REPORT
PGM3 Mutations Cause a Congenital Disorderof Glycosylation with Severe Immunodeficiencyand Skeletal Dysplasia
Asbjørg Stray-Pedersen,1,2,3,4,* Paul H. Backe,5,6,7 Hanne S. Sorte,4 Lars Mørkrid,6,7 Niti Y. Chokshi,3,8
Hans Christian Erichsen,9 Tomasz Gambin,1 Katja B.P. Elgstøen,6 Magnar Bjøras,5,7
Marcin W. Wlodarski,10 Marcus Kruger,10 Shalini N. Jhangiani,1,11 Donna M. Muzny,1,11 Ankita Patel,12
Kimiyo M. Raymond,13 Ghadir S. Sasa,8,14 Robert A. Krance,8,14 Caridad A. Martinez,8,14
Shirley M. Abraham,15 Carsten Speckmann,10 Stephan Ehl,10 Patricia Hall,16 Lisa R. Forbes,2,3,8
Else Merckoll,17 Jostein Westvik,17 Gen Nishimura,18 Cecilie F. Rustad,4 Tore G. Abrahamsen,7,9
Arild Rønnestad,9 Liv T. Osnes,19 Torstein Egeland,7,19 Olaug K. Rødningen,4 Christine R. Beck,1
Baylor-Johns Hopkins Center for Mendelian Genomics, Eric A. Boerwinkle,1,11,20 Richard A. Gibbs,1,11
James R. Lupski,1,8,11,12,21,* Jordan S. Orange,2,3,8,21 Ekkehart Lausch,10,21 and I. Celine Hanson3,8,21
Human phosphoglucomutase 3 (PGM3) catalyzes the conversion of N-acetyl-glucosamine (GlcNAc)-6-phosphate into GlcNAc-1-phos-
phate during the synthesis of uridine diphosphate (UDP)-GlcNAc, a sugar nucleotide critical to multiple glycosylation pathways. We
identified three unrelated children with recurrent infections, congenital leukopenia including neutropenia, B and T cell lymphopenia,
and progression to bone marrow failure. Whole-exome sequencing demonstrated deleterious mutations in PGM3 in all three subjects,
delineating their disease to be due to an unsuspected congenital disorder of glycosylation (CDG). Functional studies of the disease-asso-
ciated PGM3 variants in E. coli cells demonstrated reduced PGM3 activity for all mutants tested. Two of the three children had skeletal
anomalies resembling Desbuquois dysplasia: short stature, brachydactyly, dysmorphic facial features, and intellectual disability. How-
ever, these additional features were absent in the third child, showing the clinical variability of the disease. Two children received
hematopoietic stem cell transplantation of cord blood and bonemarrow frommatched related donors; both had successful engraftment
and correction of neutropenia and lymphopenia. We define PGM3-CDG as a treatable immunodeficiency, document the power of
whole-exome sequencing in gene discoveries for rare disorders, and illustrate the utility of genomic analyses in studying combined
and variable phenotypes.
Glycosylation is a ubiquitous posttranslational modifica-
tion essential for the proper functioning of a broad spec-
trum of proteins and lipids. In this process, glycans are
constructed from a cellular pool of activated monosaccha-
rides, the sugar nucleotides. The structural diversity of the
glycans ensures specific and selective molecular interac-
tions. Mammals utilize nine sugar-nucleotide donors for
glycosyltransferases: uridine diphosphate (UDP)-glucose,
UDP-galactose, guanosine diphosphate (GDP)-mannose,
GDP-fucose, UDP-xylose, UDP-glucuronic acid, cytidine
monophosphate (CMP)-sialic acid, UDP-N-acetyl-galactos-
amine (UDP-GalNAc), and UDP-N-acetyl-glucosamine
(UDP-GlcNAc) (Figure S1, available online). Glycans are
1Department of Molecular and Human Genetics, Baylor College of Medicine,
dren’s Hospital, Houston, TX 77030, USA; 3Section of Immunology, Allergy, an
Texas Children’s Hospital, Houston, TX 77030, USA; 4Department of Medica
Microbiology, Oslo University Hospital, 0424 Oslo, Norway; 6Department of M
of Clinical Medicine, University of Oslo, 0318 Oslo, Norway; 8Department of Pe
of Pediatrics, Oslo University Hospital, 0424 Oslo, Norway; 10Department of P
Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030,
lor College of Medicine, Houston, TX 77030, USA; 13Department of Laboratory
USA; 14Center for Cell and Gene Therapy and Texas Children’s Cancer and Hem
Houston, TX 77030, USA; 15Pediatric Hematology Oncology, University of N
Department of Human Genetics, Emory University, Decatur, GA 30033, USA;18Department of Pediatric Imaging, Tokyo Metropolitan Children’s Medical C
Immunology and Transfusion Medicine, Oslo University Hospital, 0424 Oslo, N
ter, Houston, TX 77030, USA21These authors contributed equally to this work
*Correspondence: [email protected] (A.S.-P.), [email protected] (J.R.L.)
http://dx.doi.org/10.1016/j.ajhg.2014.05.007. �2014 by The American Societ
96 The American Journal of Human Genetics 95, 96–107, July 3, 2014
attached to proteins via a nitrogen atom of an asparagine
(N-linked glycan) or an oxygen atom of a serine or threo-
nine (O-linked glycan). These two major glycosylation
mechanisms in eukaryotic cells differ in the protein targets
and cellular localization. Defects in genes encoding the
formation of sugar nucleotides or different steps of the
glycosylation processes result in the disruption of distinct
glycosylation pathways and might lead to congenital dis-
orders of glycosylation (CDGs).
UDP-GlcNAc, the end product of the hexosamine
biosynthetic pathway (Figure S1), is an activated precursor
for both N-linked and O-linked glycosylation of proteins1,2
and is needed for the generation of glycosaminoglycans,
Houston, TX 77030, USA; 2Center for Human Immunobiology, Texas Chil-
d Rheumatology, Department of Pediatrics, Baylor College of Medicine and
l Genetics, Oslo University Hospital, 0424 Oslo, Norway; 5Department of
edical Biochemistry, Oslo University Hospital, 0424 Oslo, Norway; 7Institute
diatrics, Baylor College ofMedicine, Houston, TX 77030, USA; 9Department
ediatrics, Freiburg University Hospital, 79106 Freiburg, Germany; 11Human
USA; 12Medical Genetics Laboratories, Molecular and Human Genetics, Bay-
Medicine and Pathology, Mayo College of Medicine, Rochester, MN 55905,
atology Centers, Baylor College of Medicine and Texas Children’s Hospital,
ew Mexico, Albuquerque, NM 87106, USA; 16Emory Genetics Laboratory,17Department of Radiology, Oslo University Hospital, 0424 Oslo, Norway;
enter, 2-8-29 Musashidai, Fuchu, Tokyo 183-8561, Japan; 19Department of
orway; 20Human Genetics Center, University of Texas Health Science Cen-
y of Human Genetics. All rights reserved.
proteoglycans, and glycolipids. Specifically, UDP-GlcNAc
is incorporated into N-glycans, O-glycans, and glycosyl-
phosphatidylinositol (GPI)-anchored proteins and is also
a donor for the reversible addition of O-GlcNAc to
proteins, i.e., in proteoglycan synthesis. In the yeast
Saccharomyces cerevisiae, as well as in higher eukaryotes,
phosphoglucomutase 3 (Pgm3) catalyzes an important
step in the synthesis of UDP-GlcNAc: the conversion of
GlcNAc-6-phosphate (GlcNAc-6-P) into GlcNAc-1-phos-
phate (GlcNAc-1-P) (Figure S1).1,3 Homozygous knockout
of Pgm3 in mice is embryonically lethal, whereas homozy-
gous hypomorphic alleles cause trilineage cytopenias
(anemia, thrombocytopenia, and leukopenia), ascribed to
decreased UDP-GlcNAc.3 The human homolog, PGM3
(MIM 172100), synonymously designated phosphoacetyl-
glucosamine mutase 1 (AGM1), is most abundantly ex-
pressed in the pancreas, prostate, and testis and is also
expressed in the bone marrow, placenta, salivary glands,
digestive tract, and liver, but not in lung tissue.4–6 In the
relevant tissue, the protein has mainly been found to
localize to the nucleus and cytoplasm and be associated
with the cytoskeleton.6 Mutations in another phosphoglu-
comutase-encoding gene, PGM1 (MIM 171900), cause hu-
man PGM1-CDG (CDG type It [MIM 614921]; Figure S1),
clinically characterized by growth retardation, hepatop-
athy, myopathy, dilated cardiomyopathy, hypoglycemia,
and the bifid uvula.7–10 Disease-causing hypomorphic
PGM3 mutations have been reported in two young-adult
cohorts with clinical presentation of eczema, recurrent in-
fections, immunoglobulin E (IgE)-mediated disease, bron-
chiectasis, variable degrees of neurocognitive impairment,
kyphoscoliosis, and CD4 or CD8 T cell lymphopenia.11,12
None of the described individuals had severe immune defi-
ciency, bone marrow failure, or skeletal dysplasia.
