- Email: [email protected]
Expanding the phenotypic spectrum of Mabry Syndrome with novel PIGO gene variants associated with hyperphosphatasia, intractable epilepsy, and complex gastrointestinal and urogenital malformations
European Journal of Medical Genetics xxx (xxxx) xxxx
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: www.elsevier.com/locate/ejmg
Expanding the phenotypic spectrum of Mabry Syndrome with novel PIGO gene variants associated with hyperphosphatasia, intractable epilepsy, and complex gastrointestinal and urogenital malformations Alexander M. Holtza, Amanda W. Harringtonb, Erin R. McNamarac, Agnieszka Kieliand, Janet S. Soula,d, Mayra Martinez-Ojedaa, Philip T. Levya,∗ a
Department of Pediatrics, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA Department of Surgery, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA Department of Urology, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA d Department of Neurology, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA b c
ARTICLE INFO
ABSTRACT
Keywords: Mabry syndrome PIGO Epilepsy Glycophosphatidylinositol VACTERL Anal atresia Esophageal atresia GPI anchor
Mabry syndrome is a glycophosphatidylinositol (GPI) deficiency characterized by intellectual disability, distinctive facial features, intractable seizures, and hyperphosphatasia. We expand the phenotypic spectrum of inherited GPI deficiencies with novel bi-allelic phosphatidylinositol glycan anchor biosynthesis class O (PIGO) variants in a neonate who presented with intractable epilepsy and complex gastrointestinal and urogenital malformations.
1. Introduction
2. Clinical report
Glycophosphatidylinositol (GPI) is a glycolipid anchor that tethers proteins to the cell surface and regulates their trafficking into membrane subdomains (Manea, 2018). GPI-anchored proteins play critical roles to modulate cell signaling during embryogenesis (Zurzolo and Simons, 2016). Mutations to the GPI biosynthetic pathway are responsible for Mabry syndrome, a condition characterized by hyperphosphatasia, anorectal anomalies, distinctive facial features, nail hypoplasia, treatment refractory seizures, developmental delay, and intellectual disability (Tanigawa et al., 2017). The objective of this report is to describe a case of a neonate with novel compound heterozygous variants in the phosphatidylinositol glycan anchor biosynthesis class O (PIGO) gene, who presented with complex gastrointestinal and urogenital abnormalities, respiratory failure, and intractable epilepsy. In addition, we review the available literature describing PIGO associated Mabry syndrome to expand the phenotypic spectrum of inherited glycophosphatidylinositol (GPI) deficiencies.
Our patient was a female infant born by normal spontaneous vaginal delivery at 37 weeks and 6 days gestation to a 24 year-old gravida 4, para 2022 mother. The prenatal course was complicated by bilateral hydronephrosis and an irregular heart rate. APGARs were 7 and 7 at 1 and 5 min, respectively. The infant's birth weight was 3195g (Z-score 0). Physical exam in the delivery room was notable for an imperforate anus. Attempts to pass an orogastric tube failed, a replogle was placed, and subsequent chest radiogram demonstrated an esophageal pouch and diagnosis of esophageal atresia. The infant developed respiratory distress and hypoxemia requiring supplemental oxygen and was admitted to the neonatal intensive care unit. Several dysmorphisms were noted on physical exam, including microcephaly, upslanted palpebral fissures, epicanthal folds, normoset ears with thickened helices, downturned corners of the mouth with a tented upper lip, microretrognathia, brachydactyly, and nail hypoplasia (Fig. 1A–F). Several additional congenital anomalies were promptly discovered consistent with VACTERL association (Vertebral defects,
Abbreviations: GPI, Glycophosphatidylinositol; PIGO, Phosphatidylinositol glycan anchor biosynthesis class O; VACTERL, Vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, renal anomalies, and limb abnormalities ∗ Corresponding author. Division of Newborn Medicine, Boston Children's Hospital, 300 Longwood Avenue | Hunnewell 436, Boston, MA, 02115, USA. E-mail address: [email protected] (P.T. Levy). https://doi.org/10.1016/j.ejmg.2019.103802 Received 13 June 2019; Received in revised form 5 October 2019; Accepted 30 October 2019 1769-7212/ © 2019 Published by Elsevier Masson SAS.
Please cite this article as: Alexander M. Holtz, et al., European Journal of Medical Genetics, https://doi.org/10.1016/j.ejmg.2019.103802
European Journal of Medical Genetics xxx (xxxx) xxxx
A.M. Holtz, et al.
Fig. 1. Dysmorphology associated with compound heterozygous PIGO variants. (A) Image at 10 weeks of age demonstrates up-slanting palpebral fissures, epicanthal folds, downturned corners of the mouth, a tented upper lip, and microretrognathia. (B) Profile reveals a large dysmorphic ear. (C) Images of the hand show distal digit hypoplasia and nail dysplasia. Note similar features in the foot (D), arrow denotes absent nail. (E) XR of the hand, arrow denotes an absent distal phalangeal ossification center of 5th finger, (F), XR of the foot, arrows show lack of ossification of the distal phalanges of 2nd through 5th toes.
