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Case Study
Congenital disorder of
glycosylphosphatidylinositol (GPI)-anchor
biosynthesisdThe phenotype of two patients with
novel mutations in the PIGN and PGAP2 genes
Aleksandra Jezela-Stanek
a,*,i
,El
_
zbieta Ciara
a,i
,
Dorota Piekutowska-Abramczuk
a,i
, Joanna Trubicka
a
,
El_
zbieta Jurkiewicz
b
, Dariusz Rokicki
c
, Hanna Mierzewska
d
,
Justyna Spychalska
e
, Małgorzata Uhrynowska
e
,
Marta Szwarc-Bronikowska
c
, Piotr Buda
c
, Abdul Rahim Said
f
,
Ewa Jamroz
g
, Małgorzata Rydzanicz
h
, Rafał Płoski
h
,
Małgorzata Krajewska-Walasek
a
, Ewa Pronicka
a,c
a
Department of Medical Genetics, The Children's Memorial Health Institute, Warsaw, Poland
b
Department of Diagnostic Imaging, The Children's Memorial Health Institute, Warsaw, Poland
c
Department of Pediatrics, Nutrition and Metabolic Diseases, The Children's Memorial Health Institute, Warsaw,
Poland
d
Department of Child and Adolescent Neurology, Institute of Mother and Child, Warsaw, Poland
e
Department of Immunology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland
f
Children's Home Hospice, Opole, Poland
g
Department of Pediatrics and Developmental Age Neurology, Medical University of Silesia, Katowice, Poland
h
Department of Medical Genetics, Warsaw Medical University, Warsaw, Poland
article info
Article history:
Received 8 October 2015
Received in revised form
10 January 2016
Accepted 11 January 2016
Keywords:
GPI-anchor deficiency
PGAP2 gene
abstract
Background: Glycosylphosphatidylinositol (GPI)-anchor deficiencies are a new subclass of
congenital disorders of glycosylation. About 26 genes are involved in the GPI-anchor
biosynthesis and remodeling pathway, of which mutations in thirteen have been reported
to date as causative of a diverse spectrum of intellectual disabilities. Since the clinical
phenotype of these disorders varies and the number of described individuals is limited, we
present new patients with inherited GPI-anchor deficiency (IGD) caused by mutations in
the PGAP2 and PIGN genes.
Patients and methods: The first girl presented with profound psychomotor retardation, low
birth parameters, and chest deformities already existing in neonatal period. The disease
*Corresponding author. Department of Medical Genetics, The Children's Memorial Health Institute, Aleja Dzieci Polskich 20, 04-730
Warsaw, Poland. Tel.: þ48 228157452; fax: þ48 22 8157457.
E-mail address: jezela@gmail.com (A. Jezela-Stanek).
i
Equal contribution.
Official Journal of the European Paediatric Neurology Society
european journal of paediatric neurology 20 (2016) 462e473
http://dx.doi.org/10.1016/j.ejpn.2016.01.007
1090-3798/©2016 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
PIGN gene
Congenital disorders of
glycosylation
Flow cytometry
course was slowly progressive with severe hypotonia, chronic fever, and respiration
insufficiency at the age of 6. The second girl showed profound psychomotor retardation,
marked hypotonia, and high birth weight (97 centile). Dysmorphy was mild or absent in
both girls. Whole exome sequencing revealed novel variants in the genes PGAP2 (c.2T>G
and c.221G>A) and PIGN (c.790G>A and c.932T>G). Impaired GPI binding were was subse-
quently uncovered, although the hyperactivity of alkaline phosphatase (a GPI-anchored
protein) occurred only in first case.
Conclusions: Based on our results we can conclude that: 1. GPI-anchor biosynthesis disorders
may represent a relatively frequent and overlooked metabolic defect; 2. The utility of GPI
binding assessment as a screening test for this group of rare diseases requires further studies.
©2016 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights
reserved.
1. Introduction
Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-
APs) are glycolipids composed of phosphatidylinositol,
carbohydrate residues, glucosamine, and mannose. About
150 proteins with various functions are GPI anchored,
including membrane-associated enzymes, adhesion mole-
cules, activation antigens, differentiation markers, immu-
nologically important proteins, including complement
regulatory proteins, and other miscellaneous glycoproteins.
The exact role of GPI-anchored proteins remains unclear.
However, while they function as hydrolytic enzymes,
adhesion molecules, receptors, protease inhibitors, and
complement regulatory proteins, they share similar char-
acteristics based on their common glycolipid membrane
anchors, and can act as receptors in cell adhesion, differ-
entiation, and hostepathogen interactions
28,31,38
Recent
studies showed the importance of GPI-APs for normal
neuronal and embryonic development.