We describe PGM3-CDG, a CDG detected by whole-
exome sequencing (WES) in three children with a similar
hematological phenotype. Compared to the phenotype
of recently published affected individuals, their unique
clinical and immunological PGM3-CDG phenotype in-
cluded recurrent infections, combined immunodeficiency,
neutropenia with progression to bone marrow failure, and
variable dysmorphic features. Importantly, two individuals
presented with a recognizable skeletal dysplasia phenotype
resembling Desbuquois dysplasia (DBQD [MIM 251450]);
Table 1). DBQD is an autosomal-recessive osteochondro-
dysplasia characterized by growth retardation, short ex-
tremities (rhizomelic and mesomelic shortening), joint
laxity, and progressive kyphoscoliosis. Affected individuals
have facial dysmorphisms, a short neck, shortened tubular
bones with metaphyseal flaring, an exaggerated trochanter
minor of the proximal femur (monkey-wrench malforma-
tion), and advanced bone age. DBQD type 1 includes
hand anomalies such as an extra ossification center distal
to the second metacarpal bone, bifid distal-thumb pha-
lanx, and dislocation of the interphalangeal joints.
Mutations in the gene encoding calcium-activated nucleo-
tidase 1 (CANT1 [MIM 613165]), located at 17q25.3, have
The
been reported in DBQD type 1, but affected individuals
without detected CANT1 mutations suggest genetic het-
erogeneity.13–15 CANT1 functions in proteoglycan meta-
bolism.16,17 Proteoglycan synthesis is also disrupted in
DBQD type 2 as a result of a deficiency in xylosyltransfer-
ase 1 (XYLT1 [MIM 608124]).18 Severe combined immuno-
deficiency (SCID) or other types of congenital immunode-
ficiencies have not been reported in DBQD.
Three unrelated children from distinct world popula-
tions—a female with Afghani parents (P1), a male with
Mexican-American parentage (P2), and another male
from Germany (P3)—were studied (Figure 2). Clinically,
P1 and P3 constitute a distinct third Desbuquois variant
we here propose to classify as DBQD type 3.
Informed consent for research studies was obtained from
the probands, siblings, and parents through protocols
approved by the institutional review boards at Baylor
College of Medicine and Universitatsklinikum Freiburg
and through institutional research protocols approved by
regional ethics committees; all followed the principles
stated in the Declaration of Helsinki. In P3 and his family,
molecular analyses were also performed in accordance
with the German Genetic Diagnosis Act (GenDG), and in
P1, analyses were performed in accordance with the
National Biotechnology Act. Specific parental releases
were obtained from parents for the use of clinical data
(from P1–P3) in this manuscript.
After birth, the female child (P1; subject A.II-2 in
Figure 2) presented with respiratory distress with radio-
graphically verified pneumonias despite antimicrobial
therapy. She had leukopenia with neutropenia and
SCID with low numbers of T lymphocytes and
B cells but normal numbers of natural killer (NK) cells
(T�B�NKþSCID phenotype) and no anemia, thrombocy-
topenia, or splenic anomalies. T cell receptor excision
circles were not low in peripheral blood at birth.19
Lymphocyte subsets as measured by flow cytometry at
older age points revealed decreasing numbers of B cells
(Table S1).19,20 Striking skeletal abnormalities were noted
clinically and by radiography at birth: rhizomelic short-
ening of tubular bones with brachydactyly, short meta-
carpal and metatarsal bones and phalanges, and pectus
carinatum (Figure 1A). Dysmorphic facial features in-
cluded downturned corners of the mouth, midface
hypoplasia, and micrognathia (Figure 1B). Other skeletal
anomalies included bilateral exaggerated trochanter
minor, coronal clefts of the caudal lumbar vertebrae,
and cranial Wormian bones (Figures 1E–1G). She did not
have microcephaly or hydrocephalus, and MRI showed
normal cerebral myelination patterns at 5 months of
age. She had eczematous skin lesions from 2 months of
age. At 4 months of age, she received a hematopoietic
stem cell transplant (HSCT) from a 6/6 matched cord
blood donor, and at 1 year of age (6 months after
HSCT), she had leukocytes within normal ranges and
no infections. She was globally developmental delayed
(4-month stage at 1 year of age).
American Journal of Human Genetics 95, 96–107, July 3, 2014 97
Table 1. Clinical Characteristics of the Three Children Presenting with PGM3-CDG
Individual
P1 P2 P3
Gender female male male
Ethnicity Afghani Mexican German
Parental consanguinity no but same clan no no
Family history one healthy older sibling,no other cases
two deceased older siblings (5 monthsand 7 months old) with a similardisease, one additional older healthysibling
two healthy younger siblings,no other cases
Birth weight (percentilea) 4,135 g (95th ) 4,080 g (90th) 3,015 g (25th)
Birth length (percentilea) 46 cm (5th) 48 cm (10th–25th) 42 cm (4 cm < 2nd)
Birth OFC (percentilea) 38 cm (85th) NA 34 cm (35th)
Follow-up weight 8.7 kg (3rd) at 18 months 23.4 kg (50th–75th) at 6 years NA
Follow-up length 73 cm (2 cm < 2nd) at 18 m 120 cm (50th) at 6 years NA
Follow-up OFC 44 cm (5th) at 12 months NA NA
Skeletal dysplasia with short-limbeddwarfism, brachydactyly, and ‘‘monkey-wrench’’ femora
þ � þ
Pectus carinatum þ � þ
Dysmorphic facial features, downturnedcorners of mouth, midface hypoplasia, andmicrognathia
þ � þ
Developmental delay and/or intellectualdisabilities
þ � þ
Other one evaluation forhydrocephalus, no shuntneeded
� seizures since 4 months,hydrocephalus verified, shuntimplantation at 5 months
Age at onset of infections and/orimmunodeficiency
birth birth birth
T�B�NKþ SCID þ þ þ
Neutropenia þ þ þ
Anemia (þ) � þ
Thrombocytopenia � � �
Splenomegaly � � �
Recurrent respiratory infections, otitismedia, and pneumonia
þ þ þ
Skin infections þ þ þ
Eczema since 2 months since 2–3 months since 2 months
Gastrointestinal problems GERD GERD, persistent diarrhea GERD
Serum immunoglobulins low IgM and IgA, normalIgG and IgE
low IgM, normal IgG and IgA, highIgE (1,233–1,768 kU/l)
normal IgM, IgA, and IgE,low IgG from 3 months
Start age for antibiotics and antifungaltherapy
birth intermittent since birth, prophylacticsince 2.5 years
birth
Start age for immunoglobulin substitution 4 weeks 2.5 years 3 months
Start age for G-CSF injections 3 weeks 1 year 2 months
RBC transfusions (SAG-M) three SAG-M in total � first SAG-M at 6 weeks,repeated every 2–3 weeks
HSCT þ þ �
HSCT recipient age 4 months 6 years no HSCT
(Continued on next page)
98 The American Journal of Human Genetics 95, 96–107, July 3, 2014
Table 1. Continued
Individual
P1 P2 P3
HSCT donor type cord blood, unrelated,6/6 HLA match
HLA-identical sibling no HSCT
HSCT outcome successfully cured successfully cured no HSCT
Age at latest evaluation 1.5 years (living) 6.5 years (living) deceased at 7 months
PGM3 mutations (RefSeq NM_015599.2) c.[737A>G];[737A>G] c.715G>C and chr6.hg19:g.(83,013,454_83,145,962)_(84,389,166_84,395,825)del
c.[737dupA];[1352A>G]
Predicted PGM3 changes p.[Asn246Ser];[Asn246Ser] p.[Asp239His];[0] p.[Asn246Lysfs*7];[Gln451Arg]
Genes tested (with normal results) bySanger sequencing prior to WES
RAG1, RAG2, JAK3, ELANE,and HAX1
NEMO, CD40L, ELANE, HAX1, SBDS,SH2D1A, WAS, FOXP3, MAGT1, STK4,IFNGR1, IFNGR2, CXCR4, and GFI1
CANT1, CHST3, IMPAD1, SBDS,and RMRP
CMA (with normal results) prior to WES CMA Agilent 180K no CMA CMA Agilent 244K
Abbreviations are as follows: CMA, chromosomal microarray; G-CSF, granulocyte colony-stimulating factor; GERD, gastresophageal reflux disease; HLA, humanleukocyte antigen; HSCT, hematopoietic stem cell transplantation; IgA, immunoglobulin A; IgE, immunoglobulin E; IgG, immunoglobulin G; IgM, immunoglob-ulin M; NA, not available; OFC, occipitofrontal circumference; RBC, red blood cell; SAG-M, saline-adenine-glucose-mannitol stored RBC unit, T�B�NKþSCID,severe combined immunodeficiency with a lack of T and B lymphocytes but presence of NK cells; and WES, whole-exome sequencing.aAge percentiles according to WHO Child Growth Standards.
