Mutations in PIGU Impair the Function of the GPI Transamidase Complex, Causing Severe Intellectual Disability, Epilepsy, and Brain Anomalies
2019; Elsevier BV; Volume: 105; Issue: 2 Linguagem: Inglês
10.1016/j.ajhg.2019.06.009
ISSN1537-6605
AutoresAlexej Knaus, Fanny Kortüm, Tjitske Kleefstra, Asbjørg Stray‐Pedersen, Dejan Đukić, Yoshiko Murakami, Thorsten Gerstner, Hans van Bokhoven, Zafar Iqbal, Denise Horn, Taroh Kinoshita, Maja Hempel, Peter Krawitz,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe glycosylphosphatidylinositol (GPI) anchor links over 150 proteins to the cell surface and is present on every cell type. Many of these proteins play crucial roles in neuronal development and function. Mutations in 18 of the 29 genes implicated in the biosynthesis of the GPI anchor have been identified as the cause of GPI biosynthesis deficiencies (GPIBDs) in humans. GPIBDs are associated with intellectual disability and seizures as their cardinal features. An essential component of the GPI transamidase complex is PIGU, along with PIGK, PIGS, PIGT, and GPAA1, all of which link GPI-anchored proteins (GPI-APs) onto the GPI anchor in the endoplasmic reticulum (ER). Here, we report two homozygous missense mutations (c.209T>A [p.Ile70Lys] and c.1149C>A [p.Asn383Lys]) in five individuals from three unrelated families. All individuals presented with global developmental delay, severe-to-profound intellectual disability, muscular hypotonia, seizures, brain anomalies, scoliosis, and mild facial dysmorphism. Using multicolor flow cytometry, we determined a characteristic profile for GPI transamidase deficiency. On granulocytes this profile consisted of reduced cell-surface expression of fluorescein-labeled proaerolysin (FLAER), CD16, and CD24, but not of CD55 and CD59; additionally, B cells showed an increased expression of free GPI anchors determined by T5 antibody. Moreover, computer-assisted facial analysis of different GPIBDs revealed a characteristic facial gestalt shared among individuals with mutations in PIGU and GPAA1. Our findings improve our understanding of the role of the GPI transamidase complex in the development of nervous and skeletal systems and expand the clinical spectrum of disorders belonging to the group of inherited GPI-anchor deficiencies. The glycosylphosphatidylinositol (GPI) anchor links over 150 proteins to the cell surface and is present on every cell type. Many of these proteins play crucial roles in neuronal development and function. Mutations in 18 of the 29 genes implicated in the biosynthesis of the GPI anchor have been identified as the cause of GPI biosynthesis deficiencies (GPIBDs) in humans. GPIBDs are associated with intellectual disability and seizures as their cardinal features. An essential component of the GPI transamidase complex is PIGU, along with PIGK, PIGS, PIGT, and GPAA1, all of which link GPI-anchored proteins (GPI-APs) onto the GPI anchor in the endoplasmic reticulum (ER). Here, we report two homozygous missense mutations (c.209T>A [p.Ile70Lys] and c.1149C>A [p.Asn383Lys]) in five individuals from three unrelated families. All individuals presented with global developmental delay, severe-to-profound intellectual disability, muscular hypotonia, seizures, brain anomalies, scoliosis, and mild facial dysmorphism. Using multicolor flow cytometry, we determined a characteristic profile for GPI transamidase deficiency. On granulocytes this profile consisted of reduced cell-surface expression of fluorescein-labeled proaerolysin (FLAER), CD16, and CD24, but not of CD55 and CD59; additionally, B cells showed an increased expression of free GPI anchors determined by T5 antibody. Moreover, computer-assisted facial analysis of different GPIBDs revealed a characteristic facial gestalt shared among individuals with mutations in PIGU and GPAA1. Our findings improve our understanding of the role of the GPI transamidase complex in the development of nervous and skeletal systems and expand the clinical spectrum of disorders belonging to the group of inherited GPI-anchor deficiencies. The linkage of over 150 different proteins to the cell surface is facilitated by the glycosylphosphatidylinositol (GPI) anchor. GPI-anchored proteins (GPI-APs) play important roles in embryogenesis, neurogenesis, signal transduction, and various other biological processes in all tissues of the human body.1Kinoshita T. Fujita M. Maeda Y. Biosynthesis, remodelling and functions of mammalian GPI-anchored proteins: Recent progress.J. Biochem. 