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The Phenotype of a Germline in PIGA: The Somatically Mutated in Paroxysmal Nocturnal Hemoglobinuria

Jennifer J. Johnston,1 Andrea L. Gropman,2 Julie C. Sapp,1 Jamie K. Teer,1 Jodie M. Martin,2 Cyndi F. Liu,3 Xuan Yuan,3 Zhaohui Ye,3 Linzhao Cheng,3 Robert A. Brodsky,3 and Leslie G. Biesecker1,4,*

Phosphatidylinositol glycan class A (PIGA) is involved in the first step of glycosylphosphatidylinositol (GPI) biosynthesis. Many , including CD55 and CD59, are anchored to the cell by GPI. Loss of CD55 and CD59 on erythrocytes causes complement- mediated lysis in paroxysmal nocturnal hemoglobinuria (PNH), a disease that manifests after clonal expansion of hematopoietic cells with somatic PIGA . Although somatic PIGA mutations have been identified in many PNH patients, it has been proposed that germline mutations are lethal. We report a family with an X-linked lethal disorder involving cleft palate, neonatal seizures, contrac- tures, central nervous system (CNS) structural malformations, and other anomalies. An X exome next-generation sequencing screen identified a single nonsense PIGA mutation, c.1234C>T, which predicts p.Arg412*. This variant segregated with disease and carrier status in the family, is similar to mutations known to cause PNH as a result of PIGA dysfunction, and was absent in 409 controls. PIGA-null mutations are thought to be embryonic lethal, suggesting that p.Arg412* PIGA has residual function. Trans- fection of a mutant p.Arg412* PIGA construct into PIGA-null cells showed partial restoration of GPI-anchored proteins. The genetic data show that the c.1234C>T (p.Arg412*) mutation is present in an affected child, is linked to the affected chromosome in this family, is rare in the population, and results in reduced, but not absent, biosynthesis of GPI anchors. We conclude that c.1234C>TinPIGA results in the lethal X-linked phenotype recognized in the reported family.

Phosphatidylinositol glycan class A (PIGA [MIM 311770]) a missense mutation was identified in PIGN (MIM is one of seven proteins involved in the first step of GPI 606097) in a large consanguineous family affected anchor biosynthesis: transferring N-acetylglucosamine by multiple-congenital-anomalies-hypotonia-seizures syn- (GlcNAc) from uridine 50-diphospho N-acetylglucosamide drome (MCAHS [MIM 614080]). We identified a nonsense (UDP-GlcNAc) to PI to form GlcNac-PI.1 Many proteins mutation in PIGA by using X chromosome exome are anchored to cells by GPI, and in the absence of GPI sequencing in a family with a male-lethal X-linked anchors, these proteins are degraded. The loss of CD55 disorder. This report both expands the phenotype for and CD59 on erythrocytes causes complement-mediated deficiencies of the GPI biosynthesis pathway and more lysis in paroxysmal nocturnal hemoglobinuria (PNH specifically identifies the phenotype for germline muta- [MIM 300818]), a disease that manifests after clonal expan- tions in PIGA. sion of hematopoietic cells with somatic PIGA muta- The family described here presented with three deceased tions.2–5 Although somatic PIGA mutations have been male children and two obligate carrier females. The identified in many patients with PNH, it has been proband (IV-2, Figures 1 and 2) was a male infant and proposed that germline PIGA mutations are lethal.6,7 the second pregnancy of a 33-year-old woman. He was Germline mutations in PIGA have never been identified, delivered by Cesarean section secondary to his having but mutations in encoding PIGM,8 PIGV,9 and been in the breech position. Prenatal ultrasound showed PIGN,10 proteins involved in GPI-anchor biosynthesis, a right pelviectasis. Apgar scores were 11 and 7.5 Birth have been reported. A promoter mutation in PIGM (MIM weight was 3540 g (~75th centile), length was 53.5 cm 610273) was identified in two families affected by in- (>90th centile), and occipital frontal circumference (OFC) herited glycosylphophatidylinosital deficiency (MIM was 37 cm (>90th centile). He had Pierre Robin sequence; 610293) that resulted in thrombosis and seizures. Muta- a prominent occiput; an enlarged fontanelle; a depressed tions in PIGV (MIM 610274) were identified in four fami- nasal bridge; a short, anteverted nose; malar flattening; up- lies affected by hyperphosphatasia mental retardation slanted palpebral fissures; an overfolded helix; gingival syndrome (HPMR), also known as Mabry syndrome overgrowth; a small mouth with downturned corners (MIM 239300). Individuals with Mabry syndrome present and a triangular shape; a short neck (Figure 1A); a globu- with elevated alkaline phosphatase levels, mental retarda- lous chest; and small nails. There was a systolic II–III/VI tion, seizures, hypotonia, and facial dysmorphism. Lastly, murmur with a fixed split S2. He had hip, knee, and elbow