HHV6, and EBV PCR). Metabolic workup was also non-diagnostic with a normal newborn screen, plasma amino acids, urine organic acids, urine bile acids, and very long chain fatty acids. Alpha-1 antitrypsin phenotyping was normal. The patient's direct hyperbilirubinemia resolved after discontinuing parental nutrition and advancing enteral feeds. However, her alkaline phosphatase levels were > 1500 U/L and there was evidence of mild osteopenia on radiography studies. Her initial chromosomal microarray and cholestasis sequencing panel were normal. Trio whole genome sequencing revealed compound heterozygous variants in the PIGO gene that were inherited in trans, including one variant of undetermined significance [NM_032634.4:c.1352T > G; p. (Met451Arg), ClinVar ID 373818] and one likely pathogenic frameshift variant [NM_032634.4:c.1392delinsGA; p.(Ile464Mfs*42); ClinVar ID 623646]. Additional findings included a heterozygous variant of undetermined significance in the KRAS gene [NM_004985.4:c.460G > T; p. (Asp154Tyr)] that was maternally inherited. A paternally inherited heterozygous variant of undetermined significance in the MFN2 gene [NM_014874.3:c.748C > T; p.(Arg250Trp); ClinVar ID 543219] was also found. Given the high degree of overlap with other children with bi-allelic PIGO variants and the lack of significant parental medical history related to the KRAS and MFN2 variants, a diagnosis of Mabry syndrome, also known as hyperphosphatasia and mental retardation syndrome (PMHRS), was made. At two months of age, the patient developed episodes of seizure-like activity with insuppressible movements of her upper extremities and trunk; however, EEG reportedly did not show an electrographic seizure correlate. Her EEG background was initially very abnormal, with generalized background slowing, and a burst suppression pattern with prolonged inter-burst interval consistent with severe diffuse cerebral dysfunction. She subsequently developed seizures characterized by tonic posturing of her shoulders and arms with intermittent arm flexion, lip smacking, hypoxemia with visible perioral/facial cyanosis. A subsequent EEG at 4 months of age confirmed accompanying electrographic seizure activity with a generalized spike and slow wave followed by electrodecrement, then runs of rhythmic focal centroparietal spikes lasting ~30–60 s. Her EEG background pattern continued to show a severe diffuse encephalopathy with frequent periods of discontinuity lasting up to 20 s, no anterior-posterior gradient or posterior dominant rhythm, a lack of state changes, and frequent multifocal sharps, spike waves and polyspikes. Clinical and subclinical seizures occurred from 4 to 5 and even up to 27 times per hour. Seizures
Anal atresia, Cardiac defects, Tracheo-Esophageal fistula, Renal anomalies, and Limb abnormalities). An echocardiogram revealed a small patent foramen ovale and a small ductus arteriosus. There were no significant vertebral or limb anomalies noted; however, skeletal survey identified an absent distal phalangeal ossification center of the left 5th finger with a tiny one on the right (Fig. 1E, arrow), in addition to a lack of ossification of the distal phalanges of the 2nd through 5th toes (Fig. 1F, arrows). A primary repair of the esophageal atresia with ligation of the trachea-esophageal fistula was performed on day 2 of age. Following this initial intervention, she required intermittent intubation in the setting of multiple procedures and diagnostic studies. She was ultimately extubated and maintained on non-invasive respiratory support, but over the course of three months she developed an esophageal stricture and severe tracheobronchomalacia with intermittent desaturations requiring suctioning, position changes, and stimulation. She ultimately required continuous BiPAP support to maintain normoxemia and normocarbia. Several anomalies were noted throughout the genitourinary system based on clinical examination, abdominal ultrasound, and voiding cystourethrogram: duplicated vagina and uterus with normal appearing cervices, bilateral echogenic kidneys with dilated pelvices and calyces, peripheral renal cysts, tortuous and dilated ureters, marked bilateral hydroureteronephrosis and a recto-vestibular fistula with imperforate anus. On VCUG there was no evidence of vesicoureteral reflux. A cystoscopy identified a normal, yet elongated urethra and an opening located posteriorly and distal to the ureteric ridge that could not be probed. Neither ureteral orifice could be identified. Her course was complicated by Klebsiella pneumoniae urosepsis with the development of massive left hydroureteronephrosis with pyonephrosis requiring insertion of a percutaneous nephrostomy tube. During the early neonatal period at 3 weeks of age, she underwent exploratory laparotomy for concerns of a small colon identified on contrast study and inability to adequately empty through the dilated rectovestibular fistula. An open gastrostomy-tube was placed with creation of a colostomy and mucous fistula. She had significant feeding intolerance requiring prolonged periods of parenteral nutrition. She developed parenteral nutrition associated cholestasis with a peak direct bilirubin of 8.0 mg/dL and a mild liver transaminase elevation. Diagnostic workup included an unremarkable liver ultrasound and negative infectious studies (normal CMV titers, negative enterovirus, 2
European Journal of Medical Genetics xxx (xxxx) xxxx
A.M. Holtz, et al.
persisted despite treatment with several antiepileptic medications with a final regimen of levetiracetam 50 mg/kg/day, lacosamide 10 mg/kg/ day, and phenobarbital 5 mg/kg/day. No EEG changes were observed after a 100 mg IV bolus of pyridoxine. Brain MRI scans at about 4 and 4.5 months of age showed similar findings of symmetric decreased diffusivity and T2 prolongation in the medial globus pallidi and thalami, red nucleus, substantia nigra, dorsal pons and cerebellar dentate nuclei, and middle cerebellar peduncles. She had a thin corpus callosum, hypoplastic vermis, optic nerves and chiasm, prominent sulci and CSF spaces and a frontotemporal subdural fluid collection. Her neurologic exam at that age was notable for borderline microcephaly, minimal spontaneous waking and eye opening, occasional blink to light but no visual fixation, significant proximal > distal weakness and hypotonia, distal appendicular spasticity, and frequent nonpurposeful movements of her face and limbs when awake. Auditory brain stem response testing revealed only wave I with no other repeatable waveforms. Further airway interventions to address the severe tracheobronchomalacia were ultimately not pursued given her critical illness, intractable seizures, and expected long-term neurologic prognosis. The patient ultimately stabilized and was discharge home. Metrics at last examination prior to transfer at 5-months of age were weight 6.71 kg (Z-score −0.1), length 59 cm (Z-score −1.9), and head circumference 39 cm (Z-score −1.68). At 9 months of age, she developed respiratory failure requiring intubation due to rhinovirus infection and Klebsiella urosepsis leading to shock with multi-organ dysfunction. Her family made the compassionate decision to defer further extreme measure and she died after withdrawing supportive measures.
defective activity of tissue-nonspecific alkaline phosphatase (TNAP, encoded by ALPL gene), a GPI-anchored protein that dephosphorylates pyridoxal phosphate to pyridoxine to cross the neuronal plasma membrane. Pyridoxine is then converted back to pyridoxal phosphate intracellularly, which is an essential cofactor for glutamate decarboxylase in the synthesis of the inhibitory neurotransmitter, GABA. Mice lacking TNAP develop seizures due to GABA depletion that are responsive to vitamin B6 administration (Waymire et al., 1995). Based on this, pyridoxine treatment was attempted and lead to complete seizure resolution in one patient with bi-allelic PIGO mutations (Kuki et al., 2013). Unfortunately, there was no clinical or electroencephalographic response to an IV pyridoxine bolus in our patient and only transient responses were observed in other patients described in the literature (Tanigawa et al., 2017; Xue et al., 2016). This highlights the importance of defining the mechanism of dysfunctional GPI-anchoring in epileptogenesis to develop more effective therapies. The PIGO gene encodes one of the ethanolamine phosphate transferase enzymes that catalyzes the addition of ethanolamine phosphate to the third mannose residue, which is the site of attachment for proteins on the GPI anchor. The young girl described in this case report represents the 18th case of bi-allelic PIGO mutations