2008; 144: 287-294Crossref PubMed Scopus (221) Google Scholar, 2Kinoshita T. Biosynthesis and deficiencies of glycosylphosphatidylinositol.Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. 2014; 90: 130-143Crossref PubMed Scopus (98) Google Scholar Hence, biosynthesis, modification, and transfer of the GPI anchor to the proteins are highly conserved processes mediated by at least 31 genes. The GPI transamidase is a heteropentameric complex (encoded by PIGK [MIM: 605087], PIGS [MIM: 610271], PIGT [MIM: 610272], GPAA1 [MIM: 603048], and PIGU [MIM: 608528, RefSeq accession number NM_080476.4]) that mediates the transfer of the protein to the GPI anchor in the endoplasmatic reticulum (ER). A recent protein-sequence analysis study showed that the ten predicted transmembrane domains (TMs) of PIGU share high sequence similarity with the mannosyltransferases PIGM, PIGV, PIGB, and PIGZ but lack functionally important motifs.3Eisenhaber B. Sinha S. Wong W.-C. Eisenhaber F. Function of a membrane-embedded domain evolutionarily multiplied in the GPI lipid anchor pathway proteins PIG-B, PIG-M, PIG-U, PIG-W, PIG-V, and PIG-Z.Cell Cycle. 2018; 17: 874-880Crossref PubMed Scopus (12) Google Scholar These ten TMs were anticipated to bind the GPI lipid anchor. In addition, it was determined that PIGU is the last component that associates with the core protein complex of the GPI transamidase formed by PIGT, PIGS, PIGK, and GPAA1.4Hong Y. Ohishi K. Kang J.Y. Tanaka S. Inoue N. Nishimura J. Maeda Y. Kinoshita T. Human PIG-U and yeast Cdc91p are the fifth subunit of GPI transamidase that attaches GPI-anchors to proteins.Mol. Biol. Cell. 2003; 14: 1780-1789Crossref PubMed Scopus (95) Google Scholar Therefore, the presumed molecular function of PIGU is the presentation of the GPI lipid anchor to the transamidase complex in a productive conformation.3Eisenhaber B. Sinha S. Wong W.-C. Eisenhaber F. Function of a membrane-embedded domain evolutionarily multiplied in the GPI lipid anchor pathway proteins PIG-B, PIG-M, PIG-U, PIG-W, PIG-V, and PIG-Z.Cell Cycle. 2018; 17: 874-880Crossref PubMed Scopus (12) Google Scholar Mutations in three genes of the GPI transamidase complex have been associated with human disease. Severe intellectual disability, global developmental delay, muscular hypotonia, seizures, and cerebellar atrophy were described in individuals with GPAA1 (GPIBD15 [MIM: 617810])5Nguyen T.T.M. Murakami Y. Sheridan E. Ehresmann S. Rousseau J. St-Denis A. Chai G. Ajeawung N.F. Fairbrother L. Reimschisel T. et al.Mutations in GPAA1, encoding a GPI transamidase complex protein, cause developmental delay, epilepsy, cerebellar atrophy, and osteopenia.Am. J. Hum. Genet. 2017; 101: 856-865Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar and PIGT mutations (GPIBD7 or multiple congenital anomalies-hypotonia-seizures syndrome 3 [MCAHS3 (MIM: 615398)]);6Kvarnung M. Nilsson D. Lindstrand A. Korenke G.C. Chiang S.C. Blennow E. Bergmann M. Stödberg T. Mäkitie O. Anderlid B.M. et al.A novel intellectual disability syndrome caused by GPI anchor deficiency due to homozygous mutations in PIGT.J. Med. Genet. 2013; 50: 521-528Crossref PubMed Scopus (98) Google Scholar PIGS deficiency leads to severe global developmental delay, seizures, hypotonia, ataxia, and dysmorphic facial features (GPIBD18 [MIM: 618143]).7Nguyen T.T.M. Murakami Y. Wigby K.M. Baratang N.V. Rousseau J. St-Denis A. Rosenfeld J.A. Laniewski S.C. Jones J. Iglesias A.D. et al.Mutations in PIGS, encoding a GPI transamidase, cause a neurological syndrome ranging from fetal akinesia to epileptic encephalopathy.Am. J. Hum. Genet. 