PGM3 mutations cause a congenital disorder of glycosylation with severe immunodeficiency and skeletal dysplasia.

Please cite this article in press as: Stray-Pedersen et al., PGM3 Mutations Cause a Congenital Disorder of Glycosylation with Severe Immunodeficiency and Skeletal Dysplasia, The American Journal of Human Genetics (2014), http://dx.doi.org/10.1016/j.ajhg.2014.05.007

REPORT PGM3 Mutations Cause a Congenital Disorder of Glycosylation with Severe Immunodeficiency and 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 Bjøra˚s,5,7 ¨ ger,10 Shalini N. Jhangiani,1,11 Donna M. Muzny,1,11 Ankita Patel,12 Marcin W. Wlodarski,10 Marcus Kru 13 Kimiyo M. Raymond, 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-phosphate 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-associated 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. However, 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 bone marrow from matched 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 modification essential for the proper functioning of a broad spectrum of proteins and lipids. In this process, glycans are constructed from a cellular pool of activated monosaccharides, the sugar nucleotides. The structural diversity of the glycans ensures specific and selective molecular interactions. 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-galactosamine (UDP-GalNAc), and UDP-N-acetyl-glucosamine (UDP-GlcNAc) (Figure S1, available online). Glycans are

attached to proteins via a nitrogen atom of an asparagine (N-linked glycan) or an oxygen atom of a serine or threonine (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 disorders 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,

1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; 2Center for Human Immunobiology, Texas Children’s Hospital, Houston, TX 77030, USA; 3Section of Immunology, Allergy, and Rheumatology, Department of Pediatrics, Baylor College of Medicine and Texas Children’s Hospital, Houston, TX 77030, USA; 4Department of Medical Genetics, Oslo University Hospital, 0424 Oslo, Norway; 5Department of Microbiology, Oslo University Hospital, 0424 Oslo, Norway; 6Department of Medical Biochemistry, Oslo University Hospital, 0424 Oslo, Norway; 7Institute of Clinical Medicine, University of Oslo, 0318 Oslo, Norway; 8Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA; 9Department of Pediatrics, Oslo University Hospital, 0424 Oslo, Norway; 10Department of Pediatrics, Freiburg University Hospital, 79106 Freiburg, Germany; 11Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA; 12Medical Genetics Laboratories, Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; 13Department of Laboratory Medicine and Pathology, Mayo College of Medicine, Rochester, MN 55905, USA; 14Center for Cell and Gene Therapy and Texas Children’s Cancer and Hematology Centers, Baylor College of Medicine and Texas Children’s Hospital, Houston, TX 77030, USA; 15Pediatric Hematology Oncology, University of New Mexico, Albuquerque, NM 87106, USA; 16Emory Genetics Laboratory, Department of Human Genetics, Emory University, Decatur, GA 30033, USA; 17Department of Radiology, Oslo University Hospital, 0424 Oslo, Norway; 18 Department of Pediatric Imaging, Tokyo Metropolitan Children’s Medical Center, 2-8-29 Musashidai, Fuchu, Tokyo 183-8561, Japan; 19Department of Immunology and Transfusion Medicine, Oslo University Hospital, 0424 Oslo, Norway; 20Human Genetics Center, University of Texas Health Science Center, Houston, TX 77030, USA 21 These 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. Ó2014 by The American Society of Human Genetics. All rights reserved.

The American Journal of Human Genetics 95, 1–12, July 3, 2014 1

Please cite this article in press as: Stray-Pedersen et al., PGM3 Mutations Cause a Congenital Disorder of Glycosylation with Severe Immunodeficiency and Skeletal Dysplasia, The American Journal of Human Genetics (2014), http://dx.doi.org/10.1016/j.ajhg.2014.05.007 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 months and 7 months old) with a similar disease, one additional older healthy sibling

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

Follow-up length

73 cm (2 cm < 2 ) at 18 m

120 cm (50 ) at 6 years

NA

Follow-up OFC

44 cm (5th) at 12 months

NA

NA

Skeletal dysplasia with short-limbed dwarfism, brachydactyly, and ‘‘monkeywrench’’ femora

þ

þ

Pectus carinatum

þ

þ

Dysmorphic facial features, downturned corners of mouth, midface hypoplasia, and micrognathia

þ

þ

Developmental delay and/or intellectual disabilities

þ

þ

Other

one evaluation for hydrocephalus, no shunt needed

seizures since 4 months, hydrocephalus verified, shunt implantation at 5 months

Age at onset of infections and/or immunodeficiency

birth

birth

birth

TBNKþ SCID

þ

þ

þ

Neutropenia

þ

þ

þ

nd

th

NA

Anemia

(þ)