14
GPI-AP deficiencies are a recently emerging group of
diseases within congenital disorders of glycosylation. They
are caused by mutations in two different classes of genes:
PIG (Phosphatidyl Inositol Glycan) involved in the biosyn-
thesis and transfer of GPI, and PGAP (Post GPI Attachment to
Proteins) involved in structural remodeling of GPI after its
attachment to proteins. About 26 PIG and PGAP genes are
involved in the GPI anchor biosynthesis and remodeling
pathway.
18
To date, inherited GPI deficiencies caused by
mutations in at least 13 PIG and PGAP genes have been re-
ported (Table 1). Only PIGA is X-linked recessive; all of the
others are autosomal recessive. Clinical symptoms of in-
dividuals with inherited GPI-anchor deficiencies (IGDs) are
within a wide range, leading to difficulties in their pheno-
typic recognition.
Herein we present the clinical, biochemical and molecular
characteristics of new cases with a congenital disorder of GPI
anchor biosynthesis and remodeling, in whom exome
sequencing revealed mutations in the genes PGAP2 and PIGN.
In comparison with the previously described PGAP2 and PIGN
affected individuals, our patients show the more severe and
the mildest phenotypes, respectively.
2. Case presentation
2.1. Patient 1
The girl, the only child of healthy unrelated parents, was born
at 39 weeks gestation following an uncomplicated pregnancy
with a birth weight of 2940 g (1.57 SD), head circumference of
32 cm (3.01 SD), and 8 points on the Apgar scale. Her family
history was unremarkable. She was born with a flat occiput
and pectus excavatum. Hyperbilirubinemia was noted during
the neonatal period (max. 18 mg/dl), but intrauterine infection
was excluded. In the routine ultrasound examination of the
brain, II degree intraventricular hemorrhage was found,
otherwise the brain structure was normal. At 3 months of life,
psychomotor retardation and generalized weakness were
observed.
Significantly elevated alkaline phosphatase activity (AP)
(1700 U/l) and low normal calcium concentration (2.3 mmol/l)
led to suspicion of rickets, which was later excluded based on
normal simultaneously measured concentrations of phos-
phates (1.85 mmol/l), parathyroid hormone, and 25-OH
vitamin D.
At 4 months of life West-like epileptic attacks with
typical hypsarrhythmia and suppression-burst EEG pattern
were noted, followed by progressive neurological deterio-
ration, hyporeflexia, and hypotonia. Moreover, from 8
months of life persistent, refractory to treatment hyper-
thermia was observed. No acquired immunodeficiency
disorder was found, as TORCH, HIV, and Mycobacterium
tuberculosis infections were excluded. Due to progressive
neurological deterioration and recurrent aspiration pneu-
monia, the patient underwent gastrostomy tube place-
ment. Nerve conduction study showed decreased neural
conduction, electromyography (EMG) examination was
normal. Further EMG examination revealed features of
european journal of paediatric neurology 20 (2016) 462e473 463
Table 1 ePhenotypes and mutations in thirteen GPI-APs genes.