1Genetic Disease Research Branch, National Research Institute, National Institutes of Health, Bethesda, MD, USA; 2Division of Neurology, Children’s National Medical Center, Washington, DC, USA; 3Division of Hematology, Department of Internal Medicine, Johns Hopkins University, Baltimore, MD, USA; 4On behalf of the National Institutes of Health Intramural Sequencing Center, National Human Genome Research Institute, Rockville, MD, USA *Correspondence: [email protected] DOI 10.1016/j.ajhg.2011.11.031. Ó2012 by The American Society of Human Genetics. All rights reserved.

The American Journal of Human Genetics 90, 295–300, February 10, 2012 295 Figure 1. Phenotype of IV-2 and IV-4 (A–C) Individual IV-2. (A) Craniofacial features including micrognathia, a promi- nent occiput, a depressed nasal bridge, a short, anteverted nose, malar flattening, upslanted palpebral fissures, a small, trian- gular mouth, downturned corners of the mouth, and a short neck. (B) Sagittal T1-weighted cranial MRI demonstrates thinning of the genu of the corpus cal- losum, mild sulcal prominence, and cere- bellar hypoplasia. (C) Axial T1-weighted MRI demonstrates white-matter or myeli- nation immaturity for the individual’s age. (D–F) Individual IV-4. (D) Facial image showing similar facial features to those of IV-2. (E) Sagittal T1 cranial MRI demon- strates a thin corpus callosum, sulcal pro- minence, and a small cerebellum. (F) Axial T1-weighted cranial MRI demonstrates white-matter immaturity.

11 weeks and died from pneumonia. No autopsy was performed. The brother (IV-4 in Figure 2)ofindi- vidual IV-2 was born at 35 weeks. Prenatal ultrasound showed polyhy- dramnios. Delivery was by Cesarean for fetal distress and breech. Apgar scores were 1,1 6,5 and 7.10 Birth weight was 3500 g (>90th centile), length was 48 cm (75th–90th centile), and OFC was 35.5 cm (~97th centile). His features were similar to those of his brother (Figure 1D). He also had a fused metopic suture, a high-arched palate, and underdeveloped gums. Poor central tone with brisk reflexes and contractures were noted with severe myoclonic seizures. Additional findings included a small PDA. Calcium was normal, but alkaline phosphatase was 412 IU/liter (39–117 IU/liter). Cranial MRI show- ed a thin corpus callosum, sulcal prominence, a small cerebellum, and contractures, broad palms with short fingers, and poor white matter immaturity (Figures 1E and 1F). An EEG central tone with brisk reflexes. showed a burst suppression pattern. Hgb was 17.3 g/dl, On evaluation, he had obstructive apnea, an atrial septal RBC was 4.74 M, MCV was 103.7 fl, and RDW was 16.2%. defect, vesicoureteral reflux, and a duplicated collecting He lived for 10 weeks and died of respiratory failure. The system. Cranial MRI showed a thin corpus callosum, autopsy showed hepatic microvesicular steatosis and bron- white-matter immaturity, no septum pellucidum, a dilated chopneumonia. Examination of the central nervous left lateral ventricle, and a small cerebellum (Figures 1B system (CNS) showed dilated superficial vasculature. The and 1C). He had myoclonic seizures requiring multiple corpus callosum was thin, and the cerebellum was small. medications, and his EEG showed burst suppression. The olfactory bulb and tracts were absent. White matter Ionized calcium was 0.97 mmol/liter (1.13–1.32 mmol/ appeared dark (reversed cortical ribbon sign) and was liter), Hgb was 12.3 g/dl (14.0–18.0 g/dl), RBC was decreased. Microscopically, abnormal cortical lamination 3.92 M (4.7–6.1 M), MCV was 91.9 fl (80–94 fl), and was present. There were many red, ischemic neurons. RDW was 14.7% (11.5%–14.5%). He lived for about The inferior olivary nucleus was simplified. The basis

296 The American Journal of Human Genetics 90, 295–300, February 10, 2012 A Figure 2. Identification and Segregation of the PIGA Mutation I (A) Pedigree showing segregation of carrier or [+/+] affected status with the presence of the mutation (þ, wild-type; M, mutant). (B) Filtering criteria used for identification of the mutation from a total of 1,360 variants identified II in individual III-2. [+/M] (C) Sanger sequence verification; an arrow shows the position of the mutation.