described in the literature to date and she presents with both overlapping and distinct features. Table 1 provides a summary of the clinical characteristics of our patient in addition to these previously reported cases (Krawitz et al., 2012; Kuki et al., 2013; Morren et al., 2017; Nakamura et al., 2014; Pagnamenta et al., 2017; Tanigawa et al., 2017; Xue et al., 2016; Zehavi et al., 2017). Universal features include global developmental delay and hyperphosphatasia, although mild alkaline phosphatase elevations were observed in some patients with only 10/18 showing levels > 1000 U/L. Seizures were present in 12/18 patients with a typical onset of < 2 years of age, although 3 of these patients were either deceased or hadn't been assessed beyond 2 years of age. Gastrointestinal anomalies (10/18) were another common feature, including anal atresia (6/18; two additional patients with anal stenosis), Hirschsprung disease (6/18), and esophageal atresia (2/18). Distal digit abnormalities including distal digit hypoplasia (14/18) and nail abnormalities (10/18) were the most common reported dysmorphisms. Hearing loss was described in 9/18 patients. Facial dysmorphisms were varied, although it is notable that 6/18 patients were described to have a tented upper lip and 8/18 children had ear abnormalities. The most unique characteristic of this case is the complex urogenital malformations. Our patient had several abnormalities including peripheral renal cysts, dilated tortuous ureters, and significant hydronephrosis that ultimately led to urosepsis and the need for a percutaneous nephrostomy tube. No other morphologic renal abnormalities have been reported in patients with bi-allelic PIGO mutations. Interestingly, congenital hydronephrosis and other renal defects have been observed in patients with PIGV and PIGN mutations, which also encode enzymes involved in the processing of sugar moieties on the GPI anchor (Fleming et al., 2015; Horn et al., 2013). The complete duplication of the vagina and uterus described above represents a novel feature of inherited GPI deficiencies. In fact, the spectrum of congenital anomalies in our patient led to an initial description of VACTERL association, meeting three criteria including anal atresia, TEF/EA, and renal abnormalities. However, VACTERL association is a diagnosis of exclusion when a formal genetic diagnosis cannot be reached. Based on the above case, we suggest that the onset of seizures in the setting of VACTERL features should prompt a workup for an inherited GPI anchor deficiency with targeted genetic sequencing panels or via whole exome sequencing. Flow cytometric analysis of GPI-anchored proteins such as CD16 can also support this diagnosis. This should be considered even in the absence of hyperphosphatasia. Our patient was found to possess two novel PIGO variants with a missense mutation (NM_032634.4:c.1352T > G; p(Met451Arg); Fig. 2A). and one pathogenic frameshift mutation
3. Discussion Glycophosphatidylinositol (GPI) is a glycolipid that anchors soluble proteins to the outer leaflet of the plasma membrane. There are over 150 GPI-anchored proteins in the mammalian genome that play critical roles in embryogenesis, cell signaling, adhesion/migration, metabolism, the immune response, and many other cellular processes (Manea, 2018). GPI-anchored proteins are preferentially sorted into sphingolipid- and cholesterol-containing membrane microdomains, or rafts, and are targeted to the apical surface of polarized cells (Zurzolo and Simons, 2016). These proteins can also be released from the cell surface via phospholipase-mediate cleavage of the GPI anchor (Fujihara and Ikawa, 2016). The biosynthesis, protein attachment, maturation, and subcellular trafficking of GPI-anchored proteins involves more than 30 genes, many of which have been implicated in human disease (BellaiDussault et al., 2018). There are over 200 patients described in the literature with defects in GPI-anchored protein biosynthesis, known as inherited GPI deficiencies. Patients with inherited GPI deficiencies present on a broad phenotypic spectrum with both overlapping and distinct features depending on the affected step in GPI-anchor biosynthesis. In general, these disorders are inherited in an autosomal recessive fashion, except for PIGA, which is X-linked recessive. A recent review of 202 patients with inherited GPI deficiencies defines many commonalities including developmental delay, intellectual disability, seizures, and dysmorphic features (Bellai-Dussault et al., 2018). These defects range from mild to severe and may depend on the level of residual enzyme activity (BellaiDussault et al., 2018; Tanigawa et al., 2017). A wide variety of congenital malformations are also associated with distinct inherited GPI disorders, including craniofacial, ocular, otologic, gastrointestinal, cardiac, renal, and skeletal anomalies. Hyperphosphatasia caused by aberrant secretion of GPI-anchored alkaline phosphatase is a variable finding in these disorders and a normal level should not exclude this diagnosis. Seizures in inherited GPI deficiencies tend to be intractable to standard therapies and are a major cause of morbidity and mortality. One proposed mechanism of epileptogenesis in these disorders involves 3
1 F 9moa p.M451R/ p.I464Mfs
Yes (4mo) Thin corpus callosum, hypoplastic cerebellar vermi, optic nerve and chiasm, T2 prolongation in basal ganglia, thalami, brain stem GDD + Anal atresia, recto- vestibular fistula, TEF/EA Duplicated vagina anduterus Dilated tortuous ureters, hydronephrosis, peripheral renal cysts – + Large dysmorphic ear – + + Upslanting palpebral fissures, epicanthal folds, downturned corners of mouth, tented upper lip, microretrognathia Yes (> 1500)
Patient# Gender Age Genotype
Seizures (onset) Brain MRI
4
– Normal
GDD
– Anal atresia (partial), Hirschsprung's disease – – –
Seizures (onset) Brain MRI
Development
Hypotonia Gastrointestinal
Gento-urinary Renal Cardiovascular
10 M 20moa p.N370S/ p.A834Cfs
9 F 17moa p.N370S/ p.A834Cfs
Patient# Gender Age Genotype
+ Anal atresia, rectourethral fistula, Hischsprung's disease Cryptorchidism – –
GDD
– Hypoplasia of cerebellar vermis and brain stem
Tanigawa et al., 2017
– – –
+ EA, Hirschsprung's disease
Yes (neonatal) Cerebellar vermis atrophy, white matter volume reduction GDD
11 F 9mo p.N370S/ p.G883fs
Yes (1872)
– – –
+ –
GDD
– – –
+ –
GDD
13 F 2yo p.M344K/ p.G883fs Yes (2yo) Normal
Yes (1381)
– – – – + + –
– –
GDD + Anal stenosis
3 F 12yo p.L957F/ p.T788Hfs – N/A
12 F 5yo p.M344K/ p.G883fs Yes (2yo) Normal
ASD, PA stenosis + – Left coronal synostosis + + Hypertelorism, long palpebral fissures, tented upper lip, broad nasal bridge
GDD + Anal atresia with fistula, Hirschsprung's disease – Vesico-ureteral reflux
Yes (1yo) N/A
2 F 22moa p.L957F/ c.3069+5G > A
Krawitz et al., 2012
Source
Hyperphosphatasia (U/ L)
Cardiovascular Hearing loss Ear anomalies Craniofacial anomalies Distal digit hypoplasia Nail hypoplasia Facial dysmorphisms
Gento-urinary Renal
Development Hypotonia Gastrointestinal
Current Report
Source
Table 1 Clinical features of 18 patients with PIGO deficiency.
Cryptorchidism – –
– High-intensity signals in periventricular white matter Language delay, mild learning difficulties + Anal atresia
Hypogonadism – –
+ Mild anal prolapse with pressure
Yes (17mo) Thin corpus callosum, cavum septum pellucidum, white matter lesions Severe intellectual disability
15 M 22yo p.H871P/ p.R604P
Morren et al., 2017
Yes (1201–5959)
Tetrology of Fallot + Low-set Cleft lip and palate + + Hypertelorism, blepharophimosis,
– –
GDD + Hirschsprung's disease
Yes (1yo) Hypomyelination, abnormal signals in basal ganglia to brainstem
5 M 9yo p.R119W/ p.A834Cfs
14 M 13yo p.K1047E/ p.Q430a
Yes (1436)
– – – – + + –
– –
GDD + Anal stenosis
4 F 15yo p.L957F/ p.T788Hfs – N/A
Kuki et al., 2013
– – –
+ –
16 F 2.7yoa p.M255I/ p.M255I Yes (10mo) Hypoplastic corpus callosum, mild cortical atrophy GDD
Zehavi et al., 2017
Yes (436–900)
– + – – – – High-arched palate, tented upper lip
– –
GDD + –
Yes (7mo) Progressive, diffuse cerebral and cerebellar atrophy
6 M 19yo p.T130N/ p.G430a
Mild/normal
– + – – – – –
– –
GDD + –
7 F 1yoa p.T130N/ p.G430a Yes (1yo) N/A
– – –
+ –
Yes (6mo) Hypoplastic corpus callosum, cortical atrophy, delayed myelination GDD
17 F 5yo p.M255I/ p.M255I
Nakamura et al., 2014
(continued on next page)
– – –
+ Hirschsprung's disease
GDD
18 M 2yo p.G238D/ p.R436W – N/A, reported microcephaly
Pagnamenta et al., 2017
Yes (739–943)
– – – Submucosal cleft + – –
– –
GDD + Anal atresia
Yes (6mo) High DWI signal in globus pallidus and dorsal brainstem
8 M 21mo p.153S/ p.A452Gfs
Xue et al., 2016
A.M. Holtz, et al.
