Received: 5 December 2016 Revised: 22 February 2017 Accepted: 23 February 2017

DOI: 10.1002/humu.23208

RESEARCH ARTICLE

Nonketotic hyperglycinemia: Functional assessment

of missense variants in GLDC to understand phenotypes
of the disease

Irene Bravo-Alonso1 Rosa Navarrete1 Laura Arribas-Carreira1

Almudena Perona2 David Abia3 María Luz Couce4 Angels García-Cazorla5
Ana Morais6 Rosario Domingo7 María Antonia Ramos8 Michael A. Swanson9
Johan L.K. Van Hove9 Magdalena Ugarte1 Belén Pérez1 Celia Pérez-Cerdá1
Pilar Rodríguez-Pombo1

1 Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biología Molecular Severo Ochoa, CBM-CSIC, Departamento de Biología Molecular, Universidad

Autónoma Madrid, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), IDIPAZ, Madrid, Spain
2 SmartLigs S.L., Parque Científico de Madrid, Madrid, Spain

3 Servicio de Bioinformática, Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain

4 Unit of Diagnosis and Treatment of Congenital Metabolic Diseases, Service of Neonatology, Department of Pediatrics, Hospital Clínico Universitario de Santiago,

CIBERER, Health Research Institute of Santiago de Compostela (IDIS), Santiago de Compostela, Spain
5 Institut de Recerca Pediàtrica-Hospital Sant Joan de Déu (IRP-HSJD), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud

Carlos III, Barcelona, Spain

6 Unidad de Nutrición Infantil y Enfermedades Metabólicas, Hospital Universitario Infantil La Paz, Madrid, Spain

7 Servicio de Pediatría, Hospital Virgen de la Arrixaca, Murcia, Spain

8 Servicio de Genética, Hospital B del Complejo Hospitalario de Navarra, Pamplona, Navarra, Spain

9 Department of Pediatrics, Section of Clinical Genetics and Metabolism, University of Colorado, Aurora, Colorado

Correspondence
Pilar Rodríguez-Pombo, Centro de Biología
Molecular Severo Ochoa, C/ Nicolás Cabrera
Nº 1, Universidad Autónoma Madrid, Madrid
28049, Spain.
Email: mprodriguez@cbm.csic.es
Contract grants sponsors: Spanish Ministerio de
Economía y Competitividad and Fondo Europeo
de Desarrollo Regional (FEDER) (PI12/02078)
and PI16/00573 MINECO-FEDER; Fundación
Ramón Areces (CIVP16A1853); Fundación Isabel
Gemio.
Communicated by David S. Rosenblatt
Abstract
The rapid analysis of genomic data is providing effective mutational confirmation in patients with
clinical and biochemical hallmarks of a specific disease. This is the case for nonketotic hyperglycin-
emia (NKH), a Mendelian disorder causing seizures in neonates and early-infants, primarily due to
mutations in the GLDC gene. However, understanding the impact of missense variants identified in
this gene is a major challenge for the application of genomics into clinical practice. Herein, a com-
prehensive functional and structural analysis of 19 GLDC missense variants identified in a cohort
of 26 NKH patients was performed. Mutant cDNA constructs were expressed in COS7 cells fol-
lowed by enzymatic assays and Western blot analysis of the GCS P-protein to assess the residual
activity and mutant protein stability. Structural analysis, based on molecular modeling of the 3D
structure of GCS P-protein, was also performed. We identify hypomorphic variants that produce
attenuated phenotypes with improved prognosis of the disease. Structural analysis allows us to
interpret the effects of mutations on protein stability and catalytic activity, providing molecular
evidence for clinical outcome and disease severity. Moreover, we identify an important number of
mutants whose loss-of-functionality is associated with instability and, thus, are potential targets
for rescue using folding therapeutic approaches.

KEYWORDS
GCS P-protein, GLDC gene, nonketotic hyperglycinemia, structure function and phenotype
correlations in GLDC