The Mexican-American male (P2; subject B.II-4 in
Figure 2) presented with recurrent infections (upper-respi-
ratory-tract infections, skin abscesses, and chronic otitis
media) soon after birth. Family history was significant for
two older full male siblings (B.II-2 and B.II-3 in Figure 2),
who died early from infection at 7 and 5 months of age,
respectively. These children exhibited persistent vomiting,
failure to thrive, pneumonia, and eczema. Neutropenia
was documented in only a single sibling. Neither P2 nor
his siblings had evidence of skeletal abnormalities, e.g.,
they had a normally configured thorax and proportionate
stature without facial dysmorphic features. This boy had
infancy-onset eczema and IgE-mediated food allergy with
associated anaphylaxis. Neutropenia diagnosed at 1 year
of age was responsive to granulocyte colony-stimulating
factor (G-CSF) therapy. NK cells, platelets, and red blood
cell (RBC) counts were normal, but his disease progressed
with loss of peripheral blood B and T cells (Table S2).
When he was 5 years old, his bone marrow aspirate was
hypocellular, had a 40% reduction of cellularity in compar-
ison to previous bone marrow samples, and showed evi-
dence of bonemarrow failure. At 6 years of age, he received
a matched-related HSCT from his human-leukocyte-
antigen-identical healthy brother. This boy had successful
engraftment and resolution of his neutropenia and return
of lymphocyte function. He currently attends school and
has mild speech delay but normal cognition.
Like the female child (P1), the most severely affected
child (P3; subject C.II-1 in Figure 2) had clinically apparent
DBQD-like disease. Short limbs and a small thoracic diam-
eter were noted on fetal ultrasound, and short-limbed
dwarfism and brachydactyly, along with pectus carinatum
and facial dysmorphism, were diagnosed after birth (Fig-
ures 1C and 1D). Skeletal radiographs demonstrated short
The
tubular bones, several phalangeal and tarsal dislocations
(Figure 1J), short femoral necks with metaphyseal beaking,
and exaggerated lesser trochanters (Figure 1K). P3 also had
leukopenia from birth, neutropenia with reduced response
to G-CSF, and a T�B�NKþSCID phenotype (Table S3) with
recurrent and severe infections. From 2 months of age, he
had a eczematous scalp and intertriginous skin lesions.
At 3 months, he required ventilatory and circulatory
support after influenza and coincident generalized bacte-
rial infection. He developed intermittent tonic seizures
with hypsarrhythmia on electroencephalography, and
MRI demonstrated internal and external hydrocephalus,
delayed myelination, and periventricular white-matter
lesions. He had complex neurological deterioration and
died at 7 months of age from overwhelming infection.
In summary, all three children had recurrent infections
since birth, congenital neutropenia, and a combined
immunodeficiency characterized by low numbers of
T cells, an increased CD4/CD8 ratio, progressive loss of
B cells with age, and persistently normal NK cells (Tables
S1–S3). None of them had thrombocytopenia or signifi-
cant anemia. Two children (P1 and P3) had skeletal
anomalies consistent with DBQD (Figure 1). The
Mexican-American boy (P2) had two older male siblings
(one with neutropenia and most likely the same disorder
and neither with skeletal dysplasia) who died in infancy
from infection. Two children (P1 and P2) were successfully
treated with HSCTwith neutrophil and T and B cell correc-
tion. The most severely affected child (P3) died prior to
receiving a transplant.
WES was performed on all three individuals with the use
of genomic DNA extracted from whole blood prior to
HSCT.21,22WES for two subjects (P1 and P2) was performed
at the Baylor College of Medicine Human Genome
American Journal of Human Genetics 95, 96–107, July 3, 2014 99
Figure 1. Dysmorphic Features andRadiological Manifestations in P1 and P3(A) P1 at 1 month of age. Note the shortfingers and short long bones (rhizomelic)and the bell-shaped thorax with pectuscarinatum.(B) P1 at 1 year of age. Note the dysmor-phic facial features, including a down-turned mouth, midface hypoplasia, andmicrognathia. The pectus carinatum is stillprominent, but body stature appears lessdisproportionate. The child is unable tosit by herself.(C and D) Neonatal photographs of P3show strikingly similar dysmorphic fea-tures and physical findings.(E and F) Skull X-rays of P1 at 3 months ofage demonstrate cranial Wormian bones(arrows).(G) A hand X-ray of P1 at 3 months of ageshows the extra ossification center, thepseudoepiphysis (arrow), proximal to thesecond metacarpal bone.(H) A hip X-ray of the pelvic bones andfemora of P1 at 3 months of age demon-strates the exaggerated trochanter minor(monkey-wrench appearance) of the prox-imal femur on both sides (arrows).(I–K) Neonatal radiographs of P3 demon-strate (I) no Wormian bones in the skull,(J) severe brachydactyly and phalangealdislocations, but no advanced skeletalmaturation or extra ossicles in the carpo-gram (also not present at 3 months), and(K) typical monkey-wrench morphology(arrows) of the proximal femora; there ismetaphyseal flaring at the distal femoralends.Specific parental releases were obtainedfrom parents for the use of photographs(of P1 and P3) in this manuscript.
Sequencing Center (BCM-HGSC) as part of the Baylor-
Johns Hopkins Center for Mendelian Genomics. WES
testing in P3 was performed by the Department of Pediat-
rics at Freiburg University Hospital with a patient-parent
trio design on a SOLiD5500xl platform as previously
described.23 The statistical summary of variants detected
by WES is summarized in Table S4. The candidate dis-
ease-associated variants identified by WES were indepen-
dently confirmed by Sanger sequencing and analyzed for
familial segregation (primers in Table S5). The WES
method used at BCM-HGSC, including the HGSC CORE
design, has been described.21,22,24–29 Annotation data
were added to the variant-call-format file with a suite of
annotation tools, designated ‘‘Cassandra.’’30 Rare variants
were selected on the basis of the NHLBI Exome Sequencing
Project (ESP) Exome Variant Server, 1000 Genomes (as
of October 2013), and two in-house-generated data-
bases that include results of whole-exome-sequenced sam-
ples from ~4,000 (Arterosclerosis Risk in Communities
[ARIC]) and >200 (Baylor College of Medicine Center for
Mendelian Genomics [BCM-CMG]) different individuals.
Variants of interest were selected on the basis of both
100 The American Journal of Human Genetics 95, 96–107, July 3, 201
rarity and evaluation by the following prediction tools:
PhyloP, SIFT, PolyPhen-2, likelihood-ratio test (LRT), and
MutationTaster. In addition, knowledge of gene function,
pathways, and expression patterns and results from other
model systems were considered. Prior to evaluation of
genes absent from publically available databases, all
variants in exonic and the captured intronic regions
of HGMD and disease-related OMIM genes were evaluated.