þ

Thrombocytopenia

Splenomegaly

Recurrent respiratory infections, otitis media, 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, normal IgG and IgE

low IgM, normal IgG and IgA, high IgE (1,233–1,768 kU/l)

normal IgM, IgA, and IgE, low IgG from 3 months

Start age for antibiotics and antifungal therapy

birth

intermittent since birth, prophylactic since 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)

2

The American Journal of Human Genetics 95, 1–12, July 3, 2014


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) by Sanger 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, human leukocyte antigen; HSCT, hematopoietic stem cell transplantation; IgA, immunoglobulin A; IgE, immunoglobulin E; IgG, immunoglobulin G; IgM, immunoglobulin M; NA, not available; OFC, occipitofrontal circumference; RBC, red blood cell; SAG-M, saline-adenine-glucose-mannitol stored RBC unit, TBNKþSCID, severe combined immunodeficiency with a lack of T and B lymphocytes but presence of NK cells; and WES, whole-exome sequencing. a Age percentiles according to WHO Child Growth Standards.

proteoglycans, and glycolipids. Specifically, UDP-GlcNAc is incorporated into N-glycans, O-glycans, and glycosylphosphatidylinositol (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-phosphate (GlcNAc-1-P) (Figure S1).1,3 Homozygous knockout of Pgm3 in mice is embryonically lethal, whereas homozygous hypomorphic alleles cause trilineage cytopenias (anemia, thrombocytopenia, and leukopenia), ascribed to decreased UDP-GlcNAc.3 The human homolog, PGM3 (MIM 172100), synonymously designated phosphoacetylglucosamine mutase 1 (AGM1), is most abundantly expressed 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 phosphoglucomutase-encoding gene, PGM1 (MIM 171900), cause human PGM1-CDG (CDG type It [MIM 614921]; Figure S1), clinically characterized by growth retardation, hepatopathy, 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 infections, immunoglobulin E (IgE)-mediated disease, bronchiectasis, variable degrees of neurocognitive impairment, kyphoscoliosis, and CD4 or CD8 T cell lymphopenia.11,12 None of the described individuals had severe immune deficiency, bone marrow failure, or skeletal dysplasia.

We describe PGM3-CDG, a CDG detected by wholeexome 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 included 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 osteochondrodysplasia characterized by growth retardation, short extremities (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 malformation), 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 phalanx, and dislocation of the interphalangeal joints. Mutations in the gene encoding calcium-activated nucleotidase 1 (CANT1 [MIM 613165]), located at 17q25.3, have been reported in DBQD type 1, but affected individuals without detected CANT1 mutations suggest genetic heterogeneity.13–15 CANT1 functions in proteoglycan metabolism.16,17 Proteoglycan synthesis is also disrupted in DBQD type 2 as a result of a deficiency in xylosyltransferase 1 (XYLT1 [MIM 608124]).18 Severe combined immunodeficiency (SCID) or other types of congenital immunodeficiencies have not been reported in DBQD. Three unrelated children from distinct world populations—a female with Afghani parents (P1), a male with Mexican-American parentage (P2), and another male



Figure 1. Dysmorphic Features and Radiological Manifestations in P1 and P3 (A) P1 at 1 month of age. Note the short fingers and short long bones (rhizomelic) and the bell-shaped thorax with pectus carinatum. (B) P1 at 1 year of age. Note the dysmorphic facial features, including a downturned mouth, midface hypoplasia, and micrognathia. The pectus carinatum is still prominent, but body stature appears less disproportionate. The child is unable to sit by herself. (C and D) Neonatal photographs of P3 show strikingly similar dysmorphic features and physical findings. (E and F) Skull X-rays of P1 at 3 months of age demonstrate cranial Wormian bones (arrows). (G) A hand X-ray of P1 at 3 months of age shows the extra ossification center, the pseudoepiphysis (arrow), proximal to the second metacarpal bone. (H) A hip X-ray of the pelvic bones and femora of P1 at 3 months of age demonstrates the exaggerated trochanter minor (monkey-wrench appearance) of the proximal femur on both sides (arrows). (I–K) Neonatal radiographs of P3 demonstrate (I) no Wormian bones in the skull, (J) severe brachydactyly and phalangeal dislocations, but no advanced skeletal maturation or extra ossicles in the carpogram (also not present at 3 months), and (K) typical monkey-wrench morphology (arrows) of the proximal femora; there is metaphyseal flaring at the distal femoral ends. Specific parental releases were obtained from parents for the use of photographs (of P1 and P3) in this manuscript.