Human Mutation. 2017;1–14. wileyonlinelibrary.com/journal/humu 

c 2017 Wiley Periodicals, Inc. 1
2 BRAVO-ALONSO ET AL .
1 INTRODUCTION
A plethora of different disease mechanisms may trigger seizures in
neonates or young infants. Recognition of the correct diagnosis is
important because prompt treatment can affect the neurological out-
come of patients (Rahman, Footitt, Varadkar, & Clayton, 2012; Van
Hove & Lohr, 2011). A monogenic cause of neonatal epilepsy is clas-
sic nonketotic hyperglycinemia (NKH) (MIM# 605899). NKH is an
autosomal-recessive disorder of glycine metabolism caused by a defi-
cient activity of the glycine cleavage system (GCS). It is biochemically
characterized by a massive accumulation of glycine in body fluids. A
higher elevation of glycine in cerebrospinal fluid (CSF) than in plasma
results in an elevated CSF/plasma glycine ratio.
Similar to other pathologies (Rodriguez-Pombo, Navarrete,
Merinero, Gomez-Puertas, & Ugarte, 2006), NKH presents a con-
tinuum of clinical and biochemical presentations. Recent data from
clinical surveys support a distinction between severe and attenuated
presentations of the disease based on the presence of a variable
degree of development (Hennermann, Berger, Grieben, Scharer, & Van
Hove, 2012).
Most patients present as neonates with hypotonia, poor feeding,
and lethargy progressing to coma, seizures, and apnea requiring ven-
tilation. Some patients die during the newborn period. The majority
of survivors exhibit severe mental retardation and intractable seizures
typical of severe NKH (Hennermann et al., 2012; Hoover-Fong et al.,
2004; Suzuki, Kure, Oota, Hino, & Fukuda, 2010). One-sixth of NKH
patients have an attenuated form, half of whom present in early-to-mid
infancy with seizures, hypotonia, and developmental delay and/or cog-
nitive impairments, behavioral problems, and impaired work or school
performance (Hennermann et al., 2012). Distinguishing different NKH
forms is important for prognosis and stratification of patients for treat-
ment. Treatment usually involves sodium benzoate to reduce plasma
glycine levels, and dextromethorphan to decrease the excessive stimu-
lating activity of glycine on N-methyl-D-aspartate receptors (Bjoraker
et al., 2016; Hamosh, Maher, Bellus, Rasmussen, & Johnston, 1998; Van
Hove et al., 2005). Biochemical parameters such as the CSF/plasma
glycine ratio can be helpful in this classification.
GCS is a mitochondrial enzymatic complex that catalyzes the
reversible oxidative cleavage of glycine in a multistep reaction (Kikuchi,
1973). In humans, GCS complex consists of four distinctive proteins.
The homodimeric, pyridoxal 5′ -phosphate (PLP)-dependent GCS P-
protein (GLDC; MIM# 238300) catalyzes the first step of glycine
cleavage, in which one carbon is released as CO2 . A tetrahydrofolate
(THF)-dependent T-protein (AMT; MIM# 238310) transfers the second
one-carbon unit to THF-generating 5,10-methylene THF. The NAD+ -
dependent L-protein and a small lipoylated H-protein (GCSH; MIM#
238330), which interacts with the P-, T-, and L-proteins via its lipoyl
arm, constitute the other two subunits.
The three-dimensional structures of individual P and T components
from different organism have been helpful to understand their cat-
alytic mechanism (Hasse et al., 2013; Lee, Kim, Ahn, Ha, & Suh, 2004;
Nakai et al., 2005; Okamura-Ikeda et al., 2010). Not much information
is available concerning the complex interacting structures, except for
the analysis of the crystal structure of the T-protein in complex with
the H-protein.
Most NKH patients have disease-causing mutations in GLDC
(around 80%) or AMT (20%) genes (Coughlin et al., 2017). The
reported GLDC and AMT mutation spectrum includes exonic copy-
number variants including different deletions involving multiple GLDC
exons, and an extensive number of missense mutations, mostly private
(Applegarth & Toone, 2001; Conter et al., 2006; Coughlin et al., 2017;
Kanno et al., 2007; Kure, 2011; Kure et al., 2004; Kure et al., 2006;
Pardal-Fernandez et al., 2009; Suzuki et al., 2010; Swanson et al.,
2015). Evidence for an important role of genotype as a predictive fac-
tor of outcome has been reported in NKH patients (Swanson et al.,
2015). However, for an important number of missense mutations,
the disease-causing nature has been established only using predictive
tools, which can produce conflicting results and lead to ambiguous
interpretation.
In this report, we expand the spectrum of GLDC or AMT genotypes
by analyzing a cohort of 26 NKH patients from Spain. We provide an in-
depth genotype–phenotype correlation by relating a comprehensive
functional and structural analysis of 19 missense GLDC mutations to
the clinical presentation and outcome of the patients.
2 PATIENTS AND METHODS
Samples from 26 patients referred from different clinical centers were
obtained in accordance with the Helsinki Declaration of 1964 as
revised in 2000. Molecular analysis was performed as part of the diag-
nostic workup with informed consent of the parents or legal represen-
tative. Clinical and biochemical data were retrospectively collected.
The protocol was approved by the institutional Ethics Committee of
the Universidad Autónoma de Madrid (CEI-28-701).
2.1 Mutation analysis
DNA and total mRNA were isolated from blood samples or from
EBV-transformed lymphoblasts using the MagnaPure system (Roche
Applied Science, Indianapolis, IN). Various methods were used for
mutation analysis: Sanger sequencing or massive parallel exome
sequencing were used to detect single-nucleotide variants and indels,
and multiplex ligation probe amplification (MLPA) analysis or whole-
gene sequencing of the entire GLDC gene (exons and introns) were used
to identify copy-number variants in the GLDC gene.
For Sanger sequencing of GLDC with RefSeq accession number
NM_000170.2; NP_000161.2, AMT with RefSeq accession number
NM_000481.3; NP_000472.2, and GCSH with RefSeq accession num-
ber NM_004483.4; NP_004474.2, the coding region and flanking
intron–exon boundaries were PCR amplified with primers based on
the Ensemble genome browser entries: ENSG00000178445 for GLDC,
ENSG00000145020 for AMT, and ENSG00000140905 for GCSH. To
confirm the breakpoints of selected GLDC deletions, we performed
long-range PCR amplification using the Extensor Hi-Fidelity PCR Mas-
ter Mix (Thermo Fisher Scientific Inc., Waltham, MA) and primers
BRAVO-ALONSO ET AL .
designed using the RepeatMasker platform (Institute for system biol-
ogy; http://www.repeatmasker.org/).
All PCR products were sequenced using BigDye Terminator v.3.1
Mix (Applied Biosystems, Foster City, CA) and analyzed by capillary
electrophoresis using an ABI Prism 3700 Genetic Analyzer (Applied
Biosystems). As a control for most identified point mutations, DNA
of the patient’s relatives and DNA of 300 European control subjects
(Human Random Control DNA Panel-1 HRC-1 [ECAA and Sigma–
Aldrich, St Louis, MO]) was analyzed by bidirectional sequencing.
For massive parallel sequencing, a customized Nextera Rapid Cap-
ture panel (Illumina, San Diego, CA) was designed to capture the
entire sequence of the GLDC gene and the exome sequence of the
AMT, GCSH, and DLD genes. Libraries prepared from 50 ng DNA
were sequenced using 250-bp paired-end reads using the Illumina
MiSeq sequencer. The Fastq files produced are examined using the
DNAnexus platform (https://platform.dnanexus.com) for mapping and
generation of variant calling files (VCFs). These VCFs were analyzed
using VariantStudio Data Analysis Software v2.1 (Illumina). For a cur-
rent estimate of population frequencies, we reviewed all identified
point mutations against the data from the Exome Aggregation Con-
sortium (ExAC) (Cambridge, MA) (URL: http://exac.broadinstitute.org)
and Exome Variant Server: http://evs.gs.washington.edu/EVS/). Minor
allele frequencies >1% were excluded.
For mRNA expression analysis, 1 𝜇g of total RNA was reverse
transcribed using oligodT as primer and SuperScript III First-Strand
enzyme (Invitrogen, Carlsbad, CA). Primers to amplify cDNA were
designed according to the cDNA GenBank sequences listed above.
The mutation nomenclature followed the format described in HGVS
nomenclature v15.11. (http://www.HGVS.org/varnomen/). The DNA
variant numbering system was based on the corresponding cDNA
sequence, taking nucleotide +1 as the A of the ATG translation initia-
tion codon in the reference sequence.
2.2 In silico analysis of variant’s pathogenicity and
frequency
Each variant was checked for their presence in disease databases such
as Human Gene Mutation Database (HGMD). For all missense vari-
ants identified, pathogenicity was evaluated using several computa-
tional predictive programs for the analysis of the impact of a missense
change on the protein structure, function, and evolutionary conserva-
tion including: PolyPhen-2 (v2.2humvar) (http://genetics.bwh.harvard.
edu/pph) (Adzhubei et al., 2010), SIFT (JCVI-SIFT v.1.03) (http://sift.
jcvi.org/) (Kumar, Henikoff, & Ng, 2009), PROVEAN (v1.1human pro-
tein) (http://provean.jcvi.org/index.php) (Choi, & Chan, 2015), and
MutationTaster (http://www.mutationtaster.org/) (Schwarz, Cooper,
Schuelke, & Seelow, 2014).
Potential 3′ and 5′ splice sites in the GLDC sequence (c.1927-4G>A,
c.2053-5C>G, and c.2665+5G>A) and in the AMT sequence (c.259-
1G>C, c.340-1G>A, and c.878-1G>A) were analyzed using the default
settings of the integrated software package Alamut
R
Visual Interac-
tive Bio software v2.7.1, which includes five different splice-site pre-
diction tools GeneSplicer (Pertea, Lin, & Salzberg, 2001), Human Splic-
ing Finder (HSF) (Desmet et al., 2009), MaxEntScan (Yeo & Burge,
3
2004), NNSplice (Reese, Eeckman, Kulp, & Haussler, 1997), and Splice-
SiteFinder like.
2.3 Functional characterization
COS7 cells, maintained in Eagle’s minimum essential medium supple-
mented with 10% fetal calf serum and 1% antibiotics, were used for
the functional characterization of mutant P-proteins. The full-length
open reading frame of human GLDC was cloned into the mammalian
pCMV plasmid -pCMV6-XL4- (OriGene Technologies, Rockville, MD),
and was used to generate recombinant plasmids for each mutation
using the QuickChange site-directed mutagenesis kit (Stratagene, La
Jolla, CA) with appropriate mutagenesis primers. The pCMV-GLDC
wild-type, pCMV-GFP, and mutant constructs were transiently trans-
fected into COS7 cells using lipofectamine transfection reagent. Cells
were harvested 48 hr after transfection, and stored at −80°C for GCS
P-protein exchange activity. Transfection efficiency was determined by
fluorescence-associated cell cytometry measuring the proportion of
cells expressing the green fluorescent protein from a pCMV-GFP alone
or cotransfected with pCMV-GLDC constructs. The GCS P-protein
enzymatic activity was determined in triplicate by the exchange reac-
tion between carbon dioxide and glycine using NaH14 CO3 as described
(Toone, Applegarth, Coulter-Mackie, & James, 2000). Cells transfected
with either wild type, GFP construct, or untransfected were used
as controls. Protein was measured according to the Lowry method
(Lowry, Rosebrough, Farr, & Randall, 1951).
2.4 Western blot analysis
Cell pellets from transfected cells were lysed with 1% Triton X-100,
10% glycerol, 150 mM NaCl, 10 mM Tris–HCl pH 7.5 (radioim-
munoprecipitation assay buffer). After centrifugation, the protein
content of the supernatants was determined by the Bradford method
using bovine serum albumin as a standard (Bio-Rad Laboratories,
Hercules, CA). Samples were separated on a precast 10% polyacry-
lamide gel (Bio-Rad) and blotted onto nitrocellulose membranes
using the iBlot Dry Blotting system (Invitrogen). For the identifi-
cation of GCS P-protein, we used a 1:1,000 polyclonal anti-GLDC
antibody (TA308885; OriGene) and a secondary 1:5,000 horseradish-
peroxidase conjugated goat anti-rabbit antibody (Santa Cruz
Biotechnology, Dallas, Texas).
2.5 Immunofluorescence and confocal imaging
microscopy
COS7 cells cultured on glass cover slips were transfected with pCMV-
GLDC wild-type, pCMV-GFP, and mutant constructs as described
above. At 48 hr after transfection, cells were incubated with the mito-