Bioinformatic prediction of copy-number variants (CNVs)
from WES data were based on BAM files analyzed by the
Integrative Genomics Viewer (IGV) and CoNIFER.31 The
Baylor College of Medicine chromosomal microarray
(BCM CMA) used in P2 was a custom-designed genome-
wide Agilent oligoarray (BCM CMA version 10) with
exon coverage of 4,200 genes, including PGM3 and 300
genes known to bemutated in various primary immunode-
ficiency diseases.32,33 This CMA readily identifies intra-
genic CNV alleles as recessive carrier states.34–36 For P1
and P3, standardized Agilent oligoarrays 180K and 244K
were performed as part of the diagnostic workup.
WES studies of P1 and P2 identified PGM3 as a candidate
gene, and adding the WES results from P3 allowed us to
4
Figure 2. PedigreesThe families of P1 (A), P2 (B), and P3 (C) are shown with segregation of mutant alleles. Partial chromatograms of Sanger confirma-tion analysis of PGM3 mutations are shown only for the probands; arrows indicate respective nucleotide changes. For homozygousand hemizygous mutations, normal control sequences are given below the mutated allele. Also below the pedigree in (B) isthe result from the chromosomal microarray; it shows the probes in the deleted region (red). The 6q14.1–q14.2 deletion(chr6.hg19:g.(83,013,454_83,145,962)_(84,389,166_84,395,825)del) encompassed PGM3 and three neighboring OMIM genes(UBE3D, ME1, and SNAP91) on P2’s paternal allele. Samples from his deceased affected brothers were not available for genetic testing.Abbreviations are as follows: del, 1.2 Mb deletion CNV; and WT, wild-type.
conclude that the described clinical phenotype in-
cluding the DBQD-like skeletal dysplasia was most likely
due to mutations in PGM3 (MIM 172100). None of the
single-nucleotide variants (SNVs) detected in PGM3 in
the three children had been reported in the NHLBI ESP
Exome Variant Server, 1000 Genomes, dbSNP, ARIC, or
BCM-CMG.
Exome sequencing of the female child (P1) identified
a homozygous nonsynonymous SNV in exon 6 of
PGM3. Asparagine was replaced by serine at residue 246
(c.737A>G [p.Asn246Ser]; RefSeq accession number
NM_015599.2]). Sanger sequencing confirmed the mis-
sense variant in the proband, heterozygous carrier status
in both parents, and homozygous wild-type status in the
healthy older sister (Figure 2A). The variant-prediction
programs (SIFT, PolyPhen-2, LRT, MutationTaster, and
PhyloP) evaluated the variant as most likely disease
causing and the affected location as conserved. The
amino acid Asn246 is highly conserved across species
(Figure S2). Although there was no known parental
consanguinity, the parents were from the same clan,
and two large genomic intervals with absence of heterozy-
gosity surrounding PGM3were observed on chromosome 6
only (Figure S3). Neither CANT1 mutations nor CHST3
(MIM 603799) disease-causing mutations were detected
in the WES results of P1. The WES coverage of these
two genes was adequate, except for exons 1 and 2 of
CANT1 (RefSeq NM_00159772.1) and exon 1 of CHST3
(RefSeq NM_004273.4), which were subsequently Sanger
sequenced.
In P2, WES identified a nonsynonymous SNV also
located in exon 6 in PGM3. This rare variant was confirmed
by Sanger sequencing (Figure 2B) and is predicted to cause
aspartic acid replacement by histidine at position 239
(c.715G>C [p.Asp239His); RefSeqNM_015599.2; Figure 3).
The A
Prediction programs (SIFT, PolyPhen-2, LRT, PhyloP, and
MutationTaster) evaluated the variant as most likely
disease causing, and the affected amino acid is con-
served across species (Figure S2). Asp239 is located seven
amino acids from both the active site and Asn246,
altered in P1. In segregation analyses, only the mother
was heterozygous for the SNV; however, bioinformatic
interpretation of WES (IGV and CoNIFER) provided sug-
gestive evidence of a deletion CNV involving PGM3
(Figure S3), suggesting that the proband was hemizygous
for the SNV. Chromosomal microarray (BCM CMA ver-
sion 10) confirmed a 1.2 Mb deletion of 6q14.1–q14.2,
chr6.hg19:g.(83,013,454_83,145,962)_(84,389,166_84,395,
825)del, involving the entire PGM3 and three neighboring
OMIM genes (UBE3D [MIM 612495], ME1 [MIM 154250],
and SNAP91 [MIM 607923]) on the boy’s paternal allele
(Figure 2B). The deletion was inherited from his healthy
father (individual B.I-1 in Figure 2). Samples from the
deceased affected brothers were not available for genetic
testing.
In P3, who had the most severe phenotype, com-
pound-heterozygous variants were detected in PGM3.
In exon 6, we detected a 1 bp duplication (c.737dupA
[p.Asn246Lysfs*7]; RefSeq NM_015599.2). This duplica-
tion is predicted to cause nonsense-mediated degradation
of the mutant mRNA. Accordingly, quantitative real-time
PCR analysis (primers in Table S5) of PGM3 transcripts in
P3’s blood cells revealed a reduction to approximately
50% (Figure S4), which is in keeping with degradation of
the mRNA with a premature stop codon.37 On the other
allele, a missense mutation was detected in exon 11
(c.1352A>G [p.Gln451Arg]; RefSeq NM_015599.2); this is
predicted to be disease causing (deleterious according to
MutationTaster and PolyPhen but tolerated according to
SIFT). The affected amino acid is moderately conserved
merican Journal of Human Genetics 95, 96–107, July 3, 2014 101
Figure 3. Homology Model of Human PGM3(A) Structural overview of human PGM3. The three amino acidsAsp239 (D239), Asn246 (N246), and Gln451 (Q451) are coloredred and depicted in ball-and-stick representation. The greensphere represents the magnesium (Mg) ion.(B–D) Close-up view of the interaction between the altered aminoacids and their surroundings. (B) Interaction between Asp239(D239) and Arg222 (R222). (C) Interactions between Asn246(N246) and the active serine loop and between Asn246 and themetal-binding loop. (D) The environment around amino acidGln451 (Q451). The substrate GlcNAc-6-P is shown in magenta.Other amino acids are as follows: S64, Ser64; H65, His65; N66,Asn66; and P67, Pro67.
across species (Figure S2). Family segregation by Sanger
sequencing confirmed that the variants were trans-alleles,
fulfilling Mendelian expectations (Figure 2C).
The crystalline structure of human PGM3 has not been
determined; therefore, we performed structural analysis
of the three amino acid substitutions (p.Asp239His,
p.Asn246Ser, and p.Gln451Arg) in human PGM3 (UniProt
ID O95394) on the basis of a homology model made by
SWISS-MODEL38 and provided by the Protein Model
Portal.39 The homology model of human PGM3 (Figure 3)
is based on the experimental X-ray structure of Aspergillus
fumigatus Pgm340 (Protein Data Bank [PDB] ID 4BJU),
which has ~50% sequence identity with the human pro-
tein. A model of PGM3 in complex with the substrate
GlcNAc-6-P was obtained by superposition with the
Candida albicans (C. albicans) Pgm3-GlcNAc-6-P com-
plex41 (PDB ID 2DKC). As for the other members of this
superfamily, PGM3 consists of four domains (Figure 3A),
and the active site of the protein is made up of one loop
from each of the four: the serine loop in domain 1, the
metal-binding loop in domain 2, the sugar-binding loop
102 The American Journal of Human Genetics 95, 96–107, July 3, 201
in domain 3, and the phosphate-binding loop in domain 4.
Asn246 and Asp239 are both located on the same loop in
domain 2 and are positioned close to the active site (Fig-
ures 3A–3C). Asn246 is directed toward the active site
and most likely stabilizes both the serine loop and the
metal-binding loop by two and one hydrogen bond,
respectively (Figure 3C). The substitution of this amino
acid with serine is predicted to abolish these intramolecu-
lar interactions and result in an overly flexible active site.