European Journal of Medical Genetics xxx (xxxx) xxxx
European Journal of Medical Genetics xxx (xxxx) xxxx
(NM_032634.4:c.1392delinsGA; p(Ile464Mfs*42); Fig. 2B). inherited in trans. Neither variant is present in the genome Aggregation Database (gnomAD; Karczewski et al., 2019). SIFT, PROVEAN, PolyPhen-2, and MutationTaster all predict a deleterious effect of the p.(Met451Arg) variant. This mutation is found downstream of the catalytic GPI ethanolamine phosphatase transferase domain at the N-terminal portion of the 2nd transmembrane domain and affects a highly conserved methionine residue (Fig. 2A–C). Missense mutations both within and outside of the GPI ethanolamine phosphatase transferase domain can cause reduced enzyme activity and compromise protein stability (Tanigawa et al., 2017). Thus, the p.(M451R) variant likely impairs enzyme activity, especially considering the high degree of phenotypic overlap between the patient described above and other children with bi-allelic PIGO variants. Interestingly, the severity of the phenotypic presentation correlates with the residual PIGO enzyme activity as defined in functional assays; however, this activity does not correlate with serum alkaline phosphatase levels or the levels of GPI-anchored proteins measured by flow cytometry (Tanigawa et al., 2017). The maternally inherited KRAS variant and paternally inherited MFN2 variant are unlikely to be contributing to the patient's presentation given the lack of features typical of Noonan syndrome and no parental history related to RASopathies or Charcot-Marie-Tooth Neuropathy type 2A. Patients typically present with one frameshift or nonsense variant, likely acting as a null allele, and one missense variant with variable residual enzyme function (Tanigawa et al., 2017). The lack of two null alleles in a patient suggests that residual enzyme activity is required for viability. Our patient certainly fits this trend; however, it is important to note that four recently described patients were found to possess either homozygous or compound heterozygous missense variants with relatively severe presentations (Morren et al., 2017; Pagnamenta et al., 2017; Zehavi et al., 2017). All four patients had severe developmental delay and 3/4 had seizure disorders (Morren et al., 2017; Zehavi et al., 2017). The patient in Pagnamenta et al. (p.Gly23Asp/p.Arg436Trp) did not have epilepsy as of their last evaluation at 2-years of age (Pagnamenta et al., 2017). Enzyme activity assays demonstrated that the PIGO p.Gly238Asp variant had no appreciable enzyme activity while the p.Arg436Trp had some residual function (Pagnamenta et al., 2017). Further functional characterization of PIGO missense variants will help to define this genotype-to-phenotype correlation. In conclusion, we describe a novel bi-allelic PIGO mutations presenting with overlapping and distinct features compared to other patients described with Mabry syndrome. The severity of the patient's genital and urinary tract malformations extends the phenotypic spectrum of inherited GPI deficiencies. The mechanisms by which mutations in GPI anchor biosynthesis affect tissue development and promote seizure activity remains largely unknown. Further animal models will be critical to better define the consequences of aberrant GPI-anchoring in disease pathogenesis and provide preclinical models to explore novel therapeutic approaches. Financial disclosure None of authors have financial relationships relevant to this article to disclose. Declaration of competing interest The authors declare no conflicts of interest. Acknowledgements We would like to acknowledge the family of the young girl described for their loving and compassionate advocacy for their child. We also thank them for asking us to write this manuscript to share her story and to help other children. We also acknowledge all of the physicians, nurses, and other staff at Boston Children's Hospital for their dedication
a
Mild (217–241) Mild
Mild
Yes (2816–5229)
Yes (5131) Yes (3079) Yes (2594) Yes (2816–5229) Hyperphosphatasia (U/L)
denotes the patient is deceased. GDD: global developmental delay, TEF/EA: trachea-esophageal fistula/esophageal atresia, ASD: atrial septal defect, PA: pulmonary artery.
Yes (418–624)
+ + N/A
– – Hypertelorism, upslanting palpebral fissures, broad nasal bridge, blue sclera, elongated lashes Mild (260–490) + – Tented upper lip
+ – Tented upper lip
+ – Thick eye brows, epicanthal folds, high-arched palate
+ + – + + Tented upper lip + + Upslanting palpebral fissures, anteverted nares, micrognathia + + Upslanting palpebral fissures, anteverted nares, micrognathia
– – –
– – –
–
Submucosal cleft
– Cleft lip and palate Cleft palate
Craniofacial anomalies Distal digit hypoplasia Nail hypoplasia Facial dysmorphisms
– Malformed – Malformed Hearing loss Ear anomalies
Source
Table 1 (continued)
–
+ – – – – – + Malformed + Large dysmorphic ear
+ Large dysmorphic ear Cleft palate
– –
Morren et al., 2017 Tanigawa et al., 2017
Zehavi et al., 2017
– Low-set
Pagnamenta et al., 2017
A.M. Holtz, et al.
5
European Journal of Medical Genetics xxx (xxxx) xxxx
A.M. Holtz, et al.
Fig. 2. Mapping of known PIGO variants. Diagram of PIGO protein showing all known missense (A) and nonsense/frameshift (B) variants described in the literature. Orange denotes transmembrane domains and blue represents the GPI ethanolamine phosphatase transferase 3 domain. The variants described in this report are denoted in red. (C) Multi-species alignment of M451 showing conservation of this methionine residue. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
and exceptional care. The variants described in this report were identified as part of the SouthSeq project (U01HG007301) and sequencing was performed at the HudsonAlpha Institute for Biotechnology in Huntsville, AL.