R
chondrial probe Mitotracker (Invitrogen) as described (Oyarzabal
et al., 2013). For double immunocytochemistry, cells were permeabi-
lized by incubating the fixed cover slips with 0.2% Triton X-100 in
phosphate-buffered saline (PBS) for 10 min, then blocking for 20 min
with blocking solution (0.5% FCS, 0.1% Triton X-100, PBS) before incu-
bating overnight with 200 𝜇l of 1:200 anti-GLDC in blocking solu-
tion. Visualization was done with a secondary anti-rabbit antibody with
4 BRAVO-ALONSO ET AL .
bound Alexa 488 (emission wavelength 519 nm) (Invitrogen) at a con-
centration of 1:500 in blocking solution for 30 min, and observed using
an LSM 710 confocal microscope coupled to Axiovertical microscopy
AxioImager.M2 (Zeiss, Jena, Germany).
2.6 Molecular dynamics
The homodimeric homology model of GCS-P protein (NP_000161.2)
was created using MODELLER9v9 (https://salilab.org/modeller/)
(Eswar et al., 2006) based on the published crystal structure of Syne-
chocystis sp. PCC 6803 (PDB 4LGL, 4LHL, 4LHD) (Hasse et al., 2013).
For the molecular dynamics analysis, residues were protonated at pH
7.4 using H++ server (Gordon et al., 2005). Standard atomic charges
were assigned according to the ff03 force field (Duan et al., 2003;
Lee & Duan, 2004) with “bondi” atomic radius (Zhao, Abraham, &
Zissimos, 2003). Dynamics was achieved, using the AMBER12 package
(Case et al., 2010) and extended during 30 ns. The root-mean-square
deviation calculated along the dynamics remained low, indicating that
the model was not destabilized.
3 RESULTS AND DISCUSSION
3.1 Biochemical profile and mutation screening of
the NKH cohort
Based on a combination of CSF glycine levels, age at onset, and clin-
ical outcome, we distinguished between attenuated NKH and severe
NKH with poor developmental outcome or neonatal fatality in combi-
nation with high levels of glycine in CSF and neonatal onset (Swanson
et al., 2015). In this study, 16 out of the 26 patients had severe NKH
with an initial onset of hypotonia, apnea, myoclonic jerks, and deceased
in the neonatal or early infancy period with a severe outcome. In this
group of patients, the CSF glycine levels were 97–295 𝜇M, and the
CSF/plasma glycine ratios ranged between 0.097 and 0.64. There were
seven patients (P19, P20, P21, P22, P23, P24, and P25) with clearly
attenuated NKH surviving with moderate psychomotor retardation. In
these cases, CSF glycine levels at diagnosis were between 26 and 127
𝜇M and the CSF–plasma glycine ratios ranged between 0.062 and 0.14
(see Table 1). Two surviving patients (P12 and P16) had either severe
or attenuated poor outcome, but could not be further distinguished
due to insufficient developmental data. Most patients excreted high
amounts of glycine in urine, and in four patients elevated short chain
acylglycines were detected in urine. Patient P26 remains unclassified
because of the lack of clinical and/or biochemical information.
Twenty-two families had mutations in the GLDC gene, whereas
the other four families carried mutations in the AMT gene (Table 1).
Sequencing of the GLDC or AMT genes identified 32 different single-
nucleotide changes (see Table 2A and B), which were rare or absent
in major databases such as the Exome Sequencing Project, dbSNP, or
ExAc consortium (Lek et al., 2016). Most have been included in a previ-
ously published survey of mutations in NKH (Coughlin et al., 2017), and
all mutations have been deposited in the LOVD database accessible
at www.lovd.nl. Most mutations were located in exonic sequence. One
nonsense mutation and two frameshift deletion mutations in GLDC
were considered as loss-of-function changes. The other 23 single-
nucleotide changes were predicted to result in missense variants, 20
in GLDC and three in AMT (Table 2A). The scoring of pathogenicity
by different prediction tools is shown in Table 2A. Most predictions
were concordant, but for p.Met1Ile, p.Thr146Lys, and p.Arg790Trp, the
conclusions were ambiguous. Six intronic changes listed in Table 2B
were evaluated for their effect on the splicing process using Alamut
prediction software. All were predicted to affect splice sites except
GLDC changes c.1927-4G>A and c.2053-5C>G, which were both cis-
carried with missense mutations. Sequencing of RT-PCR products
from lymphoblast or blood samples verified the aberrant skipping of
exons in the corresponding mRNAs: for GLDC, c.2665+5G>A resulted
in r.2570_2665del; for AMT, c.259-1G>C resulted into r.259_339del,
c.340-1G>A into r.340_471del, and c.878-1G>A into r.878_1delg.
In those patients where conventional Sanger sequencing detected
only a single pathogenic allele, MLPA analysis identified nine large
GLDC deletions in 12 alleles, several of them private (Table 1). The
length of the deletions ranged from a single exon to 15 exons (Supp.
Fig. S1A). Interestingly, in two different patients, we detected a mis-
sense variant (p.Pro267Ala (P8) or p.Gly704Val (P11) that paired on
the same allele with the deletion c.(1155+1_1156-1)_(1850+1_1851-
1)del or c.(?_-1)_(713+1_714-1)del (Table 1). To identify GLDC dele-
tions breakpoints and to improve the turnaround time for genetic diag-
nostic studies, we explored the use of whole-genomic sequencing of
the GLDC gene using a Nextera rapid capture customized panel. Three
GLDC-deficient patients (P3, P8, and P10, see Table 1) and a cohort (n
= 12) of healthy individuals were included in this analysis. This whole-
genome analysis detected the large deletions: c.(?_-1)_(470+1_471-
1)del (P3), c.(1155+1_1156-1)_(1850+1_1851-1)del (P8), and c.(?_-
1)_(1707+1_1708-1)del (P10), confirming that this single analysis is
useful as a first line test for detecting DNA deletions (Supp. Fig. S1B).
Long-range PCR and sequencing analysis through the breakpoints
for c.(1155+1_1156-1)_(1850+1_1851-1)del confirmed the change as
c.1156-2553_1850+4575del and identified Alu repeat elements at
both 5′ and 3′ ends (see Supp. Fig. S1C for details).
3.2 Overexpression of GLDC missense variants to
evaluate the functional effect
After first excluding an effect of the missense variants on mRNA splic-
ing by in silico analysis (data not shown), we investigated the func-
tional effects of 19 GLDC missense variants identified in our cohort by
expressing the wild-type and the respective mutant cDNA construct
in COS7 cells, and measuring the resulting GCS P-protein activity
(Fig. 1A). Relative to the wild type, GCS P-protein activities were
less than 1% in homogenates from cells expressing p.Thr146Lys,
p.Leu173Pro, p.His580Tyr, p.Pro581Arg, pAla624Asp, p.Gly763Asp,
p.Gly768Glu, or p.Gly994Arg, and retained measurable activities
between 1% and 10% of wild type with the p.Pro267Ala, p.Arg362Cys,
p.Arg373Trp, p.Leu548Pro, p.Asp866His, and p.Val905Gly variants.
Homogenates from cells transfected with the variants p.Met1Ile,
p.Lys376Glu, p.Arg461Trp, p.Arg790Trp, and p.Ile933Thr rendered
GCS P-protein glycine exchange activities ranging from 12% to 50%
TA B L E 1 Clinical, biochemical, and genetic analysis of NKH patients

Gly CSF (N.V.<10 Ratio CSF/PL

Patient Age of onset Gender 𝝁M) Gly PL (N.V.<300 𝝁M) (0.02±0.008) Evolution Gene Maternal allele Paternal allele
P1 S (M1) <1wk F 295 516 0.57 Died 2m GLDC c.1742C>G c.437C>A p.Thr146Lys
p.Pro581Arg
P2 S (M2) 5d M 246 817 0.30 Died 3wk GLDC c.2980G>A c.600delG
BRAVO-ALONSO ET AL .