Furthermore, the model suggests that Asp239 participates
in a hydrogen bond with Arg222, which also is likely to
have a stabilizing effect (Figure 3B). Finally, Gln451 is
located in domain 4 and is directed toward the substrate
GlcNAc-6-P (Figure 3D). If residue Gln451 is changed to
an arginine, both the alteration in electrostatic charge,
from neutral to positive, and the longer side chain will
most likely influence the interaction between the protein
and its substrate.
Clinical screening for disorders of glycosylation did not
show abnormalities. Capillary-zone electrophoresis (CZE)
of serum, taken both before and after HSCT, demonstrated
normal serum transferrin in the reported female child (P1)
in a sample collected at 1 month of age (before immuno-
globulin substitution and RBC transfusion), at 2 months
of age, and after HSCT. Likewise, mass spectrometry anal-
ysis showed normal serum transferrin and apolipopro-
tein-CIII (apoCIII) glycoforms from all serum samples
collected in this child. For CZE analysis, the serum trans-
ferrin sialoform pattern was examined by capillary electro-
phoresis (P/ACE-SYSTEM MDQ). Serum transferrin and
apoCIII glycoforms were analyzed by simultaneous online
immunoaffinity chromatography electrospray ionization
mass spectrometry (SCIEX API4000 tandem mass spec-
trometer with Turbo V spray source).31 Total serum glycan
was analyzed after the samples were denatured and di-
gested to release the N-glycans prior to clean up. After clean
up, and permethylation with iodomethane, N-glycans
were analyzed with MALDI-TOF mass spectrometry.42,43
Serum T antigen (T) and sialylated T antigen (ST) were
quantified with MALDI-TOF. The T/ST ratios were normal
in all P1’s serum samples (Table S6). In the male Mexican-
American boy (P2), N-glycan transferrin analysis was qual-
itatively and quantitatively normal, and the apoCIII profile
and T/ST ratio were normal in serum collected after HSCT.
Serum was not available for glycosylation testing prior
to HSCT. Finally, for the most severely affected male (P3),
CZE of serum sample from 1 and 4 months of age demon-
strated normal N-glycosylation of transferrin.
In order to examine the effect of the p.Asn246Ser,
p.Asp239His, and p.Gln451Arg substitutions on PGM3
activity, we generated recombinant clones containing
the mutant alleles, expressed them in Escherichia coli
(E. coli), and subsequently purified the recombinant
PGM3 proteins. Indeed, all three proteins demonstrated
reduced phosphate-group transfer from position GlcNAc-
6-P to GlcNAc-1-P, reflected by reduced substrate con-
sumption (Table 2). The p.Asn246Ser substitution was
4
Table 2. PGM3 Activities
PGM3No. ofExperiments Mean (%) SEM (%)
Wild-type 5 100 0.0
p.Asn246Ser 5 1 8.1
p.Asp239His 5 59 11.8
p.Gln451Arg 5 50 10.0
PGM3 activities were assayed in a 200 ml standard reaction mixture containing50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 10% (v/v) glycerol, 200 mM GlcNAc-6-P substrate, and 50 mg of the indicated PGM3 at 30�C for 10 min. Reactionswere then inactivated by incubation at 80�C for 5 min. The effect of the aminoacid substitutions on PGM3 was tested by mass spectrometry in ‘‘multiple re-action monitoring’’ mode; the transition from the molecular ion (m/z 300)to a fragment specific to the substrate (GlcNAc-6-P) (m/z 138) was used formeasuring substrate consumption in relation to that of the wild-type. Datawere calculated from five independent experiments.
completely inactive even with 4-fold higher enzyme
concentrations.
Our experiments in conjunction with recent reports
document that that PGM3 mutations in humans cause a
phenotypically variable CDG.11,12 From extensive clinical
studies in three families reported here, the phenotype of
PGM3-CDG might include dysmorphic facial features,
cognitive impairment, leukopenia, T�B�NKþSCID and
neutropenia, and skeletal dysplasia. Our subjects had
more pronounced immunodeficiency, including severe
neutropenia (Tables S1–S3). Only P2 had elevated serum
IgE levels, whereas the affected individuals reported by
the two other groups all had hyper IgE syndrome, and
none of them progressed to bone marrow failure.11,12
Other disorders of glycosylation are also known to cause
a wide phenotypic spectrum, from mild to severe pheno-
types.44 Both genotype-phenotype correlation and other
modifying factors, including immunodeficiency, intellec-
tual disabilities, and skeletal dysplasia, might contribute
to the variety of phenotypes observed in CDGs. The differ-
ences observed between our three subjects might be related
to specific genotypes, i.e., p.Asn246Ser causes a more
pronounced block of enzyme activity in comparison to
hypomorphic variants such as p.Asp239His, as demon-
strated by our mutant model testing. The data from enzy-
matic activity of p.Asn246Ser and p.Asp239His correspond
with the findings in the Pgm3 mouse models, where loss-
of-function mutants confer more severe outcomes.3 How
and why p.Gln451Arg conferred substrate consumption
similar to that of p.Asp239His in model testing, whereas
P3 had the same severe skeletal phenotype as P1, will
require further investigation. Our protein homology
model predicts that p.Gln451Arg, the variant detected in
P3, might alter the substrate affinity, and high-affinity
binding of substrate might completely block the conver-
sion to product GlcNAc-1-P, even if our mutant model
testing showed half enzyme activity with regard to sub-
strate consumption. Of the six variants reported by the
other two research groups, five (p.Leu83Ser, p.Asp325Glu,
p.Asp502Tyr, p.Glu529Gln, and p.Glu340del) were dem-
The A
onstrated to be hypomorphic with sustained enzyme
activity.11,12
Other in vivo modifying factors, such as infections and
autoinflammatory changes, might also have contributed
to the severity and phenotypic spectrum observed in the
PGM3-CDG individuals. Compared to the other disorders
of carbohydrate metabolism associated with immunodefi-
ciency, such as SLC37A4-CDG (severe congenital neutro-
penia type 4 [MIM 612541], caused by mutations in
G6PC3 [MIM 611045]) and SLC35C1-CDG (CDG type IIc
[MIM 266265], caused by mutations in SLC35C1 [MIM
605881]), the involvement of lymphocytes in addition
to neutrophils demonstrates expanded immunological
impact. Unlike ALG12-CDG (CDG type Ig [MIM
607143])-affected individuals, who have B cell deficiency
and a defect in N-glycosylation, our PGM3-CDG probands
did not show any alterations in serum transferrin or
apoCIII profiles (data not shown) or T/ST ratios (Table
S6), leading us to conclude that N- and O-glycosylation
in the liver for transferrin and apoCIII is not affected by
the PGM3 defect. Thus, this might reflect the tissue- and
organ-specific functions of PGM3, as well as a critical role
for certain hematopoietic lineages. Another explanation
for the normal apoCIII profile is that it only tests core 1
O-glycosylation, which is not dependent on UDP-GlcNAc.
The first monosaccharide attached in the synthesis of
O-linked glycans is GalNAc. A core 1 structure is generated
by the addition of galactose. A core 2 structure is generated
by the addition of GlcNAc to the GalNAc of the core 1
structure. Core 3 and core 4 structures are generated by
the addition of a single GlcNAc to the original GalNAc
and by the addition of a second GlcNAc to the core 3 struc-
ture, respectively. Hence, formation of cores 2–4 is depen-
dent on UDP-GlcNAc. Reduced levels of UDP-GlcNAc in
C. albicans do not block N-glycosylation but might cause
reduced and shorter glycosylation branching,45,46 which
wewere unable to demonstrate in the sera from affected in-
dividuals. Both Sassi et al. and Zhang et al. reported a
normal transferrin pattern in their subjects with PGM3
mutations. Zhang et al. reported high serum T antigen
levels and elevated T/ST ratios, but we could not demon-
strate the same.12 Interestingly, Sassi et al. detected reduced
bi-, tri-, and tetra-antennary N-glycan branching in
leukocytes from affected individuals and suggested a geno-
type-phenotype correlation on the basis of their study sub-
jects with three different mutations.11 Homozygotes for
p.Glu340del had the most altered glycosylation branching
pattern. Our study subjects had either received bone
marrow transplantation or died before the CGD diagnosis
was made, and no leukocytes were available for further
functional studies.