Schleicher, M., Soto, J., Tibbetts, K., Tolonen, C., Wade, G., Talkowski, M.E., Neale, B.M., Daly, M.J., Macarthur, D.G., 2019. Variation across 141,456 Human Exomes and Genomes Reveals the Spectrum of Loss-Of-Function Intolerance across Human Protein-Coding Genes. bioRxiv 531210. Krawitz, P.M., Murakami, Y., Hecht, J., Krüger, U., Holder, S.E., Mortier, G.R., Delle Chiaie, B., De Baere, E., Thompson, M.D., Roscioli, T., Kielbasa, S., Kinoshita, T., Mundlos, S., Robinson, P.N., Horn, D., 2012. Mutations in PIGO, a member of the GPI-anchor-synthesis pathway, cause hyperphosphatasia with mental retardation. Am. J. Hum. Genet. 91, 146–151. https://doi.org/10.1016/j.ajhg.2012.05.004. Kuki, I., Takahashi, Y., Okazaki, S., Kawawaki, H., Ehara, E., Inoue, N., Kinoshita, T., Murakami, Y., 2013. Vitamin B6-responsive epilepsy due to inherited GPI deficiency. Neurology 81, 1467–1469. https://doi.org/10.1212/WNL.0b013e3182a8411a. Manea, E., 2018. A step closer in defining glycosylphosphatidylinositol anchored proteins role in health and glycosylation disorders. Mol. Genet. Metab. Rep. 16, 67–75. https://doi.org/10.1016/j.ymgmr.2018.07.006. Morren, M.-A., Jaeken, J., Visser, G., Salles, I., Van Geet, C., Simeoni, I., Turro, E., Freson, K., 2017. PIGO deficiency: palmoplantar keratoderma and novel mutations. Orphanet J. Rare Dis. 12 (1 12), 101. https://doi.org/10.1186/s13023-017-0654-9. 2017. Nakamura, K., Osaka, H., Murakami, Y., Anzai, R., Nishiyama, K., Kodera, H., Nakashima, M., Tsurusaki, Y., Miyake, N., Kinoshita, T., Matsumoto, N., Saitsu, H., 2014. PIGO mutations in intractable epilepsy and severe developmental delay with mild elevation of alkaline phosphatase levels. Epilepsia 55, e13–e17. https://doi.org/10.1111/epi. 12508. Pagnamenta, A.T., Murakami, Y., Taylor, J.M., Anzilotti, C., Howard, M.F., Miller, V., Johnson, D.S., Tadros, S., Mansour, S., Temple, I.K., Firth, R., Rosser, E., Harrison, R.E., Kerr, B., Popitsch, N., Kinoshita, T., Taylor, J.C., Kini, U., 2017. Analysis of exome data for 4293 trios suggests GPI-anchor biogenesis defects are a rare cause of developmental disorders. Eur. J. Hum. Genet. 25, 669–679. https://doi.org/10.1016/ j.ajhg.2016.03.024. Tanigawa, J., Mimatsu, H., Mizuno, S., Okamoto, N., Fukushi, D., Tominaga, K., Kidokoro, H., Muramatsu, Y., Nishi, E., Nakamura, S., Motooka, D., Nomura, N., Hayasaka, K., Niihori, T., Aoki, Y., Nabatame, S., Hayakawa, M., Natsume, J., Ozono, K., Kinoshita, T., Wakamatsu, N., Murakami, Y., 2017. Phenotype-genotype correlations of PIGO deficiency with variable phenotypes from infantile lethality to mild learning difficulties. Hum. Mutat. 38, 805–815. https://doi.org/10.1002/humu. 23219. Waymire, K.G., Mahuren, J.D., Jaje, J.M., Guilarte, T.R., Coburn, S.P., MacGregor, G.R., 1995. Mice lacking tissue non-specific alkaline phosphatase die from seizures due to defective metabolism of vitamin B-6. Nat. Genet. 11, 45–51. https://doi.org/10. 1038/ng0995-45. Xue, J., Li, H., Zhang, Y., Yang, Z., 2016. Clinical and genetic analysis of two Chinese
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ejmg.2019.103802. References Bellai-Dussault, K., Nguyen, T.T.M., Baratang, N.V., Jimenez-Cruz, D.A., Campeau, P.M., 2018. Clinical variability in inherited glycosylphosphatidylinositol deficiency disorders. Clin. Genet. https://doi.org/10.1111/cge.13425. Fleming, L., Lemmon, M., Beck, N., Johnson, M., Mu, W., Murdock, D., Bodurtha, J., Hoover-Fong, J., Cohn, R., Bosemani, T., Barañano, K., Hamosh, A., 2015. Genotypephenotype correlation of congenital anomalies in multiple congenital anomalies hypotonia seizures syndrome (MCAHS1)/PIGN-related epilepsy. Am. J. Med. Genet. 170, 77–86. https://doi.org/10.1056/NEJMoa1306555. Fujihara, Y., Ikawa, M., 2016. GPI-AP release in cellular, developmental, and reproductive biology. J. Lipid Res. 57, 538–545. https://doi.org/10.1073/pnas. 0710608105. Horn, D., Wieczorek, D., Metcalfe, K., Baric, I., Paležac, L., Ćuk, M., Petković Ramadža, D., Krüger, U., Demuth, S., Heinritz, W., Linden, T., Koenig, J., Robinson, P.N., Krawitz, P., 2013. Delineation of PIGV mutation spectrum and associated phenotypes in hyperphosphatasia with mental retardation syndrome. Eur. J. Hum. Genet. 22, 762–767. https://doi.org/10.1002/ajmg.a.34102. Karczewski, K.J., Francioli, L.C., Tiao, G., Cummings, B.B., Alföldi, J., Wang, Q., Collins, R.L., Laricchia, K.M., Ganna, A., Birnbaum, D.P., Gauthier, L.D., Brand, H., Solomonson, M., Watts, N.A., Rhodes, D., Singer-Berk, M., England, E.M., Seaby, E.G., Kosmicki, J.A., Walters, R.K., Tashman, K., Farjoun, Y., Banks, E., Poterba, T., Wang, A., Seed, C., Whiffin, N., Chong, J.X., Samocha, K.E., Pierce-Hoffman, E., Zappala, Z., O'Donnell-Luria, A.H., Minikel, E.V., Weisburd, B., Lek, M., Ware, J.S., Vittal, C., Armean, I.M., Bergelson, L., Cibulskis, K., Connolly, K.M., Covarrubias, M., Donnelly, S., Ferriera, S., Gabriel, S., Gentry, J., Gupta, N., Jeandet, T., Kaplan, D., Llanwarne, C., Munshi, R., Novod, S., Petrillo, N., Roazen, D., Ruano-Rubio, V., Saltzman, A.,
6
European Journal of Medical Genetics xxx (xxxx) xxxx
A.M. Holtz, et al. infants with Mabry syndrome. Brain Dev. 38, 807–818. https://doi.org/10.1016/j. braindev.2016.04.008. Zehavi, Y., Renesse, von, A., Daniel-Spiegel, E., Sapir, Y., Zalman, L., Chervinsky, I., Schuelke, M., Straussberg, R., Spiegel, R., 2017. A homozygous PIGO mutation associated with severe infantile epileptic encephalopathy and corpus callosum
hypoplasia, but normal alkaline phosphatase levels. Metab. Brain Dis. 32, 2131–2137. https://doi.org/10.1016/j.braindev.2016.04.008. Zurzolo, C., Simons, K., 2016. Glycosylphosphatidylinositol-anchored proteins: membrane organization and transport. Biochim. Biophys. Acta 1858, 632–639. https:// doi.org/10.1016/j.bbamem.2015.12.018.