2018; 103: 602-611Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Here, we describe five individuals from three unrelated families with rare biallelic missense mutations in PIGU presenting with global developmental delay, severe-to-profound intellectual disability, muscular hypotonia, seizures, brain anomalies, scoliosis, and mild facial dysmorphism consistent with GPI biosynthesis deficiencies (GPIBDs).8Ng B.G. Freeze H.H. Human genetic disorders involving glycosylphosphatidylinositol (GPI) anchors and glycosphingolipids (GSL).J. Inherit. Metab. Dis. 2015; 38: 171-178Crossref PubMed Scopus (49) Google Scholar, 9Jaeken J. Péanne R. What is new in CDG?.J. Inherit. Metab. Dis. 2017; 40: 569-586Crossref PubMed Scopus (100) Google Scholar We provide clinical and functional evidence for a form of a severe autosomal-recessive GPIBD caused by pathogenic mutations in PIGU. Proband P-1 was the first daughter of healthy first-degree cousins (family 1) from Turkey; she was born at term after an uncomplicated pregnancy. Myoclonic seizures occurred within the first months and responded poorly to treatment. At the age of 7 months, a global developmental delay and severe muscular hypotonia with reduced spontaneous movements were noted. Magnetic resonance imaging (MRI) of her brain revealed delayed myelination and a small focal periventricular gliosis on the left side. Persistent focal myoclonic seizures, occurrence of generalized myoclonic-tonic seizures, and spasticity in all limbs led to a slow regression and loss of all achieved skills. Repeated MRI of her brain at the ages of 4 years and 12 years showed atrophy of the white matter. When we saw her at the age of 19 years, we saw a profoundly disabled young woman with poor interaction and without speech; she was wheelchair bound and fed by a gastric tube. X-rays of her spine revealed osteopenia and scoliosis (See case reports in Supplemental Data). Her brother (P-2) was the third child of family 1 and was born at term after a normal pregnancy. At age 7 weeks, the first generalized seizure occurred, followed by a series of therapy-resistant frequent focal myoclonic seizures. He showed profound developmental delay, muscular hypotonia, and poor eye contact. Visual evoked cortical potentials (VECPs) were absent on the right eye and diminished on the left eye. An electrocardiogram (ECG) revealed an incomplete right bundle branch block (RBBB), and echocardiography showed an atrial septal defect (ASD) type II. At the age of 4 years, he had supraventricular tachycardia followed by further episodes requiring hospitalization and cardioversion. When we saw him at the age of 12 years, we saw a wheelchair-bound boy with profound intellectual disability, poor interaction, no speech, and severe scoliosis and who was fed by a percutaneous endoscopic gastrostomy (PEG) tube. P-3 is the first son of non-consanguineous healthy parents of European descent (family 2); he was born at 42 weeks of gestation after a normal pregnancy. He was hypotonic but without feeding problems. At the age of 11 months, he developed mainly myoclonic seizures and absences that were treated but not fully controlled. A brain MRI showed progressive cerebellar atrophy. At the age of 6 years, he showed a dysmetric movement disorder and profound developmental delay. Scoliosis was surgically corrected at the age of 17 years. His brother (P-4) was born at term after a normal pregnancy. He was hypotonic. Brain MRI performed at the age of 4 months showed frontal atrophy and a Dandy Walker variant. His global development was profoundly delayed; he was able to speak two-to-three-word sentences and to walk a few steps at age of 5 years. At the age of 6 years, myoclonic epilepsy developed; this is now well controlled. An MRI done at this age showed progressive vermis hypoplasia. At his current age of 12 years, he is able to walk independently for short distances and to speak sentences of a few words. He has also developed scoliosis. P-5 was the first daughter born to non-consanguineous healthy parents from Norway (family 3); she was born at 42 weeks of gestation after a normal pregnancy. She was hypotonic and had feeding problems. A brain MRI performed at the age of 10 months showed a thin corpus callosum and an enhanced ventricular system without signs of hydrocephalus. Her global development was profoundly delayed. Focal myoclonic seizures started at 3.5 years of age and were responding well to therapy. When we saw her at 5 years of age, she was a wheelchair-bound girl who spoke only few words and had impaired cortical vision. She was considered for a surgical correction of her scoliosis. The individuals in this study were identified via the MatchmakerExchange platform10Sobreira N.L.M. Arachchi H. Buske O.J. Chong J.X. Hutton B. Foreman J. Schiettecatte F. Groza T. Jacobsen J.O.B. Haendel M.A. et al.Matchmaker Exchange ConsortiumMatchmaker Exchange.Curr. Protoc. Hum. Genet. 