III [+/M] 18,509,569 reads. Of these, 52.1% could be uniquely aligned to the X exome target. IV This yielded an overall coverage of 1073. [M] Coverage was R13 for 80.4% of the X B C exome, and 72.9% had R203 coverage. 11 Filter Variants The MPG genotype-calling software Control made calls on 69.2% of the X exon Total variants 1,360 sequence. Coding changes were predicted Nonsynonymous/Nonsense/ with custom-designed software.12 We Frameshift/Splice 168 found 1,271 substitutions and 89 insertions III-2 Heterozygous 100 or deletions. Not in dbSNP 20 The genotypes were filtered for several attributes (Figure 2). We reasoned that the Not in Controls 6 IV-4 variant should be severe because it caused Nonsense 1 a lethal phenotype in hemizygous males, c.1234C>T so we filtered for nonsynonymous, splice- p.Arg412X site, frame-shift, and stop alleles. We used heterozygosity because the test subject was pontis had multiple dysplastic changes. The diagnosis was an unaffected female carrier for an X-linked trait. We hypoxic ischemic encephalopathy and brain malforma- sequenced eight control samples by using the same meth- tion with arhinencephaly. Muscles and bones showed odology and excluded variants that were present in noted variation in myofibril diameter and trilineage hema- multiple samples. Additionally, we filtered against variants topoiesis, respectively. present in dbSNP or the ClinSeq cohort (572 control In addition to individuals IV-2 and IV-4, an uncle (III-3 individuals).13 This left six variants, including a single in Figure 2) was reported to have had similar characteris- nonsense mutation, c.1234C>TinPIGA (RefSeq accession tics. The uncle died at one month due to ‘‘stroke.’’ There number NM_002641.2), which predicts p.Arg412*. This are no medical or autopsy records available. Neither obli- variant was confirmed in the other individuals by Sanger gate carrier female had any of the clinical manifestations sequencing (Figure 2), and mutation status segregated that were seen in the affected males. with disease and carrier status. The maternal grand- The pedigree suggested X-linked inheritance in this mother (II-2 in Figure 2) was a carrier of c.1234C>T, but family, and X-chromosome-inactivation studies (Green- the great-grandmother (I-2) was not. Analyses of marker wood Genetics Center) showed the pattern of X inactiva- GATA175D03, 14 kb telomeric to PIGA, showed that I-2 tion to be 100% skewed in both obligate carrier females, did not share an allele with her affected great-grandchild supporting X linkage. On the basis of the evidence for (IV-4). The great-grandfather (I-1) was not available for X-linked inheritance, one sample from an obligate carrier testing. We conclude that the mutation most likely arose female was included in a sequencing screen of all X exons. in II-2 on the X chromosome inherited from her father. For the purpose of this project, ‘‘X exons’’ were defined Additionally, no frameshift, nonsense, or splice-site muta- as all exons between positions 2,675,000 bp and tions in PIGA were identified in the eight independent 154,500,000 bp of known coding and noncoding genes samples sequenced with the same methodology, 572 in build 36 of the UCSC Genome Browser. We used solu- samples from the ClinSeq exome cohort13 (the whole- tion hybridization for X exons (Sure Select, Agilent, Santa exome capture included PIGA), or a survey of 208 patients Clara, CA) to generate a single-end sequencing platform with X-linked mental retardation.14 The c.1234C>T library (Illumina, San Diego, CA). The X exon oligonucleo- p.Arg412* variant found in the family was not identified tide library was designed to select 81.3% of the target. The in the 1000 Genomes data set.15 Human studies were per- library was sequenced on one lane of an Illumina GAII in formed according to an approved human subjects research single-end 36 bp configuration. The sequencing yielded protocol.

The American Journal of Human Genetics 90, 295–300, February 10, 2012 297 Figure 3. Reduced Activity of the p.Arg412* PIGA Mutant in Restoring Surface Expression of GPI-Anchored after Transfection into PIGA-Null Cell Lines Flow-cytometry analysis showing that cell- surface expression of CD59, a GPI-anchored protein, is absent in both LD cells (A, left panel) and TF1-PNH cells (A, right panel). Seventy-two hours after transfection, the cells that received vector expressing wild-type PIGA (pPB-wtPIGA) significantly upregulated surface CD59 expres- sion (C) in comparison to those that received empty vector controls (B). The expression of mutant PIGA (pPB-mutPIGA) partially restored the surface expression of CD59 (D).