7
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: www.elsevier.com/locate/ejmg
Expanding the phenotypic spectrum of Mabry Syndrome with novel PIGO gene variants associated with hyperphosphatasia, intractable epilepsy, and complex gastrointestinal and urogenital malformations Alexander M. Holtza, Amanda W. Harringtonb, Erin R. McNamarac, Agnieszka Kieliand, Janet S. Soula,d, Mayra Martinez-Ojedaa, Philip T. Levya,∗ a
Department of Pediatrics, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA Department of Surgery, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA Department of Urology, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA d Department of Neurology, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA b c
ARTICLE INFO
ABSTRACT
Keywords: Mabry syndrome PIGO Epilepsy Glycophosphatidylinositol VACTERL Anal atresia Esophageal atresia GPI anchor
Mabry syndrome is a glycophosphatidylinositol (GPI) deficiency characterized by intellectual disability, distinctive facial features, intractable seizures, and hyperphosphatasia. We expand the phenotypic spectrum of inherited GPI deficiencies with novel bi-allelic phosphatidylinositol glycan anchor biosynthesis class O (PIGO) variants in a neonate who presented with intractable epilepsy and complex gastrointestinal and urogenital malformations.
1. Introduction
2. Clinical report
Glycophosphatidylinositol (GPI) is a glycolipid anchor that tethers proteins to the cell surface and regulates their trafficking into membrane subdomains (Manea, 2018). GPI-anchored proteins play critical roles to modulate cell signaling during embryogenesis (Zurzolo and Simons, 2016). Mutations to the GPI biosynthetic pathway are responsible for Mabry syndrome, a condition characterized by hyperphosphatasia, anorectal anomalies, distinctive facial features, nail hypoplasia, treatment refractory seizures, developmental delay, and intellectual disability (Tanigawa et al., 2017). The objective of this report is to describe a case of a neonate with novel compound heterozygous variants in the phosphatidylinositol glycan anchor biosynthesis class O (PIGO) gene, who presented with complex gastrointestinal and urogenital abnormalities, respiratory failure, and intractable epilepsy. In addition, we review the available literature describing PIGO associated Mabry syndrome to expand the phenotypic spectrum of inherited glycophosphatidylinositol (GPI) deficiencies.
Our patient was a female infant born by normal spontaneous vaginal delivery at 37 weeks and 6 days gestation to a 24 year-old gravida 4, para 2022 mother. The prenatal course was complicated by bilateral hydronephrosis and an irregular heart rate. APGARs were 7 and 7 at 1 and 5 min, respectively. The infant's birth weight was 3195g (Z-score 0). Physical exam in the delivery room was notable for an imperforate anus. Attempts to pass an orogastric tube failed, a replogle was placed, and subsequent chest radiogram demonstrated an esophageal pouch and diagnosis of esophageal atresia. The infant developed respiratory distress and hypoxemia requiring supplemental oxygen and was admitted to the neonatal intensive care unit. Several dysmorphisms were noted on physical exam, including microcephaly, upslanted palpebral fissures, epicanthal folds, normoset ears with thickened helices, downturned corners of the mouth with a tented upper lip, microretrognathia, brachydactyly, and nail hypoplasia (Fig. 1A–F). Several additional congenital anomalies were promptly discovered consistent with VACTERL association (Vertebral defects,
Abbreviations: GPI, Glycophosphatidylinositol; PIGO, Phosphatidylinositol glycan anchor biosynthesis class O; VACTERL, Vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, renal anomalies, and limb abnormalities ∗ Corresponding author. Division of Newborn Medicine, Boston Children's Hospital, 300 Longwood Avenue | Hunnewell 436, Boston, MA, 02115, USA. E-mail address: [email protected] (P.T. Levy). https://doi.org/10.1016/j.ejmg.2019.103802 Received 13 June 2019; Received in revised form 5 October 2019; Accepted 30 October 2019 1769-7212/ © 2019 Published by Elsevier Masson SAS.
Please cite this article as: Alexander M. Holtz, et al., European Journal of Medical Genetics, https://doi.org/10.1016/j.ejmg.2019.103802
European Journal of Medical Genetics xxx (xxxx) xxxx
A.M. Holtz, et al.
Fig. 1. Dysmorphology associated with compound heterozygous PIGO variants. (A) Image at 10 weeks of age demonstrates up-slanting palpebral fissures, epicanthal folds, downturned corners of the mouth, a tented upper lip, and microretrognathia. (B) Profile reveals a large dysmorphic ear. (C) Images of the hand show distal digit hypoplasia and nail dysplasia. Note similar features in the foot (D), arrow denotes absent nail. (E) XR of the hand, arrow denotes an absent distal phalangeal ossification center of 5th finger, (F), XR of the foot, arrows show lack of ossification of the distal phalanges of 2nd through 5th toes.
HHV6, and EBV PCR). Metabolic workup was also non-diagnostic with a normal newborn screen, plasma amino acids, urine organic acids, urine bile acids, and very long chain fatty acids. Alpha-1 antitrypsin phenotyping was normal. The patient's direct hyperbilirubinemia resolved after discontinuing parental nutrition and advancing enteral feeds. However, her alkaline phosphatase levels were > 1500 U/L and there was evidence of mild osteopenia on radiography studies. Her initial chromosomal microarray and cholestasis sequencing panel were normal. Trio whole genome sequencing revealed compound heterozygous variants in the PIGO gene that were inherited in trans, including one variant of undetermined significance [NM_032634.4:c.1352T > G; p. (Met451Arg), ClinVar ID 373818] and one likely pathogenic frameshift variant [NM_032634.4:c.1392delinsGA; p.(Ile464Mfs*42); ClinVar ID 623646]. Additional findings included a heterozygous variant of undetermined significance in the KRAS gene [NM_004985.4:c.460G > T; p. (Asp154Tyr)] that was maternally inherited. A paternally inherited heterozygous variant of undetermined significance in the MFN2 gene [NM_014874.3:c.748C > T; p.(Arg250Trp); ClinVar ID 543219] was also found. Given the high degree of overlap with other children with bi-allelic PIGO variants and the lack of significant parental medical history related to the KRAS and MFN2 variants, a diagnosis of Mabry syndrome, also known as hyperphosphatasia and mental retardation syndrome (PMHRS), was made. At two months of age, the patient developed episodes of seizure-like activity with insuppressible movements of her upper extremities and trunk; however, EEG reportedly did not show an electrographic seizure correlate. Her EEG background was initially very abnormal, with generalized background slowing, and a burst suppression pattern with prolonged inter-burst interval consistent with severe diffuse cerebral dysfunction. She subsequently developed seizures characterized by tonic posturing of her shoulders and arms with intermittent arm flexion, lip smacking, hypoxemia with visible perioral/facial cyanosis. A subsequent EEG at 4 months of age confirmed accompanying electrographic seizure activity with a generalized spike and slow wave followed by electrodecrement, then runs of rhythmic focal centroparietal spikes lasting ~30–60 s. Her EEG background pattern continued to show a severe diffuse encephalopathy with frequent periods of discontinuity lasting up to 20 s, no anterior-posterior gradient or posterior dominant rhythm, a lack of state changes, and frequent multifocal sharps, spike waves and polyspikes. Clinical and subclinical seizures occurred from 4 to 5 and even up to 27 times per hour. Seizures