2017; 95: 1-, 15Google Scholar using data that originated from the University Medical Center Hamburg-Eppendorf, Germany (family 1, P-1 and P-2), the Radboud University Hospital, Nijmegen, the Netherlands, (family 2, P-3 and P-4) and the Norwegian National Unit for Newborn Screening, Division of Pediatric and Adolescent Medicine, Oslo, Norway (family 3, P-5). All samples were obtained after written informed consent was given by the guardians of the affected individuals. The study was performed in accordance with the Declaration of Helsinki protocols and approved by the ethics committees of the respective institutions. Whole-exome sequencing (WES) revealed two rare homozygous variants in PIGU (GenBank: NM_080476.4): c.209T>A (p.Ile70Lys), exon 3 in the two affected siblings (P-1 and P-2) of family 1, and c.1149C>A (p.Asn383Lys), exon 11 in families 2 and 3 (Figures 1 and 2A ). Sanger sequencing confirmed biparental inheritance of the variants in all families; pedigrees and genotypes are shown in Figure 1. The variant c.209T>A (CADD score 25.9) has not been observed in gnomAD,11Lek M. Karczewski K.J. Minikel E.V. Samocha K.E. Banks E. Fennell T. O'Donnell-Luria A.H. Ware J.S. Hill A.J. Cummings B.B. et al.Exome Aggregation ConsortiumAnalysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Scopus (6587) Google Scholar while c.1149C>A (CADD score 26.5) has been observed only in a heterozygous state, in individuals from a European population, and with an allele frequency of 7/277194. Both variants were predicted to be pathogenic by MutationTaster,12Schwarz J.M. Cooper D.N. Schuelke M. Seelow D. MutationTaster2: Mutation prediction for the deep-sequencing age.Nat. Methods. 2014; 11: 361-362Crossref PubMed Scopus (2437) Google Scholar UMD Predictor,13Langmead B. Trapnell C. Pop M. Salzberg S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.Genome Biol. 2009; 10: R25Crossref PubMed Scopus (15054) Google Scholar SIFT,14Li M.X. Kwan J.S. Bao S.Y. Yang W. Ho S.L. Song Y.Q. Sham P.C. Predicting mendelian disease-causing non-synonymous single nucleotide variants in exome sequencing studies.PLoS Genet. 2013; 9: e1003143Crossref PubMed Scopus (107) Google Scholar and PolyPhen2.14Li M.X. Kwan J.S. Bao S.Y. Yang W. Ho S.L. Song Y.Q. Sham P.C. Predicting mendelian disease-causing non-synonymous single nucleotide variants in exome sequencing studies.PLoS Genet. 2013; 9: e1003143Crossref PubMed Scopus (107) Google Scholar The exchange of the hydrophobic amino acid isoleucine at position 70 to a hydrophilic lysine was predicted to cause a conformational change of the first ER luminal domain between TM1 and TM2 (Grantham score 102). The exchange of the amino acid asparagine to lysine at position 383 is also positioned in an ER luminal domain before TM10 (Grantham score 94) (Figures 2B and 3C ).Figure 2Position of Mutations in PIGU Gene and ProteinShow full caption(A) Exon-intron structure and mutational landscape of PIGU.(B) Conservation (black to gray shading) of PIGU protein sequence over multiple species and prediction of transmembrane (red arrows) domains.(C) Transmembrane domain structure and location of missense variants in PIGU.