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 Universita¨tsklinikum 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 radiographically 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

4


(TBNKþSCID phenotype) and no anemia, thrombocytopenia, 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 shortening of tubular bones with brachydactyly, short metacarpal and metatarsal bones and phalanges, and pectus carinatum (Figure 1A). Dysmorphic facial features included 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


Figure 2. Pedigrees The families of P1 (A), P2 (B), and P3 (C) are shown with segregation of mutant alleles. Partial chromatograms of Sanger confirmation analysis of PGM3 mutations are shown only for the probands; arrows indicate respective nucleotide changes. For homozygous and hemizygous mutations, normal control sequences are given below the mutated allele. Also below the pedigree in (B) is the 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.

no infections. She was globally developmental delayed (4-month stage at 1 year of age). The Mexican-American male (P2; subject B.II-4 in Figure 2) presented with recurrent infections (upper-respiratory-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 comparison to previous bone marrow samples, and showed evidence of bone marrow failure. At 6 years of age, he received a matched-related HSCT from his human-leukocyteantigen-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 diameter 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 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 TBNKþSCID 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 bacterial 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 significant 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 HSCT with neutrophil and T and B cell correction. 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.21,22 WES for two subjects (P1 and P2) was performed at the Baylor College of Medicine Human Genome



Sequencing Center (BCM-HGSC) as part of the BaylorJohns Hopkins Center for Mendelian Genomics. WES testing in P3 was performed by the Department of Pediatrics 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 disease-associated variants identified by WES were independently 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 databases that include results of whole-exome-sequenced samples 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 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 genomewide Agilent oligoarray (BCM CMA version 10) with exon coverage of 4,200 genes, including PGM3 and 300 genes known to be mutated in various primary immunodeficiency diseases.32,33 This CMA readily identifies intragenic 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 conclude that the described clinical phenotype including 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 missense variant in the proband, heterozygous carrier status

6


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 heterozygosity surrounding PGM3 were 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); RefSeq NM_015599.2; Figure 3). Prediction programs (SIFT, PolyPhen-2, LRT, PhyloP, and MutationTaster) evaluated the variant as most likely disease causing, and the affected amino acid is conserved 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 suggestive evidence of a deletion CNV involving PGM3 (Figure S3), suggesting that the proband was hemizygous for the SNV. Chromosomal microarray (BCM CMA version 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, compound-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 duplication 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


Figure 3. Homology Model of Human PGM3 (A) Structural overview of human PGM3. The three amino acids Asp239 (D239), Asn246 (N246), and Gln451 (Q451) are colored red and depicted in ball-and-stick representation. The green sphere represents the magnesium (Mg) ion. (B–D) Close-up view of the interaction between the altered amino acids 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 the metal-binding loop. (D) The environment around amino acid Gln451 (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 protein. 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 complex41 (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

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 (Figures 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 intramolecular 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 immunoglobulin substitution and RBC transfusion), at 2 months of age, and after HSCT. Likewise, mass spectrometry analysis showed normal serum transferrin and apolipoprotein-CIII (apoCIII) glycoforms from all serum samples collected in this child. For CZE analysis, the serum transferrin sialoform pattern was examined by capillary electrophoresis (P/ACE-SYSTEM MDQ). Serum transferrin and apoCIII glycoforms were analyzed by simultaneous online immunoaffinity chromatography electrospray ionization mass spectrometry (SCIEX API4000 tandem mass spectrometer with Turbo V spray source).31 Total serum glycan was analyzed after the samples were denatured and digested 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 MexicanAmerican boy (P2), N-glycan transferrin analysis was qualitatively 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 demonstrated 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 GlcNAc6-P to GlcNAc-1-P, reflected by reduced substrate consumption (Table 2). The p.Asn246Ser substitution was



PGM3 Activities

PGM3

No. of Experiments

Mean (%)

Wild-type

5

100

p.Asn246Ser

5

1

8.1

p.Asp239His

5

59

11.8

p.Gln451Arg

5

50

10.0

SEM (%) 0.0

PGM3 activities were assayed in a 200 ml standard reaction mixture containing 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 10% (v/v) glycerol, 200 mM GlcNAc6-P substrate, and 50 mg of the indicated PGM3 at 30 C for 10 min. Reactions were then inactivated by incubation at 80 C for 5 min. The effect of the amino acid substitutions on PGM3 was tested by mass spectrometry in ‘‘multiple reaction 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 for measuring substrate consumption in relation to that of the wild-type. Data were 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, TBNKþSCID 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 phenotypes.44 Both genotype-phenotype correlation and other modifying factors, including immunodeficiency, intellectual disabilities, and skeletal dysplasia, might contribute to the variety of phenotypes observed in CDGs. The differences 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 demonstrated by our mutant model testing. The data from enzymatic activity of p.Asn246Ser and p.Asp239His correspond with the findings in the Pgm3 mouse models, where lossof-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 conversion to product GlcNAc-1-P, even if our mutant model testing showed half enzyme activity with regard to substrate 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-