The skeletal abnormalities, including X-ray findings, in
two children (P1 and P3) resembled DBQD; however, the
linear growth was less restricted in the subject with trans-
planted PGM3 deficiency than has previously been re-
ported in classical DBQD.13 Unlike in classical DBQD,
bone age was not advanced in P3. Another difference of
merican Journal of Human Genetics 95, 96–107, July 3, 2014 103
note in these children with PGM3-CDG is the extra ossifi-
cation center placed proximal rather than distal to the sec-
ond metacarpal bone, as is typical in DBQD type 1. The
similarities between the skeletal dysplasia in CANT1 defi-
ciency and PGM3 deficiency might be due to their com-
mon effect on the proteoglycan synthesis. Others have
shown that CANT1 deficiency causes reduced availability
of UDP-GlcNAc and thereby reduced glycosyltransferase
activities.13 Proteoglycan synthesis is also disrupted in
DBQD2 as a result of xylosyltransferase 1 (XYLT1 [MIM
608124]) deficiency.18 Occurrences of skeletal dysplasia
have been reported in other CDGs, such as PMM2-CDG
(CDG type Ia [MIM 212065], caused by mutations in
PMM2 [MIM 601785]),9,10,47 ALG6-CDG (CDG type Ic
[MIM 603147], caused by mutations in ALG6 [MIM
604566]),48 ALG12-CDG (caused by mutations in ALG12
[MIM 607144]),49 COG1-CDG (CDG type IIg [MIM
611209], caused by mutations in COG1 [MIM 606973]),50
COG7-CDG (CDG type IIe [MIM 608779], caused bymuta-
tions in COG7 [MIM 606978]),51,52 and TMEM165-CDG
(CDG type IIk [MIM 614727], caused by mutations in
TMEM165 [MIM 614726]).53 The kyphoscoliosis noted in
one-quarter of the individuals with PGM3 mutations re-
ported by Sassi et al. and Zhang et al. might represent
the milder spectrum of the PGM3-related skeletal
dysplasia.11,12 Zhang et al. reported decreased PGM3 cata-
lytic activity and reduced intracellular UDP-GlcNAc levels
in fibroblasts from three of their PGM3-CDG-affected indi-
viduals. The severity and variability of the phenotypes
observed between the persons with different PGM3 muta-
tions might be directly correlated with UDP-GlcNAc levels.
A genotype-phenotype correlation has been shown in
mice: mice compound heterozygous for mutant Pgm3
alleles with a radical effect on the protein or a loss-of-func-
tion allele are more severely affected than mice homozy-
gous for a mild mutant allele.3 Pgm3�/� mice die in early
embryogenesis and show a dramatic reduction in UDP-
GlcNAc. With defects restricted to the salivary glands,
pancreas, testis, kidney, and hematopoietic cells, mice
with partial PGM3 activity are viable. Mice with partial
PGM3 deficiency have profound B cell defects (a normal
number of naive B cells but a loss of mature B cells), an
increased CD4/CD8 ratio, and mild anemia and thrombo-
cytopenia but normal numbers of neutrophils, eosino-
phils, and monocytes.3 Whether PGM3-CDG individuals
presenting in infancy with immunodeficiency will develop
the same symptoms as mice (such as male infertility,
exocrine pancreatic insufficiency, and glomerulonephritis)
is questionable. For instance, neutropenia was a profound
hallmark in these PGM3-CDG-affected children, but not in
the mouse model or the described cohorts of older individ-
uals. The potential neurological abnormalities in PGM3-
CDG remain to be defined, and whether it is present at
birth and/or progressive with age is unclear. Interestingly,
brain microglia are derived from hematopoietic precursors
of mesoderm origin and can be replaced by blood-derived
monocytes. It is currently not clear which of the other dis-
104 The American Journal of Human Genetics 95, 96–107, July 3, 201
ease manifestations, in addition to the hematological
defects, HSCT rescues. Skeletal dysplasia has not been re-
ported in mouse models but is perhaps reflected by the
growth restriction observed in mutant mice.3 Genetic
studies, including maternal and zygotic loss-of-function
screens in Drosophila, have revealed that mutations in
nesthocker (nst), the fruit fly’s PGM3 ortholog, block meso-
dermal and tracheal development. This is interesting
because the mesoderm gives rise to bone, cartilage, and
hematopoietic precursors, including microglia. Embryos
lacking maternal and zygotic nst products show low
amounts of intracellular UDP-GlcNAc and defective
O-GlcNAcylation of fibroblast growth factor receptor
(FGFR)-specific adaptor protein, which impairs FGFR-
dependent migration of mesodermal and tracheal cells.54
The identification of a role for nst in FGFR signaling is
compelling in the light of the skeletal dysplasia observed
in our affected subjects.
Some CDGs are treatable with supplements of substrates
for the defective glycosylation pathway. For instance, indi-
viduals deficient in mannose-6-phosphate isomerase (Fru-
6-P to Man-6-P conversion) lack sufficient Man-6-P for
complete physiologic N-glycosylation, and daily supple-
ments of mannose can correct this glycosylation defi-
ciency.55 Given that PGM3 catalyzes an important step
in the synthesis of glycans, it is possible that substitution
of a compound that enhances the enzymatic reaction per-
formed by PGM3 (GlcNAc-6-P to GlcNAc-1-P conversion)
or bypasses the block, such as N-acetyl-galactosamine
(GalNAc; Figure S1), might ameliorate the pathologic
phenotype. For example, supplemental therapy with
galactose was recently found to be effective for the homol-
ogous disease PGM1-CDG (Figure S1).44 However, treat-
ment with HSCT is lifesaving because it corrects the
immunodeficiency, and two of our described children
were successfully cured, whereas the others (P3 and the
two affected brothers of P2) died of infectious complica-
tions (presumably resulting from immunodeficiency)
before transplantation was initiated.
We provide evidence that PGM3 mutations in children
can cause a CDG with leukopenia, skeletal dysplasia, dys-
morphic facial features, and cognitive impairment. The
immunological abnormality is further defined as severe
neutropenia, T and B cell lymphopenia, and progression
to complete bone marrow failure. Genotype together with
other modifying factors might contribute to the pheno-
typic variation and disease severity observed in this CDG,
as is the case in other glycosylation disorders. Our study
demonstrates PGM3-CDG as a severe infancy-onset immu-
nodeficiency in which HSCT is lifesaving and defines the
power of WES in gene discoveries for rare disorders.
Supplemental Data
Supplemental Data include three figures and six tables and can be
found with this article online at http://dx.doi.org/10.1016/j.ajhg.
2014.05.007.
4
Acknowledgments
The authors are grateful to the families for their participation
in this study. Special thanks go to Eric A. Smith for his technical
contributions. The study was performed at the Baylor-Johns
Hopkins Center for Mendelian Genomics, funded by the NIH
National Human Genome Research Institute (U54HG006542
and U54HG003273). The Centers for Mendelian Genomics repre-
sents a cooperative, international research effort to determine the
genetic cause(s) of Mendelian disorders. The German branch of
this study was supported by a grant from the German Federal Min-
istry of Education and Research to E.L. (FACE consortium TP1,
01GM1109A). E.L. was also supported by the European Commis-
sion Seventh Framework Programme (the SYBIL consortium, grant
agreement 602300). P.H.B. was supported by the South-Eastern
Norway Regional Health Authority’s Technology Platform for
Structural Biology and Bioinformatics (grant 2012085). J.R.L.
holds stock ownership in 23andMe Inc. and is a coinventor on
multiple United States and European patents related to molecular
diagnostics. The Department of Molecular and HumanGenetics at
Baylor College of Medicine derives revenue from molecular ge-
netic testing offered in the Medical Genetics Laboratories.