Gene defect Alkaline phosphatase Disease Inheritance Clinical and biochemical
features
Reference
PIGA (MIM 311770) Not high/mildly elevated MCAHS2 (multiple congenital
anomalies-hypotonia-seizures
syndrome 2)
XLR Seizures, brain malformations,
cleft palate, hypotonia, neonatal
seizures, contractures
17
PIGL (MIM 605947) Not high CHIME syndrome (Zunich
neuroectodermal syndrome)
AR Colobomas, Heart disease,
Ichthyosiform dermatosis,
Mental retardation, and Ear
anomalies (CHIME)
34
PIGM (MIM 610273) Not high PIGMeCDG (PIGMecongenital
disorder of glycosylation)
AR Portal venous thrombosis,
absence seizures
1
PIGV (MIM 610274) High HPMRS (hyperphosphatasia with
mental retardation syndrome,
Mabry syndrome)
AR Brachytelephalangy,
hyperphosphatasia, intellectual
disability, seizures, facial
dysmorphism
13
PIGN (MIM 606097) Not high MCAHS (multiple congenital
anomalies-hypotonia-seizures
syndrome)
AR Hypotonia, seizures, multiple
congenital anomalities,
progressive cerebellar atrophy
26
PIGO (MIM 614730) High HPMRS (Mabry syndrome) AR Brachytelephalangy, intellectual
disability, seizures
19
PGAP2 (MIM 614207) High PGAP2eCDG (PGAP2econgenital
disorders of glycosylation)
AR Non-syndromic intellectual
disability, seizures, distinctive
facial gestalt
10
PGAP3 (MIM 615716) High PGAP3eCDG (PGAP3econgenital
disorders of glycosylation)
AR Intellectual disability, seizures,
distinctive facial gestalt
14
PIGT (MIM 615395) Not high/low MCAHS3 (multiple congenital
anomalies-hypotonia-seizures
syndrome 3)
AR Intellectual disability, hypotonia,
seizures, and dysmorphic facial
features in combination with
abnormal skeletal, endocrine,
and ophthalmologic findings,
hypophosphatasia
22
PIGW (MIM 616025) High HPMRS5 (Hyperphosphatasia
with mental retardation
syndrome 5)
AR Delayed development, West
syndrome, dysmorphic facial
features
5
PIGQ (MIM 614749) High HPMRS2 (Hyperphosphatasia
with mental retardation
syndrome 2)
AR Mental retardation, hypotonia,
generalized seizures, nail
hypoplasia, dysmorphic
features, congenital heart defect
25
PGAP1 (MIM 615802) No data MRT42 (Mental retardation,
autosomal recessive 42)
AR Neonatal hypotonia, severely
delayed psychomotor
development, epilepsy with
stereotypic movements,
dysmorphic features
32
PIGY (MIM 610662) Mildly elevated Different phenotypes AR Developmental delay, seizures,
cataracts, dysmorphy,
microcephaly
16
european journal of paediatric neurology 20 (2016) 462e473464
neurogenic damage; sensory-evoked potentials showed
features of demyelination. Lysosomal storage disorders,
peroxisomal diseases, and organic acidurias were excluded
in further investigations. Gas chromatographyemass
spectrometry urine test showed ketosis with marked
C6eC10 dicarboxylic aciduria. Plasma lactate concentra-
tions were normal or high borderline. The transferrin
glycosylation pattern was normal.
Muscle biopsy was done at the age of 26 months. Histo-
chemistry revealed a generalized decrease in cytochrome
oxidase activity, lipid accumulation, and unspecific changes
of muscle fiber differentiation.
At the age of 6 years, respiratory insufficiency with carbon
dioxide accumulation gradually developed. Tracheostomy
had to be performed and assisted breathing was begun under
a national program of mechanical ventilation in the home
(Fig. 1).
The girl has remained in recumbent position from birth.
She has never sat or begun to speak. Presently at the age of 13,
she is still on artificial ventilation, fed by gastrostomy,
continuously treated for drug-resistant epilepsy. According to
the mother's observations, introduction of a ketogenic diet
positively influenced her quality of life, decreasing anxiety
and thermoregulation dysfunction. The occurrence of hyper-
phosphatasemia was never taken into account in differential
diagnostics before finding PGAP2 mutations. Reassessment of
her medical documentation showed that AP activity was
continuously elevated (1496e2780 U/l, ref. 100e550 U/l, 11
measurements on follow-up).
2.2. Patient 2
This proband is the first child of healthy and non-
consanguineous parents with an unremarkable family his-
tory. The pregnancy was complicated by recurrent urinary
tract infections and bacterial pharyngitis at 7 weeks gesta-
tion. The girl was born at term with a birth weight of 4300 g
(þ0.98 SD), head circumference of 34 cm (1.41 SD) , and 10
points on the Apgar scale. Pericranial hematoma was noted.
At the age of 2 months she was hospitalized because of
myoclonic jerks of limbs and suspicion of epilepsy. Brain MRI
revealed slightly widened extra axial spaces around the
frontal lobe; the EEG pattern at that time was normal. The
proband was then diagnosed for gastroesophageal reflux
with a negative result.
Neurological examination during subsequent hospitaliza-
tion at the age of 5 months revealed: head circumference of
42 cm (75e90 centile), generalized hypotonia, vertical
nystagmus, tongue tremor, multiple dyskinesias, and pres-
ence of tendon and postural reflexes. Laboratory tests results
were within normal ranges, except for plasma lactate con-
centrations (max 3.7 mmol/l; ref. <2.0 mmol/l). The EEG
pattern revealed generalized paroxysmal bioelectric activity,
consistent with video-EEG where correlation of myoclonic
seizures with bioelectric abnormalities was found. Subse-
quent MRI showed cerebellar atrophy, particularly significant
cerebellar vermis atrophy, and enlarged lateral ventricles,
more pronounced on the left side. Brain MRI scans are pre-
sented in Figs. 2 and 3, and Table 2.