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Relative Cell Surface Expression of GPI-APs on Granulocytes and B CellsShow full captionOn granulocytes of affected individuals (P-1, P-3, P-4, and P-5) a significant relative reduction of cell surface expression of FLEAR, CD16, and CD24 was observed compared to controls. But expression of CD55 and CD59 was not significantly altered on granulocytes. An increased expression of free GPI anchors was detected based on the presence of T5 antibody on B cells in affected individuals compared to controls. Values represent mean + SD. Error bars: n = 3 (parents and one healthy unrelated control), significance was verified by Student's t-test, ns = not significant, ∗p<0.05, ∗∗p<0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Exon-intron structure and mutational landscape of PIGU. (B) Conservation (black to gray shading) of PIGU protein sequence over multiple species and prediction of transmembrane (red arrows) domains. (C) Transmembrane domain structure and location of missense variants in PIGU. On granulocytes of affected individuals (P-1, P-3, P-4, and P-5) a significant relative reduction of cell surface expression of FLEAR, CD16, and CD24 was observed compared to controls. But expression of CD55 and CD59 was not significantly altered on granulocytes. An increased expression of free GPI anchors was detected based on the presence of T5 antibody on B cells in affected individuals compared to controls. Values represent mean + SD. Error bars: n = 3 (parents and one healthy unrelated control), significance was verified by Student's t-test, ns = not significant, ∗p<0.05, ∗∗p<0.01. To assess the functional implications of the missense variants in PIGU for the GPI anchoring process, we performed flow cytometry on peripheral blood samples collected in CytoChex blood collection tubes (BCTs). For multicolor flow cytometry, cells were stained with fluorescently labeled antibodies against GPI-APs (CD16, CD24, CD55, and CD59), as well as with fluorescein-labeled proaerolysin (FLAER), which binds to the GPI anchor itself. Use of the T5 4E10 antibody allowed detection of free GPI anchors.15Azzouz N. Shams-Eldin H. Niehus S. Debierre-Grockiego F. Bieker U. Schmidt J. Mercier C. Delauw M.F. Dubremetz J.F. Smith T.K. Schwarz R.T. Toxoplasma gondii grown in human cells uses GalNAc-containing glycosylphosphatidylinositol precursors to anchor surface antigens while the immunogenic Glc-GalNAc-containing precursors remain free at the parasite cell surface.Int. J. Biochem. Cell Biol. 2006; 38: 1914-1925Crossref PubMed Scopus (22) Google Scholar, 16Hirata T. Mishra S.K. Nakamura S. Saito K. Motooka D. Takada Y. Kanzawa N. Murakami Y. Maeda Y. Fujita M. et al.Identification of a Golgi GPI-N-acetylgalactosamine transferase with tandem transmembrane regions in the catalytic domain.Nat. Commun. 2018; 9: 405Crossref PubMed Scopus (26) Google Scholar, 17Striepen B. Tomavo S. Dubremetz J.F. Schwarz R.T. Identification and characterisation of glycosyl-inositolphospholipids in Toxoplasma gondii.Biochem. Soc. Trans. 1992; 20: 296SCrossref PubMed Scopus (11) Google Scholar, 18Wang Y. Hirata T. Maeda Y. Murakami Y. Fujita M. Kinoshita T. Free, unlinked glycosylphosphatidylinositols on mammalian cell surfaces revisited.J. Biol. Chem. 2019; 294: 5038-5049Crossref PubMed Scopus (18) Google Scholar Relative reduction of GPI-AP was calculated as a ratio of the staining index (SI) of an affected individual to the SIs of healthy parents and healthy unrelated controls. It is noteworthy that there were only subtle differences in GPI-AP staining between heterozygous carriers of pathogenic mutations (parents) and unrelated healthy controls. As revealed by reduced expression of FLAER, the relative expression of GPI-APs CD16 and CD24 was significantly reduced on granulolcytes compared to controls. But CD55 expression was not significantly altered and CD59 expression was only slightly increased in three out of four individuals with mutations in PIGU compared to controls. (Figure 3). However, CD55 expression was not altered from that of controls, whereas CD59 expression was higher than that in controls in three out of four individuals with mutations in PIGU. This characteristic pattern of marker staining was observed in some individuals with mutations in PIGT, but this was not addressed by the authors.19Nakashima M. Kashii H. Murakami Y. Kato M. Tsurusaki Y. Miyake N. Kubota M. Kinoshita T. Saitsu H. Matsumoto N. Novel compound heterozygous PIGT mutations caused multiple congenital anomalies-hypotonia-seizures syndrome 3.Neurogenetics. 