p.Gly994Arg p.Thr201Leufs*30
P3 S (M3) 1d F 234 1,346 0.17 Died 7d GLDC c.1738C>T c.(?_-1)_(470+1_471-
p.His580Tyr 1)del
p.?
P4 S (M4) 4d F 229 396 0.58 Died 3m GLDC c.1666G>T p.Gly556* c.2714T>G p.Val905Gly
P5 S (M5) 1d M 227 1,387 0.16 Died 1m GLDC c.2288G>A; c.1381C>T p.Arg461Trp
1927-4G>A
p.Gly763Asp; ?
P6 S (M6) 4d F 188 899 0.21 Died 1m GLDC c.518T>C c.924delT
p.Leu173Pro p.Phe308Leufs*3
P7 S (M7) 1d F 182 987 0.18 Died 2m GLDC c.(?_-1)_(334+1_335- c.(?_-1)_(334+1_335-
1)del 1)del
p.? p.?
P8 S (M8) 2d F 181 283.5 0.64 Died at 3y. Severe GLDC c.2596G>C c.[799C>G;
PMR; microcephaly p.Asp866His (1155+1_1156-
hypotonia; seizures 1)_(1850+1_1851-
1)del] p.Pro267Ala;
?
P9 S (M10) 1d F 160 1,640 0.097 Alive 2y. Severe PMR GLDC c.924delT c.924delT
Seizures p.Phe308Leufs*3 p.Phe308Leufs*3
P10 S (M25) 2d M 161 208 0.77 Died at 6y. Severe GLDC c.(?_- c.2303G>A;
PMR; seizures 1)_(1707+1_1708- c.2053-5C>G
Seizures 1)del p.Gly768Glu
P11 S (M11) 6d M 159 1,044 0.15 Died 1m GLDC c.[2111G>T; c.[2111G>T; (?_-
(?_-1)_(713+1_714- 1)_(713+1_714-1)del]
1)del] p.Gly704Val; p.Gly704Val; ?
?
P12 S (M12) 3d F 158 712 0.22 Alive at 2.5y. PMR AMT c.886C>T c.340-1G>A
p.Arg296Cys p.Gly114_Gln157del
P13 S 2d F 148 1,042 0.142 Died 1m GLDC c.1643T>C c.(?_-1)_(470+1_471-
p.Leu548Pro 1)del
p.?
P14 S (M13) 15d M 141 600 0.23 Alive 7y. Severe PMR GLDC c.2666+5G>A c.(2919+1_2920-
Seizures. IQ 40 p.Tyr858_Gly889del 1)_(*3063_?)del
p.?
P15 S (M14) 7d M 141 979 0.14 Died 1m GLDC c.1742C>G c.2368C>T p.Arg790Trp
p.Pro581Arg
P16 S (M15) 2m M 127 871 0.145 Alive at 4y. AMT c.280C>T p.Arg94Trp c.280C>T p.Arg94Trp
Hypotonia;
seizures
5

(continues)
 6

TA B L E 1 (Continued)

Gly CSF (N.V.<10 Ratio CSF/PL

Patient Age of onset Gender 𝝁M) Gly PL (N.V.<300 𝝁M) (0.02±0.008) Evolution Gene Maternal allele Paternal allele
S(
P17 M16) 7d M 112 243 0.46 Died 1m GLDC c.(?_- c.(?_-1)_(1850+1_1851-
1)_(1850+1_1851- 1)del
1)del p.?
p.?
P18 S (M17) 5d F 97 168 0.4 Died 3m AMT c.878-1G>A c.259-1G>C
p.Lys294fs p.Thr87_Gln113del
P19 A (M18) 4d M 91 730 0.12 Alive at 17y GLDC c.3G>T p.Met1Ile c.3G>T p.Met1Ile
Moderate PMR
Autism. No seizures
P20 A (M19) 3m F 88 751 0.12 Alive at 12y GLDC c.799C>G c.1126A>G p.Lys376Glu
Moderate PMR p.Pro267Ala
Seizures.
Hypothyroidism
Premature
adrenarche
P21 A (M20) 11m F 62 780 0.079 Alive at 8y. Moderate GLDC c.1117C>T c.(¿_-1)_(334+1_335-
PMR; hypoacusia p.Arg373Trp 1)del
Hypoacusia No p.?
seizures; IQ 55
P22 A 15y F 56 900 0.063 Alive at 16y Mild GLDC c.1117C>T c.3G>T p.Met1Ile
cognitive p.Arg373Trp
impairment ADHD
No seizures IQ 65
P23 A (M23) 3y 6m M 57 1,154 0.049 Alive at 8y. Autism GLDC c.1084C>T c.1084C>T p.Arg362Cys
Language delay p.Arg362Cys
Hyperactivity. No
seizures; IQ 85
P24 A (M21) 2y F 26 414 0.063 Alive at 17y PMR; AMT c.664C>T c.664C>T p.Arg222Cys
ADHD. Language p.Arg222Cys
delay. No seizures
P25 A (M22) 4m F – 585 – Died 18y. Moderate GLDC c.2798T>C c.2798T>C p.Ile933Thr
PMR; left p.Ile933Thr
Hemiparesis
Movement
disorder. No
seizures
P26 NA – M – – – – GLDC c.1871C>A c.(635+1_636-
(M24) p.Ala624Asp 1)_(1850+1_1851-
1)del p.
?
(S), severe NKH; (A), attenuated NKH, (NA) phenotype not available; NV, normal values; d, day; wk, week; m, month; y, year; F, female; M, male; -, no data available; PMR, psychomotor retardation; ADHD, attention-
deficit hyperactivity disorder; IQ, intelligence quotient.
Notes: In parenthesis, patients’ number, as appears in Coughlin et al. (2017).
Mutations nomenclature was according to http://varnomen.hgvs.org/.
BRAVO-ALONSO ET AL .
TA B L E 2 Point mutations in GLDC and AMT genes

A. Exonic mutations
Predicted amino
acid change in
GLDC GCS P-protein SIFT score PolyPhen-2 score PROVEAN score MutationTaster
Exon (NM_000170.2) (NP_000161.2) (prediction) (prediction) (prediction) prediction Location/conservation References
BRAVO-ALONSO ET AL .

1 c.3G>T p.Met1Ile 0 (damaging) 0.016 (benign) −0.509 (neutral) Disease causing Coughlin et al. (2017)
3 c.437C>A p.Thr146Lys 0 (damaging) 0.332 (benign) −3.496 (deleterious) Disease causing 𝛼-helix 6. Conserved Coughlin et al. (2017)
residue
4 c.518T>C p.Leu173Pro 0 (damaging) 0.999 (probably −6.65 (deleterious) Disease causing 𝛼–helix 8. Conserved Coughlin et al. (2017)
damaging) residue
4 c.600delG p.Thr201Leufs*30 Disease causing Coughlin et al. (2017)
6 c.799C>G p.Pro267Ala 0.02 (damaging) 0.989 (probably −7.87 (deleterious) Disease causing Strictly conserved Coughlin et al. (2017)
damaging)
7 c.924delT p.Phe308Leufs*3 Disease causing Coughlin et al. (2017)
8 c.1084C>T p.Arg362Cys 0 (damaging) 1 (probably −7.90 (deleterious) Disease causing 𝛽-strand 10 Coughlin et al. (2017)
damaging) Conserved residue
8 c.1117C>T p.Arg373Trp 0 (damaging) 1 (probably −7.90 (deleterious) Disease causing 𝛼-helix 13. Strictly Toone et al. (2000)
damaging) conserved
8 c.1126A>G p.Lys376Glu 0 (damaging) 0.97 (probably −3.80 (deleterious) Disease causing 𝜂9 (310-helix.) Coughlin et al. (2017)
damaging) Strictly conserved
10 c.1381C>T p.Arg461Trp 0 (damaging) 1 (probably −7.89 (deleterious) Disease causing 𝛽-strand 14 Conter et al. (2006)
damaging) Conserved residue
13 c.1643T>C p.Leu548Pro 0 (damaging) 0.959 (probably −6.785 (deleterious) Disease causing Conserved residue In this study
damaging)
14 c.1666G>T p.Gly556* Disease causing Coughlin et al. (2017)
15 c.1738C>T p.His580Tyr 0 (damaging) 0.999 (probably −5.91 (deleterious) Disease causing Strictly conserved Coughlin et al. (2017)
damaging)
15 c.1742C>G p.Pro581Arg 0 (damaging) 1 (probably −8.87 (deleterious) Disease causing Strictly conserved Coughlin et al. (2017)
damaging)
16 c.1871C>A p.Ala624Asp 0 (damaging) 0.975 (probably −5.12 (deleterious) Disease causing 𝛼-helix 21 Coughlin et al. (2017)
damaging)
18 c.2111G>T p.Gly704Val 0 (damaging) 0.998 (probably −8.68 (deleterious) Disease causing Coughlin et al. (2017)
damaging)
19 c.2288G>A p.Gly763Asp 0 (damaging) 0.998 (probably −6.63 (deleterious) Disease causing Strictly conserved Coughlin et al. (2017)
damaging)
19 c.2303G>A p.Gly768Glu 0 (damaging) 0.997 (probably −7.57 (deleterious) Disease causing Strictly conserved Coughlin et al. (2017)
damaging)
20 c.2368C>T p.Arg790Trp 0 (damaging) 0.013 (benign) −1.23 (neutral) Polymorphism Not conserved Kure et al. (2004)
22 c.2596G>C p.Asp866His 0 (damaging) 0.994 (probably −6.49 (deleterious) Disease causing 𝛽-strand 23. Not Coughlin et al. (2017)
damaging) conserved
(continues)
7
 8