Although no specific facial dysmorphy was seen (Fig. 4),
taking into account the clinical course of the patient, Angel-
man and Rett syndrome were considered in differential di-
agnoses, but not confirmed by genetic tests.
The patient has undergone extensive diagnostics,
including searching for inborn errors of metabolism. The
performed genetic tests allowed exclusion of spinal muscular
atrophy (no deletion in the SMN1 gene), chromosomal aber-
rations (normal result of 135k array CGH), as well as mito-
chondrial DNA pathology (negative screening for common
mutations in mitochondrial DNA). Krabbe disease, suspected
due to low galactosylceramidase activity (1.1 and 2.7 mmol/
mg protein/18 h [ref. 6.7 ±2.7]), was also excluded by the lack
of the most frequent mutation in the GALC gene.
Both patients were included into a whole exome
sequencing (WES) study based on suspicion of mitochondrial
pathology (MD). The probability of MD was assessed according
to the twelve-step Nijmegen scale
30
and was found to be 3
points for Patient 1 and 4 points for Patient 2, which corre-
sponds to a moderate level of probability (“mitochondrial
disease possible”).
Fig. 1 eFacial phenotype of Patient 1 with PGAP2 mutations (at the age of 13 y and in early infancy).
european journal of paediatric neurology 20 (2016) 462e473 465
This investigation conforms to the principles outlined in
the Declaration of Helsinki, and the study protocol was
approved by the Bioethics Commission of The Children's Me-
morial Health Institute. Written informed consent was ob-
tained from the parents of both affected girls.
2.3. Methods and results
2.3.1. Whole exome sequencing
DNA was extracted from the peripheral blood and used for
massively parallel exome sequencing. WES Library
Fig. 2 eMRI examination in Patient 2 (with PING mutations). eat the age of 9 weeks, upper row. a., b. sagittal and c. coronal
T2-weighted images show cerebellar hemispheres and vermis in normal limits, d. axial T2-weighted image shows slightly
widened extra axial spaces around the frontal lobes (more visible on the left side), and anterior part of the interhemispheric
fissure. Minimal asymmetry of the lateral ventricles eL>R. efollow-up at the age of 27 months, bottom row. e, f. sagittal T2-
weighted and g. coronal T1-weighted images show significant atrophy of the vermis. Mild atrophy of the cerebellar
hemispheres with enlarged and deep cerebellar fissures. Widening of the fourth ventricle and cisterna magna. Thin
superior cerebellar peduncles. Thin corpus callosum. h. axial T2-weighted image shows progressive frontal cortical atrophy
with enlarged fissures in the frontal lobes and widened extra axial spaces around the frontal lobes. Asymmetric widening of
the lateral ventricles eL>R.
Fig. 3 eAxial diffusion-weighted MR images of Patient 2 (PING mutations) at the age of 5 months show bilateral symmetrical
hyperintensity of the central tegmental tract.
european journal of paediatric neurology 20 (2016) 462e473466
preparation was performed using Nextera Rapid Capture
Exome kits (Illumina). The samples were run on ¼lane each
on HiSeq 1500 using 2 75 bp paired-end reads. Bioinfor-
matics analysis was performed as previously described.
36
Briefly, after initial processing by CASAVA, the generated
reads were aligned to the hg19 reference genome with the
Burrows-Wheeler Alignment Tool
23
and further processed by
the Genome Analysis Toolkit.
27
Base quality score recalibra-
tion, indel realignment, duplicate removal, and SNP/INDEL
calling were done as described.
8
The detected variants were
annotated using Annovar
40
and converted to Microsoft Access
format date for final manual analyses. Alignments were
viewed with the Integrative Genomics Viewer.
37
2.3.2. PGAP2 gene molecular results
The sequencing run for Patient 1's sample achieved 78,755,538
reads, the 10-fold target coverage was 92.2%, and the 20-fold
coverage was 78.2%, yielding a total of 280,442 revealed variants.
Two novel substitutions in the PGAP2 gene
(NM_001256240.1) were identified in this proband, including
c.2T>G (p.?) in exon 2 and c.221G>A (p.Arg74His) in exon 3
resulting in a compound heterozygous state (Fig. 5A, Table 3).
The c.2T>G molecular variant affects the initiating methio-
nine and results in start loss and possible activation of a new
potential translation initiation site or no protein translation.