Received: January 14, 2014
Accepted: May 16, 2014
Published: June 12, 2014
Web Resources
The URLs for data presented herein are as follows:
1000 Genomes Browser, http://browser.1000genomes.org/index.
html/
Arteriosclerosis Risk in Communities (ARIC) Study, http://www2.
cscc.unc.edu/aric/
Baylor-Hopkins Center for Mendelian Genomics, https://
mendeliangenomics.org/
Centers for Mendelian Genomics, http://www.mendelian.org/
dbGaP, http://www.ncbi.nlm.nih.gov/gap/
dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/
Enzyme Nomenclature, http://www.chem.qmul.ac.uk/iubmb/
enzyme/
HUGO Gene Nomenclature Committee (HGNC), http://www.
genenames.org/
The Human Protein Atlas, PGM3, http://www.proteinatlas.org/
ENSG00000013375/tissue/
Integrative Genomics Viewer (IGV), http://www.broadinstitute.
org/igv/
Likelihood-ratio test, http://www.genetics.wustl.edu/jflab/lrt_
query.html
Medical Genetics Laboratories at Baylor College of Medicine,
http://www.bcm.edu/geneticlabs/
MutationTaster, http://www.mutationtaster.org
NHLBI Exome Sequencing Project (ESP) Exome Variant Server,
http://evs.gs.washington.edu/EVS/
Online Mendelian Inheritance in Man (OMIM), http://www.
omim.org/
PhenoDB, https://mendeliangenomics.org/
PhyloP, http://compgen.bscb.cornell.edu/phast/
PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/
Protein Data Bank (PDB), http://www.rcsb.org/pdb/home/
home.do
RefSeq, http://www.ncbi.nlm.nih.gov/RefSeq
The A
SIFT, http://sift.jcvi.org
UCSC Genome Browser, http://genome.ucsc.edu/
UniProt, http://www.uniprot.org/
WHOChild Growth Standards, http://www.who.int/childgrowth/
en/
Accession Numbers
The PhenoDB accession numbers for the phenotype data reported
in this paper are BH3596 in P1 and BH2704 in P2.
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The American Journal of Human Genetics, Volume 95
Supplemental Data
PGM3 Mutations Cause a Congenital Disorder
of Glycosylation with Severe Immunodeficiency
and Skeletal Dysplasia
Asbjørg Stray-Pedersen, Paul H. Backe, Hanne S. Sorte, Lars Mørkrid, Niti Y. Chokshi,
Hans Christian Erichsen, Tomasz Gambin, Katja B.P. Elgstøen, Magnar Bjørås, Marcin
W. Wlodarski, Marcus Krüger, Shalini N. Jhangiani, Donna M. Muzny, Ankita Patel,
Kimiyo M. Raymond, Ghadir S. Sasa, Robert A. Krance, Caridad A. Martinez, Shirley M.
Abraham, Carsten Speckmann, Stephan Ehl, Patricia Hall, Lisa R. Forbes, Else
Merckoll, Jostein Westvik, Gen Nishimura, Cecilie F. Rustad, Tore G. Abrahamsen, Arild
Rønnestad, Liv T. Osnes, Torstein Egeland, Olaug K. Rødningen, Christine R. Beck,
Baylor-Johns Hopkins Center for Mendelian Genomics, Eric A. Boerwinkle, Richard A.
Gibbs, James R. Lupski, Jordan S. Orange, Ekkehart Lausch, and I. Celine Hanson
Figure S1
Man$Glc$
Glycosyla)on$Disorder$
Fru262PO4$NH3$Glutamine$
Glutamate$
Glucosamine262PO4$AcetylCoA$
CoA$
GlcNAc262PO4$
GlcNAc212PO4$
ADP$
ATP$
Glucosamine$
GlcNAc$
ADP$
ATP$
UTP$
PPi$
UDP2GlcNAc$
UDP$
N2Acetylmannosamine$
ManNAc262PO4$PEP$
UDP2GalNAc$
Neu5Ac$CTP$
CMP2Neu5Ac$NADPH$
Fuc$
Fuc212PO4$
GTP$
GDP2Fuc$
NADP+$
GDP242keto262$deoxygalactose$
GDP242keto262$deoxymannose$
Dol2P2Man$
Dol2P$GDP2Man$
Man212PO4$Man262PO4$PEP$
KDN$CTP$
CMP2KDN$
PPi$
Pi$
Pi$ PPi$
GDP$
ATP$
ADP$
N2Acetylneuraminic262PO4$
KDN292PO4$NADP+$
NADPH$
NADPH$
PPi$
ADP$
ATP$
GTP$
PPi$
PPi$
NADP+$
CMP2Neu5Gc$
GalNAc212PO4$GalNAc$ADP$ATP$ UTP$
Glc262PO4$
ADP$
ATP$
Glc212PO4$UTP$
PPi$
UDP2Glc$
UDP2$Gal$Dol2P2Glc$
Dol2P$
UDP$NADH$ NAD$
6PG$
NADPH$ NADP+$
UDP2GlcA$
UDP2Xylose$
CO2$
PGM1$
PGM3$
Glc212PO4$
UDP2Glc$
Gal212PO4$Gal$
ADP$ATP$ ATP$
ADP$
Pi$
Glc262PO4$ Glc$+$PO4$Endoplasmic$Re)culum$
G6PT1$ G6PC3$
CP$
CP$
CP$
CP$
CP$
Pi$
PPi$
UTP$
PMM2$
DPM1$
GNE$
GNE$
GALT$
GALE$
GALK1$
MPI$
GFAT$
Figure S1. Monosaccharide metabolism in mammals and human glycosylation disorders. Biosynthesis and interconversion of monosaccharides. Figure modified from the figures published by Freeze, H.H.,1,2 and presented here with permission from Freeze, H.H and J. Biol. Chem. with copyright © 2013,
by the American Society for Biochemistry and Molecular Biology.
Enzymes marked red, nucleotide sugars (activated forms of monosaccharides) in light blue boxes, and monosaccharide substrates with proven treatment
effects or potential disease modifying effects are bold and marked blue. Symbols and abbreviations: CP, Control points; Dol, Dolichol; PDM1, Dolichyl-
phosphate mannosyltransferase 1 (EC 2.4.1.83); Fuc, Fucose; G6PT1, Glucose-6-phosphate translocase subunit 1; G6PC3, Glucose-6-phosphatase 3 (EC
3.1.3.9); Gal, Galactose; GalNAc, N-acetyl-galactosamine; GALE, UDP-galactose-4-epimerase (EC 5.1.3.2); GALK1, Galactokinase 1 (EC 2.7.1.6); GALT,
Galactose 1 phosphate uridyltransferase (EC 2.7.7.12); Glc, Glucose; GlcA, Glucoronic acid; GlcNAc, N-acetyl-glucosamine; GFPT1 (GFAT), Glutamine
fructose 6-amidotransferase (EC 2.6.1.16); GNE, UDP-N-acetyl-glucosamine 2-epimerase/N-acetyl-mannosamine kinase (EC 3.2.1.183/EC 2.7.1.60); KDN,
2-keto-3-deoxy-D-glycero-D-galactonononic acid (synonym 2-Keto-3-deoxynononic acid); Man, Mannose; ManNAc, N-acetyl-mannosamine; MPI,
Mannosephosphate isomerase (EC 5.3.1.8); Neu5Ac, N-acetyl-neuraminic acid; Neu5Gc, N-glycolyl-neuraminic acid; PEP, Phosphoenolpyruvate; PGM1,
Phosphoglucomutase 1 (EC 5.4.2.2); PGM3, Phosphoglucomutase 3 (EC 5.4.2.3); PMM2, Phosphomannomutase 2 (EC 5.4.2.8); 6PG, 6-phosphogluconate.
Figure S2
p.D239H p.N246S p.N246Kfs*7
p.Q451R
Figure S2. Alignment in different species. Sequences of PGM3 homologs were aligned using the alignment feature of UniProt using the aligner clustalo. The alignment shows that all three reported
disease-causing missense mutations are highly conserved in the selected species, p.N246 completely conserved through yeast.
Figure S3
A PGM3 GPRC6A PGM3
B
1.2 Mb deletion
UBE3D PGM3 ME1 SNAP91
Figure S3. Genomic regions with absence of heterozygosity (AOH) predicted from the exome data, with focus on chromosome 6 (lower panel). A) P1, B) P2. Blue dots and lines indicate AOH. Lowest panel: Prediction of copy number variant (CNV) deletion from exome data in P2. CoNIFER3 was used
as a bioinformatics tool comparing the P2 data (red) with other samples (black).