TA B L E 2 (Continued)

A. Exonic mutations
Predicted amino
acid change in
GLDC GCS P-protein SIFT score PolyPhen-2 score PROVEAN score MutationTaster
Exon (NM_000170.2) (NP_000161.2) (prediction) (prediction) (prediction) prediction Location/conservation References
23 c.2714T>G p.Val905Gly 0 (damaging) 0.312 (benign) −6.49 (deleterious) Disease causing 𝛽-strand 25 Kure et al. (2006)
Conserved residue
23 c.2798T>C p.Ile933Thr 0 (damaging) 0.948 (probably −4.245 (deleterious) Disease causing Coughlin et al. (2017)
damaging)
25 c.2980G>A p.Gly994Arg 0 (damaging) 1 (probably −7.14 (deleterious) Disease causing 𝛼-helix 34, C-terminal Coughlin et al. (2017)
damaging) peptideNot
conserved
Predicted amino
acid change in
AMT GCST protein SIFT score PolyPhen-2 score PROVEAN score MutationTaster Location/
Exon (NM_000481.3) (NP_000472.2) (prediction) (prediction) (prediction) prediction conservation References
3 c.280C>T p.Arg94Trp 0.01 (damaging) 0.991 (probably −5.48 (deleterious) Disease causing 𝛼-helix Coughlin et al. (2017)
damaging)
6 c.664C>T p.Arg222Cys 0 (damaging) 1 (probably −7.77 (deleterious) Disease causing Coil Coughlin et al. (2017)
damaging)
8 c.886C>T p.Arg296Cys 0 (damaging) 1 (probably −7.52 (deleterious) Disease causing 𝛼-helix Coughlin et al. (2017)
damaging)
B. Splicing mutations
SSFL MaxEntScan NNSplice GeneSplicer HSF Predicted amino acid
Intron GLDC (0–100) (0–12) (0–1) (0–15) (0–100) change References
IVS 17 c.1927-4G>C 89.91 (wt) 9.72 (wt) 0.99 (wt) 12.60 (wt) 85.6 (wt)
89.91 (mut) 10.52 (mut) 1 (mut) 13.57 (mut) 86.2 (mut)
IVS 18 c.2053-5C>G 95.63 (wt) 12.69 (wt) 1 (wt) 13.00 (wt) 95.6 (wt)
95.58 (mut) 11.76 (mut) 0.99 (mut) 11.39 (mut) 92.9 (mut)
IVS 22 c.2665+5G>A 91.18 (wt) 11.01 (wt) 0.83 (wt) 4.99 (wt) 94.3 (wt) p.Tyr858_Gly889del Coughlin et al. (2017)
79.03 (mut) 6.41 (mut) 0.75 (mut) 1.79 (mut) 82.1 (mut)
Intron AMT SSFL MaxEntScan NNSplice GeneSplicer HSF Predicted amino acid References
(0–100) (0–12) (0–1) (0–15) (0–100) change
IVS 2 c.259-1G>C 82.6 (wt) 9.10 (wt) 0.75 (wt) 12.60 (wt) 91.1 (wt) p.Tyr87_Gln113del Pardal-Fernandez
et al. (2009)
(continues)
BRAVO-ALONSO ET AL .
 BRAVO-ALONSO ET AL .

TA B L E 2 (Continued)

SSFL MaxEntScan NNSplice GeneSplicer HSF Predicted amino acid

Intron AMT (0–100) (0–12) (0–1) (0–15) (0–100) change References
- (mut) - (mut) - (mut) - (mut) - (mut)
IVS 3 c.340-1G>A 78.4 (wt) 7.30 (wt) 0.54 (wt) 4.13 (wt) 83.7 (wt) p.Gly114_Gln157del Coughlin et al. (2017)
- (mut) - (mut) - (mut) - (mut) - (mut)
IVS 7 c.878-1G>A 88.0 (wt) 9.42 (wt) 0.90 (wt) 8.21 (wt) 91.3 (wt) p.Lys294fs Toone et al. (2000)
- (mut) - (mut) - (mut) - (mut) - (mut)

HSF, Human Splicing Finder; SSFL, SpliceSiteFinder like.

Notes: DNA variant numbering system was based on cDNA sequence of GLDC (RefSeqGene LRG_643) and AMT (RefSeqGene LRG_537) genes. Nucleotide numbering uses +1 as the A of the ATG translation initiation
codon in the reference sequence, with the initiation codon as codon 1. The likely pathogenicity of missense changes were calculated by four in silico prediction methods. In SIFT (v1.03; http://sift.jcvi.org/), the cut-
off for damaging prediction of 0.05. In PolyPhen-2 (v2.2.2r398; http://genetics.bwh.harvard.edu/pph2/), using HumVar-trained model scores above 0.9 were predicted as “probably damaging,” and those below 0.5
were predicted as “benign.” In PROVEAN, the cut-off was 2.5. MutationTaster (http://www.mutationtaster.org/) employs a Bayes classifier, model simple_aae (single amino acid changes) or complex_aae (frameshift or
premature stop codon).
R 
The potential effect on splice sites of the intronic changes was evaluated using five splice-site prediction tools; all included in the integrated software package Alamut Visual. SSFL, NNSplice, and HSF predictors use
the weight matrix model; GeneSplicer is based on Markov model. MaxEntScan score is based on the maximum entropy distribution. The range of scores is between brackets. The DNA variant numbering system was
based on cDNA sequence of GLDC (RefSeqGene LRG_643) and AMT (RefSeqGene LRG_537). The nucleotide numbering uses +1 as the A of the ATG translation initiation codon in the reference sequence, with the
initiation codon as codon 1.
9
10 BRAVO-ALONSO ET AL .

F I G U R E 1 The functional effect of GLDC missense variants. A: Exchange GCS P-protein activity of GLDC missense variants. pCMV-GLDC wild-
type or pCMV-GLDC variant constructs were transfected into COS7 cells for the evaluation of the resulting GCS P-protein exchange activity. Bars
represent the relative activity of mutants measured as percentage of that obtained in COS7 cells expressing the wild-type construct, which was
set to 100%. Data are from at least two different transfections assays measured in triplicate. Wild-type bar represents the mean ± standard error
of n = 10 independent transfections experiments. Differences were considered to be significant at *** P < 0.001. B: Representative immunoblot of
lysates from COS7 cells, transfected with pCMV-GLDC wild type, pCMV-GLDC variants, or pCMV-GFP (Ø) and blotted with anti-GCS P-protein
(1:1,000). Tubulin was used as loading control. C: Immunofluorescence and laser scanning confocal imaging. Representative merged images (60×
objective lens) showing localization of Mitotracker (red) and GCS P-protein (green) in COS7 cells transfected with pCMV-GLDC wild type, or vari-
ants: pCMV-GLDC c.799C>G (p.P267A); pCMV-GLDC c.1381C>T (p.R461W). For untransfected cells (Ø) or cells transfected with pCMV (scram-
ble), no GCS P-protein was detected. Nuclei were counterstained with DAPI. Arrows show GCS P-protein location. WT, wild type
BRAVO-ALONSO ET AL . 11

and were identified as very mild, similar to previously published data

(Swanson et al., 2015). Due to the nature of this functional assay, far
from the natural biological environment of patient’s cells, it is possi-
ble that the effect of certain variants could be missed; this could be the
case for p.Met1Ile, which affects the initiation start codon.
Comparing functional test with the pathogenicity prediction gen-
erated by the bioinformatics tools, the four programs were fairly pre-
dictive, with 73% (SIFT and Poly-Phen2), 79% (Mutation Taster), and
84% (PROVEAN) of predictions consistent with the functional results.
Among the 16 variants deemed pathogenic by the four in silico analy-
ses, three retained residual activities higher than 20%. For 15 changes,
the functional analysis rendered data closely related to those of at least
two of the predictions.