Additionally, it was predicted to be deleterious by five in silico
predictive software packages, i.e., CADD, PolyPhen-2, LRT,
MutationTaster, and SIFT. The c.221G>A variant is a missense
change from arginine to histidine at residue 74 of the protein,
predicted as deleterious by six algorithms (CADD, PolyPhen-2,
MutationAssessor, LRT, MutationTaster, and SIFT). The
c.221G>A variant was reported in the ExAC database (http://
exac.broadinstitute.org) with minor allele frequency (MAF)
0.00001626, and in ESP 6500 (http://evs.gs.washington.edu/
EVS) with MAF 0.00008. Both variants were validated by
Sanger sequencing confirming their parental inheritance, the
c.2T>G variant was present in the mother and the c.221G>A
variant was inherited from the father.
2.3.3. PIGN gene molecular results
The sequencing run for Patient 2's sample achieved 55,799,742
reads with a total of 110,665 detected variants. Above 81% and
Table 2 eMRI changes observed in Patient 2 with mutation in PIGN gene.
Proband's age Brain MRI results
9 weeks Slightly widened of the extra axial spaces around frontal lobes,
Slightly widened of the both lateral fissures and anterior part of the
interhemispheric fissure,
Myelination appropriate for age,
Cerebellar hemispheres and vermis within normal limits,
Corpus callosum without pathology,
Minimal asymmetry of the slight widening of the lateral ventricle eL>R,
Symmetrical abnormal signal of the central tegmental tracts seen on T2-
weighted and diffusion weighted images in the midbrain and pons.
5 months Slightly progression:
Symmetrical abnormal signal of the central tegmental tracts seen on T2-
weighted and diffusion weighted images ein the midbrain, pons and
medulla oblongata (olivary nuclei).
Widened of the extra axial spaces around frontal lobes.
Widened of the both lateral fissures and anterior part of the
interhemispheric fissure.
Cerebellar hemispheres and vermis within normal limits
Myelination appropriate for age
7 months Without differences
27 months Progression:
Significant atrophy of the vermis
Mild atrophy of the cerebellar hemispheres
Slightly enlargement of the ventricular system, asymmetry of the lateral
ventricles L>R), IV ventricle is also enlarged
Widened of the extra axial spaces around frontal lobes, both lateral
fissures and anterior part of the interhemispheric fissure as in the
previous examination.
Myelination appropriate for age
Abnormal signal of the central tegmental tracts is very week
Fig. 4 eFacial phenotype of Patient 2 with PIGN mutations
(at the age of 3 4/12 y).
european journal of paediatric neurology 20 (2016) 462e473 467
91% of the target region was covered min. 20 and 10 times,
respectively.
Two nucleotide substitutions c.790G>A (p.Gly264Arg) and
c.932T>G (p.Leu311Trp) in the PIGN gene (NM_176787.4) were
identified by WES and confirmed by bidirectional Sanger
sequencing (Fig. 5B, Table 3). Parental studies determined that
the c.790G>A variant was inherited from the unaffected father
and the c.932T>G variant was inherited from the unaffected
Fig. 5 eIntegrative Genomics Viewer view of heterozygous mutations found in the PGAP2 gene (A) and the PIGN gene (B) by
whole-exome sequencing. Nomenclature of mutations following the guidelines of the Human Genome Variation Society
using NM_001256240.1 as a reference cDNA sequence for PGAP2 and NM_176787.4 for PIGN.
european journal of paediatric neurology 20 (2016) 462e473468
mother, consistent with a trans allelic pattern of inheritance
and compound heterozygosity for these changes. Both of the
variants are apparently novel missense mutations that affect
highly or mostly conserved amino acids and are located
within the phosphodiesterase functional domain of the PIGN
gene. In addition, they were predicted by in silico analysis
(Table 3) as probably damaging GPI-anchor biosynthesis,
which suggests that they are disease-causing genetic factors.
The variants were not found in the ClinVar, dbSNP, LOVD, or
PubMed database. Only the c.790G>A substitution had been
observed with a frequency of 0.00007458 in the ExAC database,
indicating that these changes are rare.
2.3.4. GPI functional impairment
The surface expression of GPI molecules and various GPI-APs
was tested in EDTA anticoagulated peripheral blood samples
collected from the patients and as a control esix healthy
blood donors (reference level). Samples were transported and
stored at þ4C. Flow cytometric tests were performed within
48 h following blood collection. The expression of GPI anchors
was determined by staining granulocytes and monocytes with
fluorescently-labeled aerolysin (FLAER Alexa 488, Cedarlane).