Footnote Figure S3 A lower panel: Regarding AOH and GPRC6A, P1 has skeletal abnormalities in addition to immunodeficiency, which were not observed in
P2. The initial hypothesis was that the skeletal dysplasia was an additional trait under the assumption of a potential digenic model. A homozygous GPRC6A
variant (Table S5) detected in WES in P1 was regarded as a candidate, since reduced bone mass and impaired osteoblast function is reported in GPRC6A
null mice.4 The analysis of P3, who also had the same skeletal phenotype, allowed us to exclude GPRC6A as a contributing gene because this child was
proven wild-type for GPRC6A, and thus conclude that the skeletal phenotype is indeed due to autosomal recessive inherited mutations in PGM3.
rela
tivePGM3
mRN
A
CTL
patien
t 30.0
0.5
1.0
1.5
Control Patient 3
Figure S4
Figure S4. Quantitative real-time PCR of PGM3 mRNA
Amount of PGM3 mRNA detected in P3’s blood cells relative to control sample. Mean ± SEM of column Control: 1,040 ± 0.03055 (N=3), Mean ± SEM of
column P3: 3617 ± 0.04262 (N=3), Difference between means 0,6783 ± 0.05244, p = 0.002, two-tailed unpaired t test. Primer sequences in Table S5
Abbreviation: SEM, Standard Error of the Mean.
Table S1. Immunological evaluations and neutrophil counts P1
4 weeks 5 weeks 6 weeks 7 weeks 8 weeks 9 weeks 10 weeks 11 weeks 12 weeks 13 weeks Mean of 4-13 weeks
1 year of age, (6 months
post HSCT)
Age matched references* 0-3 months
Age matched references*
1-2 years B CD19+ (cells/microL) 418 380 489 694 477 NA NA NA NA 398 476 1540 300-2000 720-2600 T
CD3+ (cells/microL) 367 325 623 734 664 367 325 623 734 752 551 3316 2500-5500 2100-6200 CD4+ (cells/microL) 193 204 435 492 499 193 204 435 492 592 374 2333 1600-4000 1300-3400 CD8+ (cells/microL) 67 52 75 91 57 NA NA NA NA 55 66 805 560-1700 620-2000
NK CD16/CD56+ (cells/microL) 327 214 227 355 152 NA NA NA NA 179 242 411 170-1100 180-920
Neutrophils (cells/microL) NA 300 300 200 300 200 1100 600 500 300 <500 7100 1500-8000 1500-8000
*Normal vales (10th-90th percentiles) for lymphocytes obtained from Shearer et al JACI Nov 2003.5
Abbreviations: NA, Not available
Table S2. Immunological evaluations and neutrophil counts P2
3 months 14 months 27 months 35 months 4¼ years 5 years 6 years
(2 months post HSCT)
Age matched references* 0-3 months
Age matched references*
1-2 years
Age matched references*
2-6 years
Age matched references* 6-12 years
B CD19+ (cells/microL) 114 32 2 3 1 4 126 300-2000 720-2600 390-1400 270-860 T
CD3+ (cells/microL) 318 352 312 326 97 311 1154 2500-5500 2100-6200 1400-3700 1200-2600 CD4+ (cells/microL) 253 293 239 217 65 50 865 1600-4000 1300-3400 700-2200 650-1500 CD8+ (cells/microL) 30 32 61 103 28 243 284 560-1700 620-2000 490-1300 370-1100
NK CD16/CD56+ (cells/microL) 144 123 111 69 31 17 137 170-1100 180-920 130-720 100-480
Neutrophils (cells/microL) 700 NA 190 230 450 450 1660 1500-8000 1500-8000 1500-8000 1500-8000
*Normal vales (10th-90th percentiles) for lymphocytes obtained from Shearer et al JACI Nov 2003.5
Abbreviations: NA, Not available
Table S3. Immunological evaluations and neutrophil counts P3
Day 5 3 weeks
3 months (20 µg/kg G-CSF)
4.5 months (20 µg/kg G-CSF)
Age matched references* 0-3 months
Age matched references* 3-6 months
B CD19+ (cells/microL) 138 764 228 198 300-2000 430-3000 T
CD3+ (cells/microL) 478 1190 347 455 2500-5500 2500-5600 CD4+ (cells/microL) 277 699 315 365 1600-4000 1800-4000 CD8+ (cells/microL) 71 188 11 42 560-1700 590-1600
NK CD16/CD56+ (cells/microL) 150 688 89 399 170-1100 170-830
Neutrophils (cells/microL) 0 0 898 1066 1500-8000 1500-8000
*Normal vales (10th-90th percentiles) for lymphocytes obtained from Shearer et al JACI Nov 2003.5
Abbreviations: NA, Not available
Table S4. Whole exome sequencing statistical summary for P1-3
Individual P1 P2 P3 Total captured regions size 52Mb 52Mb 52Mb
% of captured regions with coverage>10 93.78 91.75 92.81 Average coverage 149x 96x 98x
% of bases covered by 1x 96.89 96.09 96.18 % of target hits 98.5 98.32 98.29
Total numbers of SNPs 99,531 112,575 98,751 Total numbers of INDELs 8,533 12,398 8,945
N rare* homozygous (n confirmed by Sanger) 15 (2) 20 (1) 18 (3)
N rare* compound heterozygous (n confirmed by Sanger) 11 pairs (0) 10 pairs (0) 8 pairs (1)
N X linked (n confirmed by Sanger) 20 het,1 hom (0) 9 hemi (0) 12 hemi (0) N shared genes with deleterious variants
(n confirmed by Sanger) 1 (1) 1 (1) 1 (1)
* Rare defined as < 0.003 in ESP, ARIC, and BCM-CMG.
Abbreviations: ESP, NHLBI GO Exome Sequencing Project (ESP) server; ARIC, Arterosclerosis Risk In Communities contains WES results from ~4000
individuals; BCM-CMG, in-house database for Baylor College of Medicine-Center for Mendelian Genomics >200 individuals.
Table S5. Sequence of the primers used for Sanger verification, and qPCR of the detected variants.
Primer name RefSeq transcript Primer sequence (5’-3’) Variants and regions of interest
PGM3_ex6F NM_015599.2
CCTTTGTAGGCTTCTTGCAGTGG [chr6.hg19:g.83891505T>C, c.737A>G, p.Asn246Ser]; [chr6.hg19:g.83891505dupT; c.737dupA, p.Asn246Lysfs*7]; [chr6.hg19:g.83891527C>G; c.715G>C, p.Asp239His] PGM3_ex6R TGTGGCAGAGCCAGGAATAGC
PGM3 qPCR rqf NM_015599.2 CACATGAAGTGAGCTTGGCA PGM3 exon 13 PGM3 qPCR rqr CAATCCAGTAGGCTGCATTG PGM3_ex11F NM_015599.2 ATTTGTTTCCCCATTTGCAG [chr6.hg19:g.83881669T>C; c.1352A>G; p.Gln451Arg] PGM3_ex11R TGTCAGTGAGATATAATGAGAATTGG GPRC6A_ex6F NM_148963.2 GCATTTGGCACCATGCTGGGC [chr6.hg19:g.117113762_117113766delinsGGTAATTTCCT;
c.2320_2324delinsAGGAAATTACC;p.Lys774_Tyr775delinsArgLysLeuPro] GPRC6A_ex6 CCAGTGAAGCAGGACTCAGGGC
Table S6. Matrix-assisted laser desorption ionization-time of flight mass spectrometry quantification of O-linked glycan profiles in two individuals with CDG-PGM3
Individual Age Therapy T antigen (micromol/L)
Sialyl-T antigen (micromol/L)
Ratio T/ST
P1 1m Pre HSCT 0.55 12.2 0.05 P1 2m Pre HSCT 0.57 17.7 0.03 P1 2y Post HSCT 0.53 17.7 0.03 P2 6y Post HSCT 0.55 16.3 0.03
Reference ranges 0.22 - 1.4 11.7 - 31.4 0 - 0.06 Symbols and abbreviations: HSCT, Hematopoietic stem cell transplantation; IgG, Immunoglobulin G, m, months; T/ST, T antigen/sialyated-T antigen; y, years.
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