3.3 Stability and subcellular location of missense

variants
To evaluate the stability of the expressed GLDC mutant, we performed
Western blot analysis of overexpressed mutant GCS P-proteins. Of
the 19 missense mutants analyzed, 11 showed a diminished amount
or total absence of normally sized GCS P-protein. Lower levels of
expressed mutant proteins at steady state could be the result of incor-
rect folding, increased protein aggregation, or degradation. For the
remaining mutations, the GCS P-protein detected was close to normal
in quantity and size (Fig. 1B). If mutations would affect the localiza-
tion to the mitochondria, then this could reduce the amount of func-
tional enzyme activity in the native form beyond what would be mea-
sured in the whole-cell lysate of the expression system. Immunoflu-
orescence microscopy of transfected COS7 cells using anti-GCS P-
protein antibody showed that wild-type GCS P-protein was located
F I G U R E 2 The molecular dynamic structure of a homodimeric
in mitochondria, whereas for selected mutations such as p.Pro267Ala homology model of GCS P-protein mutants. A: Mapping of amino
and p.Arg461Trp both with residual activity, we detected some signal acid residues analyzed. Color code depicts the effect of the changes
in mitochondria but also diffusely throughout the cytosol (Fig. 1C). analyzed over total structure of GCS P-protein. In brown, mutations
predicting large structural changes (T146K, L173K, H580Y, P581R,
and G994R). In blue, mutations affecting the structural conformation
3.4 GCS P-protein modeling and GLDC mutations pyridoxal 5′ -phosphate (PLP)-binding site (R373W, A624D, G763D,
and G768E). In pink, mutations causing intermediate or small struc-
We analyzed the harmful effect of most of the missense GLDC muta- tural changes (P267L, R362C, K376E, R461W, L548P, R790W, D866H,
tions identified in our series by in silico mutagenesis on the 3D I933T, and V905H). B-D: Enlarged images over specific mutations pre-
structure of the GCS P-protein model (Fig. 2A). An overview of the pared with PyMol. The hydrogen bond interactions appear as black-
homodimeric model of the GCS P-protein created from the known dashed lines. B: THR-146-LYS (large structure change): THR-146 panel
shows hydrogen bond interactions of GLU-531 at the final molec-
structure of GCS P-protein from Synechocystis sp. PCC 6803 (PDB
ular dynamic structure of homodimeric homology model of GCS P-
4LGL, 4LHL, 4LHD) (Hasse et al., 2013) and visualized with PyMol protein. Main residues in the protein are highlighted in wheat and pale
is shown in Supp. Figure S2. Most disease-causing variations iden- cyan sticks, and THR-146 or LYS-146 are shown in green sticks. C:
tified in our series affect conserved and buried residues disturb- ARG-461-TRP (intermediate structure change): ARG-461 panel shows
ing the protein structure (Table 2). In our analysis, there exists an hydrogen bond interactions of ARG-461, HIS-580, and PRO-267 at the
final molecular dynamic structure of homodimeric homology model of
apparent relationship between the type of intramolecular interac-
GCS P-protein. Main residues in the protein are highlighted in wheat
tions in which the mutated residue is involved and the severity of
sticks, and ARG-461 or TRP-461, HIS-580 and PRO-267 are shown in
the changes, classified in terms of the residual GCS P-protein activ- green sticks. D: GLY-768-GLU (PLP-binding site): GLY-768 panel shows
ity measured after overexpressing the mutant GLDC cDNA. The muta- hydrogen bond interactions of PLP at the final molecular dynamic
tions with undetectable residual activity and undetectable or severely structure of homodimeric homology model of GCS P-protein. Main
decreased protein levels were those predicted to cause large struc- residues in the protein are highlighted in wheat sticks; GLY-768 or
GLU-768 are shown in green sticks
tural changes either affecting the dimer GCS P-protein packaging
(p.Thr146Lys), or modifying important junctions between structural
domains (p.Leu173Pro, p.His580Pro, p.Pro581Arg, and p.Gly994Arg).
12
Threonine 146 is located in the 𝛼-helix 6 at the junction between
the two GCS P-protein monomers (Supp. Fig. S3). When mutated to
a lysine, it changes from a small bulky neutral residue to a large basic
hydrophilic residue that may not fit into the assigned pocket. This
forces a major rearrangement of neighboring residues changing the
hydrogen-bond map (Fig. 2B). The predicted final effect will be insta-
bility of the monomer, and a failure in dimer production. The muta-
tions p.Leu173Pro and p.Gly994Arg both affect interactions between
𝛼-helix and 𝛽-sheet structural domains. Other changes also affecting
important junctions between structural domains were p.Pro581Arg,
p.His580Pro, p.Arg461Trp, and p.Pro267Ala. The residues proline 581,
histidine 580, and arginine 461 are all located in an internal loop.
The corresponding changes have a clear impact on the structural sta-
bility of the monomer by affecting the interaction of several struc-
tural loops (Fig. 2C). The residual GCS P-protein activities for these
mutations were highly variable and ranged between undetectable
and 20%. Finally, mutations p.Gly763Asp, p.Gly768Glu, p.Arg373Trp,
p.Ala624Asp induced rearrangements of the protein structure in the
PLP-binding site. Thus, p.Gly768Glu disrupts energy interactions with
residues Ser617 and Gly618 whose back-bone’s nitrogen atoms are
engaged by hydrogen bonding with the oxygen atoms of the phos-
phate group of PLP (Hasse et al., 2013) (Fig. 2D). In the same way,
p.Ala624Asp and p.Gly763Asp decreased the catalytic activity of the
protein by destabilization of the tight PLP fit into the active site. Our
data suggest that many mutations identified in our series could pro-
voke misfolding because of conformational destabilization of the GCS
P-protein and result in decreased enzymatic function.
3.5 Structural and functional analysis of missense
variants mostly correlates with diseases phenotypes
The identification of homozygous carriers of missense mutations or
of compound heterozygous combinations of a missense mutation with
either a frameshift or a nonsense mutation allowed us to establish a
correlation between the phenotypes of the patients in whom these
mutations were found and the results of the expression studies and
structural changes caused by specific missense mutations. Patients
P25 and P24 both had a mild attenuated phenotype and carried the
homozygous missense variants p.Ile933Thr and p.Arg362Cys in GLDC.
Residue Ile933 is located at the end of the 𝛼-30 helix, and establishes
Van Der Waals interactions with the loop at the end of the helix and
with the neighboring helix. The change to Thr might preserve part of
this interaction, and result in only a mild reduction in the decarboxy-
lation activity (Fig. 1A). Similarly, the amine lateral group of Arg362
located in a 𝛽-sheet interacts with the Gly994, located in the 𝛼-helix
34 at a small terminal peptide, and binds a turn of the 𝛼-helix to this
𝛽-sheet to keep the structural stability (Supp. Fig. S4). The change to
Cys in p.Arg362Cys mutant might preserve part of this interaction and
thus retained measurable decarboxylation activity. In contrast, high-
lighting the importance of these interactions for the stability of the P-
protein monomer structure, when Gly 994 changes to Arg, the charge
repulsion between the lateral chains causes a major disruption of these
interactions. That results in a nondetectable GCS P-protein activity,
nondetectable P-protein production, and thus the predictably severe
BRAVO-ALONSO ET AL .
phenotype observed in patient P2 with genotype of p.Gly994Arg in
combination with the frameshift mutation p.Thr201Leufs*30.