In addition to the genetic data, we studied the possible functional consequences of this genetic variant. This was challenging because of the X inactivation status of the heterozygous carriers (see above) and the fact that the affected males were deceased. We tested the hematopoietic cells of the mother (III-2) of the two affected individ- uals for evidence of a small proportion of cells displaying absent GPI-anchored proteins. We tested her erythrocytes for evidence of CD59-negative, glycophorin- A-positive cells but could find none (data not shown). We also attempted to deplete her GPI-positive cells by exposing them to aerolysin, but this did not allow detection of any glycophorin-A-positive, CD59-nega- tive cells (data not shown). To assess the pathogenicity of the c.1234C>T (p.Arg412*) mutant, we per- formed transient transfection experiments in two previously characterized PIGA- deficient cell lines (LD17 and TF118) and compared vectors containing wild-type or mutant PIGA to empty vectors (Figure 3). PIGA mRNA was amplified from lympho- blast RNA (One-Step RT-PCR, QIAGEN), and products were cloned into a PCR4- TOPO vector (Invitrogen, Carlsbad, CA). A single full-length cDNA clone was identi- fied, and sequence confirmation identified p.Arg412* occurs in the last exon of PIGA and is predicted three nonsynonymous alterations that were corrected by to result in a truncated protein missing the final C-terminal site-directed mutagenesis (Quick Change, Agilent). Addi- 109 amino acids. Similar mutations are known to cause tionally, we removed an EcoRI site for cloning purposes dysfunction of PIGA16 in PNH, and both frame-shift and without altering the predicted amino acid sequence. For nonsense mutations have been identified in the 30 region the mutant cDNA, the c.1234C>T mutation was intro- of the gene. Together, the genetic data show that the duced. Wild-type and mutant cDNAs were subcloned c.1234C>T p.Arg412* variant is a severe change in the gene, into the EcoRI site of the pPB-Ubc plasmid (Dr. Kosuke is rare in the population, is present in an affected child, and Yusa, Sanger Institute, Cambridge, UK). The resultant is linked to the affected chromosome in this family. We pPB-wtPIGA and pPB-mutPIGA vectors express wild-type conclude that it caused the phenotype in this family. and mutant PIGA, respectively, under the control of

