subunits and share a similar architecture. Lipoate synthesis in mitochondria involves mitochondrial fatty acid synthesis up to octanoyl-acyl-carrier protein; and three lipoate-specific steps, including octanoic acid transfer to glycine cleavage H protein by lipoyl(octanoyl) transferase 2 (putative) (LIPT2), lipoate synthesis by lipoic acid synthetase (LIAS), and lipoate transfer by lipoyltransferase 1 (LIPT1), which is necessary to lipoylate the E2 subunits of the 2-oxoacid dehydrogenases. The reduced form dihydrolipoate is reactivated by dihydrolipoyl dehydrogenase (DLD). Mutations in LIAS have been identified that result in a variant form of nonketotic hyperglycinemia with early-onset convulsions combined with a defect in mitochondrial energy metabolism with encephalopathy and cardiomyopathy. LIPT1 deficiency spares the GCS, and resulted in a combined 2-oxoacid dehydrogenase deficiency and early death in one patient and in a less severely affected individual with a Leigh-like phenotype. As LIAS is an iron-sulphur-cluster-dependent enzyme, a number of recently identified defects in mitochondrial iron-sulphur cluster synthesis, including NFU1, BOLA3, IBA57, GLRX5 presented with deficiency of LIAS and a LIAS-like phenotype. As in DLD deficiency, a broader clinical spectrum can be anticipated for lipoate synthesis defects depending on which of the affected enzymes is most rate limiting.
    Full-text · Article · Apr 2014
    • "Biochemically, the glycine cleavage enzyme system is composed of the P-protein (GLDC gene, MIM# 238300), which removes CO 2 from glycine and transfers the amino-methyl group to lipoate on the H-protein (GCSH gene, MIM# 238330), and the T-protein (AMT gene, MIM# 238310) which releases ammonia and forms methylenetetrahydrofolate, after which the reduced lipoate is reoxidized by the L-protein (Fig. 1) (Kikuchi et al., 2008). In typical NKH, 72% of patients have a causative mutation in GLDC, and 24% in AMT, with no mutations identified in GCSH (Kure et al., 2006; Hamosh et al., 2009). In 4% of patients, no mutations were identified in a gene encoding a constituent of the glycine cleavage enzyme, despite proven deficient glycine cleavage enzyme activity and elevated glycine levels. "
    [Show abstract] [Hide abstract] ABSTRACT: Patients with nonketotic hyperglycinemia and deficient glycine cleavage enzyme activity, but without mutations in AMT, GLDC or GCSH, the genes encoding its constituent proteins, constitute a clinical group which we call 'variant nonketotic hyperglycinemia'. We hypothesize that in some patients the aetiology involves genetic mutations that result in a deficiency of the cofactor lipoate, and sequenced genes involved in lipoate synthesis and iron-sulphur cluster biogenesis. Of 11 individuals identified with variant nonketotic hyperglycinemia, we were able to determine the genetic aetiology in eight patients and delineate the clinical and biochemical phenotypes. Mutations were identified in the genes for lipoate synthase (LIAS), BolA type 3 (BOLA3), and a novel gene glutaredoxin 5 (GLRX5). Patients with GLRX5-associated variant nonketotic hyperglycinemia had normal development with childhood-onset spastic paraplegia, spinal lesion, and optic atrophy. Clinical features of BOLA3-associated variant nonketotic hyperglycinemia include severe neurodegeneration after a period of normal development. Additional features include leukodystrophy, cardiomyopathy and optic atrophy. Patients with lipoate synthase-deficient variant nonketotic hyperglycinemia varied in severity from mild static encephalopathy to Leigh disease and cortical involvement. All patients had high serum and borderline elevated cerebrospinal fluid glycine and cerebrospinal fluid:plasma glycine ratio, and deficient glycine cleavage enzyme activity. They had low pyruvate dehydrogenase enzyme activity but most did not have lactic acidosis. Patients were deficient in lipoylation of mitochondrial proteins. There were minimal and inconsistent changes in cellular iron handling, and respiratory chain activity was unaffected. Identified mutations were phylogenetically conserved, and transfection with native genes corrected the biochemical deficiency proving pathogenicity. Treatments of cells with lipoate and with mitochondrially-targeted lipoate were unsuccessful at correcting the deficiency. The recognition of variant nonketotic hyperglycinemia is important for physicians evaluating patients with abnormalities in glycine as this will affect the genetic causation and genetic counselling, and provide prognostic information on the expected phenotypic course.
    Full-text · Article · Dec 2013
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