As previously described (Coughlin et al., 2017), mutations affect-
ing residues located near the active-site pocket, such as p.Arg373Trp,
p.Ala624Asp, or p.Gly768Glu, result in stable protein with variable
residual activities. At the entrance site of glycine into the catalytic tun-
nel, the exchange of a polar-positive amino acid such as Arg373 by the
large aliphatic residue Trp modified the predicted interaction pattern
between Arg373 and Asp995, although it retained part of these inter-
actions through Van der Waals forces. This data correlate with the 4%
of residual activity detected and the attenuated phenotype of patient
P21 (see Table 1).
4 CONCLUSIONS
Genetic testing has become a formidable tool in the diagnosis of
NKH. A growing challenge is the correct interpretation of the clini-
cal significance of identified variants. It is important to identify cor-
rectly: first, which genetic variants are pathogenic and provide diag-
nostic information; second, which variants can be actionable tar-
gets for novel therapeutic approaches based on genotype. Functional
and structural analysis of missense variants can address this ques-
tion. Herein, we extend the number of natural GLDC missense muta-
tions with a functional analysis by the overexpression of 19 missense
changes identified in NKH patients in a mammalian expression sys-
tem. We found that in silico predictions using four platform tools were
valuable in predicting pathogenic nature of mutations for our mutant
series, correlating in over 75% when used in combination, but nev-
ertheless failed to cover the continuum of severity observed in this
disease.
Our results strongly agree with a previous study that the total
loss of GCS P-protein activity causes a classic severe NKH pheno-
type, whereas milder phenotypes correspond to variants maintaining
reduced but present activities, with the mildest phenotype observed
in patients with both alleles with mutations that confer residual activ-
ity such as P23 in this study (Swanson et al., 2015). Data from West-
ern blot and protein structure analysis suggest that for an important
number of mutants in our series, the loss-of-function character could
correlate with a decreased stability, thus identifying in many patients
classic NKH as a conformational disease. Mislocalization outside the
native organelle the mitochondrion, such as demonstrated here for
p.Pro267Ala and p.Arg461Trp, will further decrease the amount of P-
protein available for enzyme activity.
Therefore, most of these changes could be targeted by pharmaco-
logical chaperones that bind to the mutant enzyme stabilizing the pro-
tein in its native conformation, or for proteostasis regulators, able to
increase the folding capacity of the cell (Muntau, Leandro, Staudigl,
Mayer, & Gersting, 2014; Powers, Morimoto, Dillin, Kelly, & Balch,
2009), and also thereby accelerating the mature processing and trans-
port to the final cellular destination of the protein. The searching for
possible chaperones for GCS P-protein by using a high-throughput
screening of low-molecular-weight compounds from a commercial
library could be a starting point to this approach. Encouraging results
BRAVO-ALONSO ET AL .
have been recently obtained for other metabolic disorders (Jorge-
Finnigan et al., 2013; Yuste-Checa et al., 2017). The high overall rate
of missense changes that could cause instability in the GCS P-protein
(Coughlin et al., 2017; Swanson et al., 2015) support the exploration of
these therapeutic options.
This compendium of new 19 GLDC missense mutations analyzed in
the context of a genotype–phenotype correlation will be helpful for
clinicians when evaluating the benefit of specific interventions dur-
ing the neonatal period. Patients who have at least one mutation that
allows for residual enzyme activity, many of them described here, have
the potential to develop attenuated NKH, and data from a sibling
study indicate that early treatment improves outcome (Bjoraker et al.,
2016; Swanson et al., 2015). This extended study of expressed muta-
tions helps clinicians identify more patients with residual activity that
best benefit from early treatment. Further, these data can also guide a
drug design based on structural data and help to prioritize GLDC mis-
sense mutations amenable to future preclinical investigations of resid-
ual activity enhancing interventions in NKH patients.
On the other hand, given that it is unrealistic to propose functional
assays of all nucleotide variants identified during the genetic diagno-
sis labor, this new set of data that associate amino acid substitutions
with phenotype characteristics could help to improve the predictive
capacity of bioinformatics tools to prioritize severity of novel sequence
variants.
ACKNOWLEDGMENTS
We are grateful to Drs. Elorza, Martínez-Pardo, Montejo, Omaña,
Arruza, Escribano, García Peñas, Peña, Saez de Pipaón, Mora, Alvarez
Guisasola, Arroyo, Arruza, Garcia, Murga, and Sebastián for providing
clinical data. We acknowledge the support of A.I. Vega, C. Medrano, and
F. Leal for advice with calling and filtering of exome data. We are very
grateful to the patients and their parents for supporting this study.
DISCLOSURE STATEMENT
The authors have no conflict of interest to declare.
REFERENCES
Adzhubei, I. A., Schmidt, S., Peshkin, L., Ramensky, V. E., Gerasimova, A.,
Bork, P., … Sunyaev, S. R. (2010). A method and server for predicting
damaging missense mutations. Nature Methods, 7, 248–249.
Applegarth, D. A., & Toone, J. R. (2001). Nonketotic hyperglycinemia
(glycine encephalopathy): Laboratory diagnosis. Molecular Genetics and
Metabolism, 74, 139–146.
Bjoraker, K. J., Swanson, M. A., Coughlin, C. R., 2nd, Christodoulou, J., Tan,
E. S., Fergeson, M., … Van Hove, J. L. (2016). Neurodevelopmental out-
come and treatment efficacy of benzoate and dextromethorphan in sib-
lings with attenuated nonketotic hyperglycinemia. Journal of Pediatrics,
170, 234–239.
Case, D. A., Darden, T. A., Cheatham, T. E. III, Simmerling, C. L., Wang, J., …
Kollman, P. A. (2010). AMBER 11. University of California, San Francisco.
Conter, C., Rolland, M. O., Cheillan, D., Bonnet, V., Maire, I., & Froissart,
R. (2006). Genetic heterogeneity of the GLDC gene in 28 unrelated
patients with glycine encephalopathy. Journal of Inherited Metabolic
Disease, 29, 135–142.
13
Coughlin, C. R., 2nd, Swanson, M. A., Kronquist, K., Acquaviva, C., Hutchin,
T., Rodriguez-Pombo, P., … Van Hove, J. L. (2017). The genetic basis of
classic nonketotic hyperglycinemia due to mutations in GLDC and AMT.
Genetics in Medicine, 19, 104–111.
Choi, Y., & Chan, A. P. (2015). PROVEAN web server: A tool to predict the
functional effect of amino acid substitutions and indels. Bioinformatics,
31, 2745–2747.
Desmet, F. O., Hamroun, D., Lalande, M., Collod-Beroud, G., Claustres, M., &
Beroud, C. (2009). Human Splicing Finder: An online bioinformatics tool
to predict splicing signals. Nucleic Acids Research, 37, e67.
Duan, Y., Wu, C., Chowdhury, S., Lee, M. C., Xiong, G., … Kollman, P. (2003). A
point-charge force field for molecular mechanics simulations of proteins
based on condensed-phase quantum mechanical calculations. Journal of
Computational Chemistry, 24, 1999–2012.
Eswar, N., Webb, B., Marti-Renom, M. A., Madhusudhan, M. S., Eramian, D.,
Shen, M. Y., … Sali, A. (2006). Comparative protein structure modeling
using MODELLER. Current Protocols in Bioinformatics, Chapter 5, Unit 5.6.
Gordon, J. C., Myers, J. B., Folta, T., Shoja, V., Heath, L. S., & Onufriev, A.
(2005). H++: a server for estamiting pKas and adding missing hydrogens
to macromolecules. Nucleic Acids Research, 33, W368–W371.
Hamosh, A., Maher, J. F., Bellus, G. A., Rasmussen, S. A., & Johnston, M. V.