For determination the expression of GPI-APs cells were
stained with fluorescently conjugated monoclonal antibodies:
granulocytes with CD24 PE (clone ALB9, Beckman Coulter) and
CD66b FITC (clone G10F5, BD Pharmingen), monocytes with
CD14 APC (MФP9, Becton Dickinson) and CD157 PE (clone
SY11B5, eBioscience), and erythrocytes with CD59 PE (MEM-43,
Invitrogen).
2
Acquisition and analysis stained blood cells were per-
formed on flow cytometer FACSCalibur (BD Biosciences, San
Jose) with CellQuest Pro version 5.2.1 software (Becton Dick-
inson). GPI and GPI-APs expression was quantified by the
determination of median fluorescence intensity (Md) labeled
cells. Reference level was calculated as an average of Md
fluorescence stained cells from 6 donors.
Results of flow cytometry (FACS) analysis for Patient 1 and
for Patient 2 represent Figs. 6 and 7, respectively. The surface
level GPI anchors examined by FLAER binding on granulocytes
and monocytes from the Patient 1 was similar as mean value
on the reference cells (Fig. 6A and C). The cell-surface GPI-APs
tested by monoclonal antibodies: CD24 on granulocytes,
CD157 on monocytes and CD59 on erythrocytes showed
normal expression (Fig. 6B, E and F). However the expression
CD14 GPI-AP on monocytes from Patient 1 demonstrated sig-
nificant reduction (52%) compared with the reference mono-
cytes (Fig. 6D).
The surface expression of GPI anchors tested by FLAER, and
the expression of GPI-AP CD24 and CD66b tested by mono-
clonal antibodies was significantly decreased on the Patient 2
granulocytes compared with the reference cells (34.21 and
31% respectively) (Fig. 7A, B and C). FLAER binding to
Table 3 eDetails of molecular variants identified in PIGN and PGAP2 genes.
PIGN PGAP2
Mutant allele 1 Mutant allele 2 Mutant allele 1 Mutant allele 2
Chromosome 18 18 11 11
RefSeq cDNA NM_176787.4 NM_176787.4 NM_001256240.1 NM_001256240.1
Nucleotide change c.790G>A c.932T>G c.2T>G c.221G>A
Exon 9 11 2 3
IGV reads 41/81 41/111 6/10 16/29
RefSeq protein NP_065905.2 NP_065905.2 NP_001243169.1 NP_001243169.1
Amino acid change p.Gly264Arg p.Leu311Trp p.? p.Arg74His
Mutation
Type Missense Missense Missense Missense
Stage Heterozygous Heterozygous Heterozygous Heterozygous
Status Novel Novel Novel Novel
Parental origin Paternal Maternal Maternal Paternal
Pathogenicity prediction
CADD
a
28 22 17 24
Polyphen2 Probably damaging Probably damaging Probably damaging Probably damaging
Mutationassessor High (functional) Moderate (functional) nd Moderate (functional)
LRT Deleterious Deleterious Deleterious Deleterious
Mutationtaster Disease causing Disease causing Disease causing Disease causing
MAF data
b
ExAC 65000 0.00007458 0 0 0.00001626
ESP 6500 0 0 0 0
EUR 1000 0 0 0 0
POL 300 0 0 0 0
Dept of WES 67 94 10 27
a
Higher scores are more deleterious.
b
MAF eminor allele frequency; ExAC eexome aggregation consortium; EUR 1000 e1000 genomes project; ESP 6500 eexome sequencing
project; POL eproject of 300 exomes from Polish individuals with unrelated diseases.
european journal of paediatric neurology 20 (2016) 462e473 469
monocytes was not significantly changed (85% of ref.) (Fig. 7D),
but the expression of CD14 protein on these cells tested by
monoclonal antibodies was affected (42% of ref.) (Fig. 7E). The
expression GPI-AP CD59 on erythrocytes was also reduced
(76% of ref.) (Fig. 7F).
3. Discussion
In two sporadic patients presenting with a neurogenetic dis-
order from birth, whole exome sequencing performed after
long-term observation (at 3 and 12 years of age) revealed
compound heterozygous pathogenic variants of the PIGN and
PGAP2 genes, both involved in the biosynthesis of
glycosylphosphatidylinositol-anchored proteins. In addition
to several similarities to previously reported cases, our pa-
tients differ significantly, broadening the scope of knowledge
on inherited GPI deficiency.