2014; 15: 193-200Crossref PubMed Scopus (52) Google Scholar, 20Lam C. Golas G.A. Davids M. Huizing M. Kane M.S. Krasnewich D.M. Malicdan M.C.V. Adams D.R. Markello T.C. Zein W.M. et al.Expanding the clinical and molecular characteristics of PIGT-CDG, a disorder of glycosylphosphatidylinositol anchors.Mol. Genet. Metab. 2015; 115: 128-140Crossref PubMed Scopus (33) Google Scholar The deficiency of the GPI transamidase in linking GPI-APs to the GPI anchor results in an abundance of free GPI on the cell surface. This can be assessed by the T5 antibody, which binds to the N-acetyl-galactoseamine side branch at the first mannose of the GPI anchor.16Hirata T. Mishra S.K. Nakamura S. Saito K. Motooka D. Takada Y. Kanzawa N. Murakami Y. Maeda Y. Fujita M. et al.Identification of a Golgi GPI-N-acetylgalactosamine transferase with tandem transmembrane regions in the catalytic domain.Nat. Commun. 2018; 9: 405Crossref PubMed Scopus (26) Google Scholar, 21Tomavo S. Couvreur G. Leriche M.A. Sadak A. Achbarou A. Fortier B. Dubremetz J.F. Immunolocalization and characterization of the low molecular weight antigen (4-5 kDa) of Toxoplasma gondii that elicits an early IgM response upon primary infection.Parasitology. 1994; 108: 139-145Crossref PubMed Scopus (31) Google Scholar Compared to that of controls, the MFI for T5 of affected individuals' B cells (for monocytes and granulocytes, data not shown) showed an increase of up to 3-fold (Figure 3). Thus, we conclude that the identified missense mutations in PIGU reduce the function of the GPI transamidase complex and lead to accumulation of free GPI anchor on the cell surface. Functional validation of the p.Asn383Lys variant was performed in a CHO cell line deficient for PIGU. Expression of GPI-APs was rescued less efficiently by transient expression of the p.Asn383Lys mutant than by wild-type PIGU (Figure S3). In order to analyze the phenotypic spectrum of the GPI transamidase deficiency and compare it to other GPIBDs, we conducted a systematic review of the phenotypic features of the most prevalent GPIBDs from the literature and additional cases with molecularly diagnosed GPIBDs (unpublished data). Therefore, we included individuals with mutations in PIGT (n = 26), GPAA1 (n = 10), PIGU (n = 5), and PIGS (n = 4, excluding two fetuses) for the GPI transamidase deficiencies; PIGV (n = 26) and PGAP3 (n = 28) for two types of hyperphosphatasia with mental retardation syndrome (HPMRS), HPMRS1 (MIM: 239300) and HPMRS3 (MIM: 615716), respectively; and PIGN (n = 30; MIM: 606097) and PIGA (n = 54; MIM: 311770) for two types of multiple congenital anomalies-hypotonia-seizures syndrome (MCAHS), MCAHS1 (MIM: 614080) and MCAHS3 (MIM: 300868), respectively. We grouped the reported clinical features of the cases in the following 16 feature classes: developmental delay and/or intellectual disability, muscular hypotonia, seizures, cerebellar anomalies (cerebellar hypoplasia, vermis hypoplasia, etc.), anomalies of the corpus callosum (CC)(including thin CC or hypoplasia of the CC), hearing impairment (including hearing loss), cortical visual impairment (including cortical blindness), impairment of the eyes (including strabism, nystagmus, and severe hyper- or myopia), cleft palate, genitourinary anomalies (including malformations of kidneys, urinary tract, or genitalia), heart defects (including ASD and patent ductus arteriosus), scoliosis, osteopenia (including osteoporosis), gastrointestinal anomalies (Hirschsprung disease and megacolon), brachyphalangy (including hypoplastic nails and short fingers or fingertips), and hyperphosphatasia. We counted the phenotypic features and divided by the number of cases that were assessed for each particular feature to determine the feature frequency per gene. The feature frequencies were plotted in a two-dimensional heatmap, and clustering was performed on the basis of Euclidian distance as a measure of similarity. The feature analysis revealed a high similarity among individuals with mutated genes of the GPI transamidase complex (GPAA1, PIGT, and PIGU); their shared features were cerebellar anomalies, impairments