8


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 immunodeficiency, such as SLC37A4-CDG (severe congenital neutropenia 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 structure, respectively. Hence, formation of cores 2–4 is dependent 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 we were unable to demonstrate in the sera from affected individuals. 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 demonstrate 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 genotype-phenotype correlation on the basis of their study subjects 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 transplanted PGM3 deficiency than has previously been reported in classical DBQD.13 Unlike in classical DBQD, bone age was not advanced in P3. Another difference of


note in these children with PGM3-CDG is the extra ossification center placed proximal rather than distal to the second metacarpal bone, as is typical in DBQD type 1. The similarities between the skeletal dysplasia in CANT1 deficiency and PGM3 deficiency might be due to their common 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 by mutations 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 reported 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 catalytic activity and reduced intracellular UDP-GlcNAc levels in fibroblasts from three of their PGM3-CDG-affected individuals. The severity and variability of the phenotypes observed between the persons with different PGM3 mutations 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-function allele are more severely affected than mice homozygous for a mild mutant allele.3 Pgm3/ mice die in early embryogenesis and show a dramatic reduction in UDPGlcNAc. 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 thrombocytopenia but normal numbers of neutrophils, eosinophils, 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 individuals. The potential neurological abnormalities in PGM3CDG 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-

ease manifestations, in addition to the hematological defects, HSCT rescues. Skeletal dysplasia has not been reported 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 mesodermal 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 FGFRdependent 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, individuals deficient in mannose-6-phosphate isomerase (Fru6-P to Man-6-P conversion) lack sufficient Man-6-P for complete physiologic N-glycosylation, and daily supplements of mannose can correct this glycosylation deficiency.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 performed 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 homologous disease PGM1-CDG (Figure S1).44 However, treatment 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 complications (presumably resulting from immunodeficiency) before transplantation was initiated. We provide evidence that PGM3 mutations in children can cause a CDG with leukopenia, skeletal dysplasia, dysmorphic 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 phenotypic 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 immunodeficiency 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.



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 represents 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 Ministry of Education and Research to E.L. (FACE consortium TP1, 01GM1109A). E.L. was also supported by the European Commission 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 Human Genetics at Baylor College of Medicine derives revenue from molecular genetic 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

10 The American Journal of Human Genetics 95, 1–12, July 3, 2014

SIFT, http://sift.jcvi.org UCSC Genome Browser, http://genome.ucsc.edu/ UniProt, http://www.uniprot.org/ WHO Child 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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EXTL3 mutations cause skeletal dysplasia, immune deficiency, and developmental delay.

Mutations in EXTL3 Cause Neuro-immuno-skeletal Dysplasia Syndrome.

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Skeletal Dysplasia and Congenital Malformation.

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Mutations in TRNT1 cause congenital sideroblastic anemia with immunodeficiency, fevers, and developmental delay (SIFD).

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Recessive mutations in COL25A1 are a cause of congenital cranial dysinnervation disorder.

Mutations in PCYT1A cause spondylometaphyseal dysplasia with cone-rod dystrophy.

Compound heterozygous mutations in TTC7A cause familial multiple intestinal atresias and severe combined immunodeficiency.

Autosomal recessive mutations in the COL2A1 gene cause severe spondyloepiphyseal dysplasia.

Congenital nephrotic syndrome in an infant with ALG1-congenital disorder of glycosylation.

Skeletal Dysplasia Mutations Effect on Human Filamins' Structure and Mechanosensing.

Glycosylation site occupancy in health, congenital disorder of glycosylation and fatty liver disease.

Autosomal Recessive Phosphoglucomutase 3 (PGM3) Mutations Link Glycosylation Defects to Atopy, Immune Deficiency, Autoimmunity, and Neurocognitive Impairment.

Autosomal recessive phosphoglucomutase 3 (PGM3) mutations link glycosylation defects to atopy, immune deficiency, autoimmunity, and neurocognitive impairment.

Somatic Mutations in TSC1 and TSC2 Cause Focal Cortical Dysplasia.

Loss-of-function mutations in SCN4A cause severe foetal hypokinesia or 'classical' congenital myopathy.

Mutations in human C2CD3 cause skeletal dysplasia and provide new insights into phenotypic and cellular consequences of altered C2CD3 function.

A case of congenital disorder of glycosylation ia presented with recurrent pericardial effusion.

Skeletal Dysplasia Presenting as a Neuromuscular Disorder - Report of a Family with Camurati-Engelmann Syndrome.

CLPB mutations cause 3-methylglutaconic aciduria, progressive brain atrophy, intellectual disability, congenital neutropenia, cataracts, movement disorder.