The PGAP2 gene identified in 2013 as the cause of “hyper-
phosphatasia, mental retardation syndrome-3”(HPMRS3;
OMIM 614080) has been found in nine individuals from four
families to date.
10,20
The gene is localized on chromosome
11p15.4 and encodes a Golgi/ER-resident membrane protein
that is involved in the final step of lyso-phosphatidylinositol
(lyso-PI) remodeling to phosphatidylinositol (PI) containing
saturated fatty acids. Deficiency of this protein results in
transport to the cell surface of lyso-GPI-APs that are more
prone to cleavage by phospholipase D.
24,39
Similarly as in
other individuals with this defect, continuous (although
overlooked) increases in serum alkaline phosphatase (AP)
activity were demonstrated from infancy in Patient 1 with a
PGAP2 mutation. However, compared with the others, our
patient presented the most severe disease course with
untreatable epilepsy, dependence on artificial ventilation, and
persistent pyrexia. It cannot be excluded that at least persis-
tent pyrexia can be due to a latent infection, e.g.,
osteomyelitis.
9
The PIGN gene was reported in 2011
26
as related to multiple
congenital anomalies-hypotonia-seizures syndrome 100
(MCAHS1; OMIM 614080). The PIGN (phosphatidylinositol
glycan anchor biosynthesis, class N) gene maps to chromosome
band 18q21.33 and encodes glycosylphosphatidylinositol etha-
nolamine phosphate transferase 1, a protein expressed in the
endoplasmic reticulum that is involved in biosynthesis and
transfer of phosphoethanolamine (EtNP) to the first mannose of
the GPI anchor.
26
To date 11 cases from four families have been
reported
3,6,26
and most of them presented with various severe
congenital malformations that led to death. In contrast, our
Fig. 6 ePatient 1 ethe surface expression of GPI anchors and various GPI-anchored proteins tested by flow cytometry:
binding FLAER to GPI structures itself on granulocytes (A) and monocytes (C), binding monoclonal antibodies to CD24 (B), on
granulocytes, CD14 (D) and CD157 on monocytes (E), CD59 on erythrocytes (F). Black line epatient, gray line eblood donor,
gray dotted line enegative control. Md emedian of fluorescence intensity.
european journal of paediatric neurology 20 (2016) 462e473470
Patient 2 with PIGN mutations did not present any malforma-
tions and she demonstrated the mildest disease history as
compared with the reported cases. The course of disease re-
sembles to some extent the reported Japanese siblings,
35
as
they all show severe delay in psychomotor development, hy-
potonia, vertical nystagmus, and seizures. High birth weight
(95 centile) and head circumference (90 centile) found in
several patients with PIGN mutations
26
were also seen in our
Patient 2.
Occurrence of dysmorphic features and even a distinctive
facial gestalt was suggested in some descriptions of patients
with IGD. Abnormal facial features were noted in all eleven
patients with PIGN mutations
3,6,26
and were also considered in
those with PGAP2 mutations.
10,20
This seems not to be the case
in both of our patients, as a thorough re-assessment their
photographs over the years did not document a distinctive
facial gestalt. The facial features of Patient 1 had changed with
age; they were very subtle in childhood and did not have a
specific and recognizable dysmorphic pattern. Hansen et al.
came to a similar conclusion.
10
However, it may be doubtful
whether the facial phenotype of our Patient 1 is only sec-
ondary to her neurologic condition. Longer observations of
subsequent patients with IGD are necessary to arrive at firm
conclusions.
The various congenital malformations described
frequently in IGD are evidence of prenatal onset of patholog-
ical processes at the level of organ differentiation. It is not
clear, however, whether these processes, including brain
damage, cease after birth. The brain MRI of Patient 2 revealed
progressive cerebellar atrophy over time. A similar observa-
tion was reported by Ohba et al.
35
; but more data is needed for
final conclusions.
Although IGD should be classified as an “inborn metabolic
disease”(IMD),
29
an easy and reliable metabolic test that would
improve (accelerate) the differential diagnosis in suspected pa-
tients is lacking. This was a cause of diagnostic delay in all
known cases before next-generation sequencing became avail-
able. Alkaline phosphatase activity measurement is useful for
recognition of some IGDs (Table 1), but is highly unspecific.
An attempt to measure GPI-AP levels on the cell membrane in
available tissues (blood cells, fibroblasts) by FACS shown prom-
ising results.
14,26,35
The method is used routinely in hematology
for diagnosis of paroxysmal nocturnal hemoglobinuria, a defect
caused by somatic and germline mutations in the PIGA gene.