of eyes, and osteopenia. However, osteopenia might be related to the inactivity of these severely disabled people. Scoliosis was identified in nearly all PIGU-deficient individuals and is probably a typical clinical feature of PIGU-associated GPIBD. So far, hearing impairment and hyperphosphatasia have not been described in individuals with GPI transamidase deficiency. Genitourinary malformations and heart defects without hyperphosphatasia and cleft palate were frequent in individuals with PIGT, PIGN, and PIGA mutations and are therefore considered characteristic for the MCAHS disease entity. Hypophosphatasia and cleft palate (together with brachyphalangy) were the most common features in individuals with PIGV and PGAP3 mutations, and these features represent the major symptoms in HPMRS. The dichotomy between the HPMRS and MCAHS characteristic phenotypic features is visualized in the two branches of the dendrogram in Figure 4. However, delineation of these disease entities based on the frequency of phenotypic features is challenging because of the broad phenotypic variability of MCAHS and also because elevated amounts of serum alkaline phosphatase (ALP) is not restricted to HPMRS. The major discriminatory features between HPMRS and other GPIBDs are increased levels of ALP in combination with cleft palate and brachytelephalangy. Genitourinary malformations were described in most individuals with PIGV deficiency, while anomalies of the corpus callosum were identified in most PGAP3-affected individuals. Except for in the case of brain anomalies, malformations were more prevalent in individuals with HPMRS than in individuals with the MCAHS spectrum, whereas in the latter group, a higher frequency of brain abnormalities has been documented. Cerebellar anomalies were frequent in all GPIBDs except HPMRS. All individuals with GPIBDs were affected by developmental delay and/or intellectual disability, muscular hypotonia, and seizures. A comparison of the phenotypic features of all diagnosed individuals with mutations in PIGU versus those of individuals with mutations in GPAA1, PIGT, and PIGS is presented in Table 1. More comprehensive clinical descriptions are listed in the Supplemental Data.Table 1Comparison of the Clinical Features of Individuals with Mutations in PIGU versus Previously Reported Individuals with Mutations in PIGT, PIGS, and GPAA1PIGU P-1PIGU P-2PIGU P-3PIGU P-4PIGU P-5PIGT (n = 13)19Nakashima M. Kashii H. Murakami Y. Kato M. Tsurusaki Y. Miyake N. Kubota M. Kinoshita T. Saitsu H. Matsumoto N. Novel compound heterozygous PIGT mutations caused multiple congenital anomalies-hypotonia-seizures syndrome 3.Neurogenetics. 2014; 15: 193-200Crossref PubMed Scopus (52) Google Scholar, 20Lam C. Golas G.A. Davids M. Huizing M. Kane M.S. Krasnewich D.M. Malicdan M.C.V. Adams D.R. Markello T.C. Zein W.M. et al.Expanding the clinical and molecular characteristics of PIGT-CDG, a disorder of glycosylphosphatidylinositol anchors.Mol. Genet. Metab. 2015; 115: 128-140Crossref PubMed Scopus (33) Google Scholar, 22Pagnamenta A.T. Murakami Y. Taylor J.M. Anzilotti C. Howard M.F. Miller V. 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Horn D. et al.DDD Study GroupPIGT-CDG, a disorder of the glycosylphosphatidylinositol anchor: Description of 13 novel patients and expansion of the clinical characteristics.Genet. Med. 2019; (Epub ahead of print)https://doi.org/10.1038/s41436-019-0512-3Abstract Full Text Full Text PDF Scopus (18) Google ScholarPIGS (n = 6)7Nguyen T.T.M. Murakami Y. Wigby K.M. Baratang N.V. Rousseau J. St-Denis A. Rosenfeld J.A. Laniewski S.C. Jones J. Iglesias A.D. et al.Mutations in PIGS, encoding a GPI transamidase, cause a neurological syndrome ranging from fetal akinesia to epileptic encephalopathy.Am. J. Hum. Genet. 2018; 103: 602-611Abstract Full Text Full Text PDF PubMed Scopus (35) Google ScholarGPAA1 (n = 10)5Nguyen T.T.M. Murakami Y. Sheridan E. Ehresmann S. Rousseau J. St-Denis A. Chai G. Ajeawung N.F. Fairbrother L. Reimschisel T. et al.Mutations in GPAA1, encoding a GPI transamidase complex protein, cause developmental del
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