298 The American Journal of Human Genetics 90, 295–300, February 10, 2012 human Ubc promoter. All plasmid constructs were discussed here. Cases 1 and 2 had hypotonia, seizures, confirmed by restriction digestion and sequencing. and elevated alkaline phosphatase. No thromboses were The LD17 and TF1-PNH cells18 were cultured in RPMI 1640 recognized, although their uncle, case 3, reportedly died with 10% fetal calf serum (FCS). For TF1 culture, 1 ng/ml from a perinatal stroke. Similar to PIGA p.Arg412*, re- GM-SCF was added to the medium. Transfection of LD ported PIGM, PIGV, and PIGN alterations are hypomor- and TF1-PNH cells with 1 mgofPIGA-expression plasmid phic and lead to partial loss of GPI anchor biosynthesis. or control empty vector was performed with the Nucleofec- Interestingly, hemolytic anemia, the primary characteristic tion Kit V (Lonza, Walkersville, MD). Twenty-four hours of PNH, is not reported in patients with PIGM, PIGV, or after transfection, the cells were washed with PBS and PIGN mutations and was not found in the patients re- cultured in fresh RPMI supplemented with 1 ng/ml GM-SCF. ported here. Specifically, the affected patients reported Seventy-two hours after transfection, the cells were here did not manifest clinical hemoglobinuria. Unfortu- collected and washed with PBS before being stained with nately, the affected individuals in this report died prior to CD59 monoclonal antibody (BD Biosciences, Sparks, MD) identification of the causative mutation, and the presence for analysis of restoration of surface CD59 expression in of CD59-negative erythrocytes could not be assessed. a FACScan cytometer (BD Biosciences). Both wild-type and However, autopsies did not reveal any abnormalities in mutant PIGA restored cell-surface expression of CD59, the hematopoietic system. a GPI-anchored protein. However, the percentages of A further understanding of the role of GPI anchors in þ CD59 cells and the mean fluorescence intensity (MFI) of development comes from studies of female mice obtained both PIGA-null cell lines were significantly lower when via a Cre/loxP system targeting Piga in preimplantation they were transfected with the c.1234C>T PIGA mutant embryos.6 These mice have mosaicism for cells expressing than when they were transfected with wild-type PIGA. GPI-anchored proteins due to X-inactivation. The level of Heretofore, germline mutations in PIGA have not been GPI-negative cells in different tissues indicates the impor- described in humans and were presumed to be embryonic tance of GPI-anchored proteins in those tissues. Heart, lethal on the basis of experiments in mice3 and in both lung, brain, and kidney tissues showed high levels of murine19 and human embryonic stem cells.20 Attempts GPI-positive cells, suggesting that normal development to create knock-out mice by traditional methods resulted of these tissues depends on GPI anchors. The CNS includes in low levels of chimerism and embryonic lethality.21 many GPI-anchored proteins, including neural-cell-adhe- Pluripotent human embryonic stem cells (hES) deficient sion molecules (neural CAMs)23 and glypicans24 important in PIGA are defective in trophoblast formation because for neuronal migration. Defects in neuronal migration they lack GPI-anchored BMP4 coreceptors.20 The data pre- cause CNS dysfunction, specifically seizures. We hypothe- sented here are consistent with the hypothesized embry- size that the CNS structural anomalies and dysfunction onic lethality of null mutations in PIGA and suggest that observed here were caused by reduced function of GPI- residual function of the protein is necessary for fetal devel- anchored CNS proteins. opment. The neonatal lethality seen in this family suggests The advent of next-generation sequencing technologies that even residual function of PIGA is insufficient for paired with exon capture is allowing rapid discovery in normal development and survival. the area of the genetic etiology of human disease. This Germline mutations have been described in mannosyl- study demonstrates that a single family can be used to iden- transferases in the GPI pathway, supporting an important tify the causative mutation for a disease. In a disease in role for GPI anchor biosynthesis in development and which the identified alteration is not obviously deleterious, homeostasis. A PIGM promoter mutation causing reduced or in which the function of the affected gene is not well GPI-linked protein expression was described in two fami- defined, additional cases might be necessary to allow one lies affected by portal- and hepatic-vein thrombosis and to conclude causation. In the case reported here, a nonsense persistent absence seizures.8 Missense PIGV mutations mutation was identified in a well-described gene with cause hyperphosphatasia mental retardation syndrome a function that is clearly important to normal hematopoi- (HPMR [MIM 239300]).9 Individuals with HPMR have esis and important to both development and survival in elevated alkaline phosphatase, a distinct facial gestalt, a mouse model.21 This study shows that humans with and neurological features including seizures, muscular severe PIGA mutations can survive to birth, although devel- hypotonia, and intellectual disability. A germline missense opment is aberrant. Understanding the germline pheno- mutation has also been reported in PIGN, encoding GPI type of PIGA mutations will allow identification of other ethanolamine phosphate transferase 1.10 Studies in a affected families and facilitate better understanding of the knockout mouse cell line suggest that PIGN is not essential role of PIGA in development and hematopoiesis. for surface expression of GPI-anchored proteins22 al- though surface expression of CD59 in patient fibroblasts was reduced. The resulting phenotype includes hypotonia, Acknowledgments seizures, and multiple congenital anomalies. There is The authors thank M. Abubakar, G. Bowles-Johnson, S. Covington, overlap among the phenotypes associated with these C. Gomez, A. Hutchins, F.Porter, M. Raygada, O. Rennert, and other disorders and those of affected members of the family clinicians at Georgetown University Hospital, Children’s National

The American Journal of Human Genetics 90, 295–300, February 10, 2012 299 Medical Center, and Johns Hopkins University for clinical investi- 11. Teer, J.K., Bonnycastle, L.L., Chines, P.S., Hansen, N.F., gations of this family. J. Hanover assisted with attempts to revive Aoyama, N., Swift, A.J., Abaan, H.O., Albert, T.J., Margulies, the frozen fibroblasts of patient IV-4. Julia Fekecs assisted with E.H., Green, E.D., et al; NISC Comparative Sequencing graphics. Most importantly, we gratefully acknowledge the family Program. (2010). Systematic comparison of three genomic and dedicate this work to the memory of Walker P. Carter, Jr and enrichment methods for massively parallel DNA sequencing. Michael H. Carter III. This research was supported in part by the Genome Res. 20, 1420–1431. Intramural Research Program of the National Human Genome 12. Johnston, J.J., Teer, J.K., Cherukuri, P.F., Hansen, N.F., Loftus, Research Institute, National Institutes of Health. 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300 The American Journal of Human Genetics 90, 295–300, February 10, 2012