2
In Patient 1, demonstrating constant elevation of AP ac-
tivity, the FLAER binding on granulocytes and monocytes e
which characterize the GPI expression ewas normal. The
expression of GPI-APs: CD24 on granulocytes, CD157 on
Fig. 7 ePatient 2 ethe surface expression of GPI anchors and various GPI-anchored proteins tested by flow cytometry:
binding FLAER to GPI structures itself on granulocytes (A) and monocytes (D), binding monoclonal antibodies to CD24 (B) and
CD66b (C) on granulocytes, CD14 on monocytes (E), CD59 on erythrocytes (F). Black line epatient, gray line eblood donor,
gray dotted line enegative control. Md emedian of fluorescence intensity.
european journal of paediatric neurology 20 (2016) 462e473 471
monocytes and CD59 on erythrocytes was similar as that in
healthy blood donors. By comparison, analysis the expression
GPI-APs decay accelerating factor (DAF, CD55) and CD59 on
lymphoblastoid cell lines from patients with PGAP2 mutations
also showed no significant difference compared to healthy
donors.
10
In contrast, in vitro functional analysis performed in
PGAP2-deficient Chinese hamster ovary cell lines showed
reduced DAF and CD59 expression.
10
The lower expression of
cell-surface CD14 protein on monocytes (52% of reference
level) of Patient 1 is difficult to explain. It might be resulted
from an unspecific cell activation or individual variability.
11,15
However it may be a result of release of this protein from
plasma membrane to the medium in PGAP2 mutated mono-
cytes
39
similarly to the AP release. Davis et al. demonstrated
experiments with haploid genetic screens PGAP2 knockout
cells suggesting that GPI modifications have markedly distinct
effects on individual GPI-AP pathways. Moreover, these data
indicate that incomplete GPI anchor modification can result in
intracellular retention of certain GPI-APs.
7
The GPI anchor assessment in our Patient 2 revealed
remarkable abnormalities including decreased binding of
FLAER and the decreased expression of some GPI-APs detected
by monoclonal antibodies comparing to the healthy in-
dividuals. The most significant differences were noticed in the
GPI anchors and two GPI-APs CD24 and CD66b on granulocytes
that were expressed on 21e34% of reference levels. Ohba et al.
reported similar results on PIGN mutated granulocytes; they
found expression of CD16 and CD24 decreased to 26e54% of
control values. However, they noticed no differences in the
expression of CD59, CD55, CD48, or GPI tested by FLAER
binding on B lymphoblasts in their patients as compared with
a control group.
35
Only a slight difference between our Patient 2 and healthy
individuals in FLAER binding to GPI anchors on monocytes
(85% of control expression), except CD14 (expression reduced
to 42% of reference levels) was demonstrated. Furthermore,
we found decreased (76%) CD59 expression on erythrocytes, a
finding similar to that observed by Maydan et al.
26
on fibro-
blasts in a patient with a homozygous PIGN mutation.
The flow cytometry results demonstrated functional
impairment of the PIGN mutations on GPI and GPI-anchored
protein expression in blood cells, however, the consequences
of this phenomenon are not known. They might depend on the
involved tissue or the function of the protein that is bound to
GPI. As suggested by Ohba et al., changes to a subset of GPI-
anchored proteins can be sufficient to cause neurological de-
fects and (or) an abnormal structure of the GPI moiety in PIGN
mutated cells might affect the function of these proteins.
GPI-anchor deficiencies cannot be diagnosed or even
screened with transferrin isoform or APOCIII glycosylation
assay. Furthermore results in unrecognizable and unspecific
clinical phenotype underline the importance of whole-exome
sequencing testing in diagnostic procedures.
14
However,
based on our results we suggest that the further studies
leading to the development of the screening test should be
concentrated on the analysis of granulocytes and monocytes
for expression of both GPI anchors and GPI-APs performed
simultaneously. Evaluation of erythrocytes is unlikely to be
useful (too small differences in expression between patients
and healthy controls were observed).
This study was partially financed by CMHI project S136/13
and NCN Project Harmonia 4 No. UMO-2013/08/M/NZ5/00978.
Conflict of interest
None.
Acknowledgments
The authors dedicate this work to their late colleague, Dr.
Maciej Adamowicz, a biochemist with a keen and inquisitive
mind, a humble and benevolent man who patiently taught us
all in Poland about disorders of glycosylation. We missed
Maciek so much at the time of writing this paper.
Many thanks to the parents of both girls for their invalu-
able contribution describing the details of the clinical course
of this very rare disorder.
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