(1998). Long-term use of high-dose benzoate and dextromethorphan
for the treatment of nonketotic hyperglycinemia. Journal of Pediatrics,
132, 709–713.
Hasse, D., Andersson, E., Carlsson, G., Masloboy, A., Hagemann, M., Bauwe,
H., & Andersson, I. (2013). Structure of the homodimeric glycine decar-
boxylase P-protein from Synechocystis sp. PCC 6803 suggests a mech-
anism for redox regulation. Journal of Biological Chemistry, 288, 35333–
35345.
Hennermann, J. B., Berger, J. M., Grieben, U., Scharer, G., & Van Hove,
J. L. (2012). Prediction of long-term outcome in glycine encephalopa-
thy: A clinical survey. Journal of Inherited Metabolic Disease, 35, 253–
261.
Hoover-Fong, J. E., Shah, S., Van Hove, J. L., Applegarth, D., Toone, J., &
Hamosh, A. (2004). Natural history of nonketotic hyperglycinemia in 65
patients. Neurology, 63, 1847–1853.
Jorge-Finnigan, A., Brasil, S., Underhaug, J., Ruiz-Sala, P., Merinero, B.,
Banerjee, R., … Perez, B. (2013). Pharmacological chaperones as
a potential therapeutic option in methylmalonic aciduria cblB type.
Human Molecular Genetics, 22, 3680–3689.
Kanno, J., Hutchin, T., Kamada, F., Narisawa, A., Aoki, Y., Matsubara,
Y., & Kure, S. (2007). Genomic deletion within GLDC is a major
cause of non-ketotic hyperglycinaemia. Journal of Medical Genetics, 44,
e69.
Kikuchi, G. (1973). The glycine cleavage system: Composition, reaction
mechanism, and physiological significance. Molecular and Cellular Bio-
chemistry, 1, 169–187.
Kumar, P., Henikoff, S., & Ng, P. C. (2009). Predicting the effects of coding
non-synonymous variants on protein function using the SIFT algorithm.
Nature Protocols, 4, 1073–1081.
Kure, S. (2011). Two novel laboratory tests facilitating diagnosis of glycine
encephalopathy (nonketotic hyperglycinemia). Brain & Development, 33,
753–757.
Kure, S., Ichinohe, A., Kojima, K., Sato, K., Kizaki, Z., Inoue, F., …
Matsubara, Y. (2004). Mild variant of nonketotic hyperglycinemia with
typical neonatal presentations: Mutational and in vitro expression anal-
yses in two patients. Journal of Pediatrics, 144, 827–829.
Kure, S., Kato, K., Dinopoulos, A., Gail, C., DeGrauw, T. J., Christodoulou,
J., … Matsubara, Y. (2006). Comprehensive mutation analysis of GLDC,
AMT, and GCSH in nonketotic hyperglycinemia. Human Mutation, 27,
343–352.
14
Lee, M. C. & Duan, Y. (2004). Distinguish protein decoys by using a scor-
ing function based on a new AMBER force field, short molecular dyna-
mics simulations, and the generalized born solvent model. Proteins, 55,
620–34.
Lee, H. H., Kim, D. J., Ahn, H. J., Ha, J. Y., & Suh, S. W. (2004). Crystal structure
of T-protein of the glycine cleavage system. Cofactor binding, insights
into H-protein recognition, and molecular basis for understanding non-
ketotic hyperglycinemia. Journal of Biological Chemistry, 279, 50514–
50523.
Lek, M., Karczewski, K. J., Minikel, E. V., Samocha, K. E., Banks, E.,
& Fennell, T., … Exome Aggregation Consortium. (2016). Analysis
of protein-coding genetic variation in 60,706 humans. Nature, 536,
285–291.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein
measurement with the Folin phenol reagent. Journal of Biological Chem-
istry, 193, 265–275.
Muntau, A. C., Leandro, J., Staudigl, M., Mayer, F., & Gersting, S. W. (2014).
Innovative strategies to treat protein misfolding in inborn errors of
metabolism: Pharmacological chaperones and proteostasis regulators.
Journal of Inherited Metabolic Disease, 37, 505–523.
Nakai, T., Nakagawa, N., Maoka, N., Masui, R., Kuramitsu, S., & Kamiya, N.
(2005). Structure of P-protein of the glycine cleavage system: Implica-
tions for nonketotic hyperglycinemia. EMBO Journal, 24, 1523–1536.
Okamura-Ikeda, K., Hosaka, H., Maita, N., Fujiwara, K., Yoshizawa, A. C.,
Nakagawa, A., & Taniguchi, H. (2010). Crystal structure of aminomethyl-
transferase in complex with dihydrolipoyl-H-protein of the glycine
cleavage system: Implications for recognition of lipoyl protein sub-
strate, disease-related mutations, and reaction mechanism. Journal of
Biological Chemistry, 285, 18684–18692.
Oyarzabal, A., Martinez-Pardo, M., Merinero, B., Navarrete, R., Desviat,
L. R., Ugarte, M., & Rodriguez-Pombo, P. (2013). A novel regulatory
defect in the branched-chain alpha-keto acid dehydrogenase com-
plex due to a mutation in the PPM1K gene causes a mild variant
phenotype of maple syrup urine disease. Human Mutation, 34, 355–
362.
Pardal-Fernandez, J. M., Carrascosa-Romero, M. C., de Cabo-de la Vega,
C., Iniesta-Lopez, I., Gil-Pons, E., & Martinez-Gutierrez, A. (2009). Atyp-
ical glycine encephalopathy in an extremely low birth weight infant:
Description of a new mutation and clinical and electroencephalographic
analysis. Epileptic Disorders, 11, 48–53.
Powers, E. T., Morimoto, R. I., Dillin, A., Kelly, J. W., & Balch, W. E. (2009). Bio-
logical and chemical approaches to diseases of proteostasis deficiency.
Annual Review of Biochemistry, 78, 959–991.
Rahman, S., Footitt, E. J., Varadkar, S., & Clayton, P. T. (2012). Inborn
errors of metabolism causing epilepsy. Developmental Medicine and Child
Neurology, 1, 23–36.
Pertea, M., Lin, X., Salzberg, S. L. (2001). GeneSplicer: a new computational
method for splice site prediction. Nucleic Acid Research, 29, 1185–1190.
BRAVO-ALONSO ET AL .
Reese, M. G., Eeckman, F. H., Kulp, D., & Haussler, D. (1997). Improved splice
site detection in Genie. Journal of Computational Biology, 4, 311–323.
Rodriguez-Pombo, P., Navarrete, R., Merinero, B., Gomez-Puertas, P., &
Ugarte, M. (2006). Mutational spectrum of maple syrup urine disease
in Spain. Human Mutation, 27, 715.
Schwarz, J. M., Cooper, D. N., Schuelke, M., & Seelow, D. (2014). Mutation-
Taster2: Mutation prediction for the deep-sequencing age. Nature Meth-
ods, 11, 361–362.
Suzuki, Y., Kure, S., Oota, M., Hino, H., & Fukuda, M. (2010). Nonketotic
hyperglycinemia: Proposal of a diagnostic and treatment strategy. Pedi-
atric Neurology, 43, 221–224.
Swanson, M. A., Coughlin, C. R., Jr., Scharer, G. H., Szerlong, H. J., Bjoraker,
K. J., Spector, E. B., … Van Hove, J. L. (2015). Biochemical and molecular
predictors for prognosis in nonketotic hyperglycinemia. Annals of Neu-
rology, 78, 606–618.
Toone, J. R., Applegarth, D. A., Coulter-Mackie, M. B., & James, E. R. (2000).
Biochemical and molecular investigations of patients with nonketotic
hyperglycinemia. Molecular Genetics and Metabolism, 70, 116–121.
Van Hove, J. L., & Lohr, N. J. (2011). Metabolic and monogenic causes
of seizures in neonates and young infants. Molecular Genetics and
Metabolism, 104, 214–230.
Van Hove, J. L., Vande Kerckhove, K., Hennermann, J. B., Mahieu, V.,
Declercq, P., Mertens, S., … Jaeken, J. (2005). Benzoate treatment and
the glycine index in nonketotic hyperglycinaemia. Journal of Inherited
Metabolic Disease, 28, 651–663.
Yeo, G., & Burge, C. B. (2004). Maximum entropy modeling of short
sequence motifs with applications to RNA splicing signals. Journal of
Computational Biology, 11, 377–394.
Yuste-Checa, P., Brasil, S., Gamez, A., Underhaug, J., Desviat, L. R., Ugarte,
M., … Perez, B. (2017). Pharmacological chaperoning: A potential treat-
ment for PMM2-CDG. Human Mutation, 38, 160–168.
Zhao, Y. H., Abraham, M. H., & Zissimos, A. M. (2003). Fast calculations of
van der Waals volume as a sum of atomic and bond contributions and
its application to drug compounds. Journal of Organic Chemistry, 68,
7368–7373.
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How to cite this article: Bravo-Alonso I, Navarrete R,
Arribas-Carreira L, et al. Nonketotic hyperglycinemia: Func-
tional assessment of missense variants in GLDC to understand
phenotypes of the disease. Human Mutation. 2017;00:1–14.
https://doi.org/10.1002/humu.23208