Anal atresia, Cardiac defects, Tracheo-Esophageal fistula, Renal anomalies, and Limb abnormalities). An echocardiogram revealed a small patent foramen ovale and a small ductus arteriosus. There were no significant vertebral or limb anomalies noted; however, skeletal survey identified an absent distal phalangeal ossification center of the left 5th finger with a tiny one on the right (Fig. 1E, arrow), in addition to a lack of ossification of the distal phalanges of the 2nd through 5th toes (Fig. 1F, arrows). A primary repair of the esophageal atresia with ligation of the trachea-esophageal fistula was performed on day 2 of age. Following this initial intervention, she required intermittent intubation in the setting of multiple procedures and diagnostic studies. She was ultimately extubated and maintained on non-invasive respiratory support, but over the course of three months she developed an esophageal stricture and severe tracheobronchomalacia with intermittent desaturations requiring suctioning, position changes, and stimulation. She ultimately required continuous BiPAP support to maintain normoxemia and normocarbia. Several anomalies were noted throughout the genitourinary system based on clinical examination, abdominal ultrasound, and voiding cystourethrogram: duplicated vagina and uterus with normal appearing cervices, bilateral echogenic kidneys with dilated pelvices and calyces, peripheral renal cysts, tortuous and dilated ureters, marked bilateral hydroureteronephrosis and a recto-vestibular fistula with imperforate anus. On VCUG there was no evidence of vesicoureteral reflux. A cystoscopy identified a normal, yet elongated urethra and an opening located posteriorly and distal to the ureteric ridge that could not be probed. Neither ureteral orifice could be identified. Her course was complicated by Klebsiella pneumoniae urosepsis with the development of massive left hydroureteronephrosis with pyonephrosis requiring insertion of a percutaneous nephrostomy tube. During the early neonatal period at 3 weeks of age, she underwent exploratory laparotomy for concerns of a small colon identified on contrast study and inability to adequately empty through the dilated rectovestibular fistula. An open gastrostomy-tube was placed with creation of a colostomy and mucous fistula. She had significant feeding intolerance requiring prolonged periods of parenteral nutrition. She developed parenteral nutrition associated cholestasis with a peak direct bilirubin of 8.0 mg/dL and a mild liver transaminase elevation. Diagnostic workup included an unremarkable liver ultrasound and negative infectious studies (normal CMV titers, negative enterovirus, 2
European Journal of Medical Genetics xxx (xxxx) xxxx
A.M. Holtz, et al.
persisted despite treatment with several antiepileptic medications with a final regimen of levetiracetam 50 mg/kg/day, lacosamide 10 mg/kg/ day, and phenobarbital 5 mg/kg/day. No EEG changes were observed after a 100 mg IV bolus of pyridoxine. Brain MRI scans at about 4 and 4.5 months of age showed similar findings of symmetric decreased diffusivity and T2 prolongation in the medial globus pallidi and thalami, red nucleus, substantia nigra, dorsal pons and cerebellar dentate nuclei, and middle cerebellar peduncles. She had a thin corpus callosum, hypoplastic vermis, optic nerves and chiasm, prominent sulci and CSF spaces and a frontotemporal subdural fluid collection. Her neurologic exam at that age was notable for borderline microcephaly, minimal spontaneous waking and eye opening, occasional blink to light but no visual fixation, significant proximal > distal weakness and hypotonia, distal appendicular spasticity, and frequent nonpurposeful movements of her face and limbs when awake. Auditory brain stem response testing revealed only wave I with no other repeatable waveforms. Further airway interventions to address the severe tracheobronchomalacia were ultimately not pursued given her critical illness, intractable seizures, and expected long-term neurologic prognosis. The patient ultimately stabilized and was discharge home. Metrics at last examination prior to transfer at 5-months of age were weight 6.71 kg (Z-score −0.1), length 59 cm (Z-score −1.9), and head circumference 39 cm (Z-score −1.68). At 9 months of age, she developed respiratory failure requiring intubation due to rhinovirus infection and Klebsiella urosepsis leading to shock with multi-organ dysfunction. Her family made the compassionate decision to defer further extreme measure and she died after withdrawing supportive measures.
defective activity of tissue-nonspecific alkaline phosphatase (TNAP, encoded by ALPL gene), a GPI-anchored protein that dephosphorylates pyridoxal phosphate to pyridoxine to cross the neuronal plasma membrane. Pyridoxine is then converted back to pyridoxal phosphate intracellularly, which is an essential cofactor for glutamate decarboxylase in the synthesis of the inhibitory neurotransmitter, GABA. Mice lacking TNAP develop seizures due to GABA depletion that are responsive to vitamin B6 administration (Waymire et al., 1995). Based on this, pyridoxine treatment was attempted and lead to complete seizure resolution in one patient with bi-allelic PIGO mutations (Kuki et al., 2013). Unfortunately, there was no clinical or electroencephalographic response to an IV pyridoxine bolus in our patient and only transient responses were observed in other patients described in the literature (Tanigawa et al., 2017; Xue et al., 2016). This highlights the importance of defining the mechanism of dysfunctional GPI-anchoring in epileptogenesis to develop more effective therapies. The PIGO gene encodes one of the ethanolamine phosphate transferase enzymes that catalyzes the addition of ethanolamine phosphate to the third mannose residue, which is the site of attachment for proteins on the GPI anchor. The young girl described in this case report represents the 18th case of bi-allelic PIGO mutations described in the literature to date and she presents with both overlapping and distinct features. Table 1 provides a summary of the clinical characteristics of our patient in addition to these previously reported cases (Krawitz et al., 2012; Kuki et al., 2013; Morren et al., 2017; Nakamura et al., 2014; Pagnamenta et al., 2017; Tanigawa et al., 2017; Xue et al., 2016; Zehavi et al., 2017). Universal features include global developmental delay and hyperphosphatasia, although mild alkaline phosphatase elevations were observed in some patients with only 10/18 showing levels > 1000 U/L. Seizures were present in 12/18 patients with a typical onset of < 2 years of age, although 3 of these patients were either deceased or hadn't been assessed beyond 2 years of age. Gastrointestinal anomalies (10/18) were another common feature, including anal atresia (6/18; two additional patients with anal stenosis), Hirschsprung disease (6/18), and esophageal atresia (2/18). Distal digit abnormalities including distal digit hypoplasia (14/18) and nail abnormalities (10/18) were the most common reported dysmorphisms. Hearing loss was described in 9/18 patients. Facial dysmorphisms were varied, although it is notable that 6/18 patients were described to have a tented upper lip and 8/18 children had ear abnormalities. The most unique characteristic of this case is the complex urogenital malformations. Our patient had several abnormalities including peripheral renal cysts, dilated tortuous ureters, and significant hydronephrosis that ultimately led to urosepsis and the need for a percutaneous nephrostomy tube. No other morphologic renal abnormalities have been reported in patients with bi-allelic PIGO mutations. Interestingly, congenital hydronephrosis and other renal defects have been observed in patients with PIGV and PIGN mutations, which also encode enzymes involved in the processing of sugar moieties on the GPI anchor (Fleming et al., 2015; Horn et al., 2013). The complete duplication of the vagina and uterus described above represents a novel feature of inherited GPI deficiencies. In fact, the spectrum of congenital anomalies in our patient led to an initial description of VACTERL association, meeting three criteria including anal atresia, TEF/EA, and renal abnormalities. However, VACTERL association is a diagnosis of exclusion when a formal genetic diagnosis cannot be reached. Based on the above case, we suggest that the onset of seizures in the setting of VACTERL features should prompt a workup for an inherited GPI anchor deficiency with targeted genetic sequencing panels or via whole exome sequencing. Flow cytometric analysis of GPI-anchored proteins such as CD16 can also support this diagnosis. This should be considered even in the absence of hyperphosphatasia. Our patient was found to possess two novel PIGO variants with a missense mutation (NM_032634.4:c.1352T > G; p(Met451Arg); Fig. 2A). and one pathogenic frameshift mutation
3. Discussion Glycophosphatidylinositol (GPI) is a glycolipid that anchors soluble proteins to the outer leaflet of the plasma membrane. There are over 150 GPI-anchored proteins in the mammalian genome that play critical roles in embryogenesis, cell signaling, adhesion/migration, metabolism, the immune response, and many other cellular processes (Manea, 2018). GPI-anchored proteins are preferentially sorted into sphingolipid- and cholesterol-containing membrane microdomains, or rafts, and are targeted to the apical surface of polarized cells (Zurzolo and Simons, 2016). These proteins can also be released from the cell surface via phospholipase-mediate cleavage of the GPI anchor (Fujihara and Ikawa, 2016). The biosynthesis, protein attachment, maturation, and subcellular trafficking of GPI-anchored proteins involves more than 30 genes, many of which have been implicated in human disease (BellaiDussault et al., 2018). There are over 200 patients described in the literature with defects in GPI-anchored protein biosynthesis, known as inherited GPI deficiencies. Patients with inherited GPI deficiencies present on a broad phenotypic spectrum with both overlapping and distinct features depending on the affected step in GPI-anchor biosynthesis. In general, these disorders are inherited in an autosomal recessive fashion, except for PIGA, which is X-linked recessive. A recent review of 202 patients with inherited GPI deficiencies defines many commonalities including developmental delay, intellectual disability, seizures, and dysmorphic features (Bellai-Dussault et al., 2018). These defects range from mild to severe and may depend on the level of residual enzyme activity (BellaiDussault et al., 2018; Tanigawa et al., 2017). A wide variety of congenital malformations are also associated with distinct inherited GPI disorders, including craniofacial, ocular, otologic, gastrointestinal, cardiac, renal, and skeletal anomalies. Hyperphosphatasia caused by aberrant secretion of GPI-anchored alkaline phosphatase is a variable finding in these disorders and a normal level should not exclude this diagnosis. Seizures in inherited GPI deficiencies tend to be intractable to standard therapies and are a major cause of morbidity and mortality. One proposed mechanism of epileptogenesis in these disorders involves 3
1 F 9moa p.M451R/ p.I464Mfs
Yes (4mo) Thin corpus callosum, hypoplastic cerebellar vermi, optic nerve and chiasm, T2 prolongation in basal ganglia, thalami, brain stem GDD + Anal atresia, recto- vestibular fistula, TEF/EA Duplicated vagina anduterus Dilated tortuous ureters, hydronephrosis, peripheral renal cysts – + Large dysmorphic ear – + + Upslanting palpebral fissures, epicanthal folds, downturned corners of mouth, tented upper lip, microretrognathia Yes (> 1500)
Patient# Gender Age Genotype
Seizures (onset) Brain MRI
4
– Normal
GDD
– Anal atresia (partial), Hirschsprung's disease – – –
Seizures (onset) Brain MRI
Development
Hypotonia Gastrointestinal
Gento-urinary Renal Cardiovascular
10 M 20moa p.N370S/ p.A834Cfs
9 F 17moa p.N370S/ p.A834Cfs
Patient# Gender Age Genotype
+ Anal atresia, rectourethral fistula, Hischsprung's disease Cryptorchidism – –
GDD
– Hypoplasia of cerebellar vermis and brain stem
Tanigawa et al., 2017
– – –
+ EA, Hirschsprung's disease
Yes (neonatal) Cerebellar vermis atrophy, white matter volume reduction GDD
11 F 9mo p.N370S/ p.G883fs
Yes (1872)
– – –
+ –
GDD
– – –
+ –
GDD
13 F 2yo p.M344K/ p.G883fs Yes (2yo) Normal
Yes (1381)
– – – – + + –
– –
GDD + Anal stenosis
3 F 12yo p.L957F/ p.T788Hfs – N/A
12 F 5yo p.M344K/ p.G883fs Yes (2yo) Normal
ASD, PA stenosis + – Left coronal synostosis + + Hypertelorism, long palpebral fissures, tented upper lip, broad nasal bridge
GDD + Anal atresia with fistula, Hirschsprung's disease – Vesico-ureteral reflux
Yes (1yo) N/A
2 F 22moa p.L957F/ c.3069+5G > A
Krawitz et al., 2012
Source
Hyperphosphatasia (U/ L)
Cardiovascular Hearing loss Ear anomalies Craniofacial anomalies Distal digit hypoplasia Nail hypoplasia Facial dysmorphisms
Gento-urinary Renal
Development Hypotonia Gastrointestinal
Current Report
Source
Table 1 Clinical features of 18 patients with PIGO deficiency.
Cryptorchidism – –
– High-intensity signals in periventricular white matter Language delay, mild learning difficulties + Anal atresia
Hypogonadism – –
+ Mild anal prolapse with pressure
Yes (17mo) Thin corpus callosum, cavum septum pellucidum, white matter lesions Severe intellectual disability
15 M 22yo p.H871P/ p.R604P
Morren et al., 2017
Yes (1201–5959)
Tetrology of Fallot + Low-set Cleft lip and palate + + Hypertelorism, blepharophimosis,
– –
GDD + Hirschsprung's disease
Yes (1yo) Hypomyelination, abnormal signals in basal ganglia to brainstem
5 M 9yo p.R119W/ p.A834Cfs
14 M 13yo p.K1047E/ p.Q430a
Yes (1436)
– – – – + + –
– –
GDD + Anal stenosis
4 F 15yo p.L957F/ p.T788Hfs – N/A
Kuki et al., 2013
– – –
+ –
16 F 2.7yoa p.M255I/ p.M255I Yes (10mo) Hypoplastic corpus callosum, mild cortical atrophy GDD
Zehavi et al., 2017
Yes (436–900)
– + – – – – High-arched palate, tented upper lip
– –
GDD + –
Yes (7mo) Progressive, diffuse cerebral and cerebellar atrophy
6 M 19yo p.T130N/ p.G430a
Mild/normal
– + – – – – –
– –
GDD + –
7 F 1yoa p.T130N/ p.G430a Yes (1yo) N/A
– – –
+ –
Yes (6mo) Hypoplastic corpus callosum, cortical atrophy, delayed myelination GDD
17 F 5yo p.M255I/ p.M255I
Nakamura et al., 2014
(continued on next page)
– – –
+ Hirschsprung's disease
GDD
18 M 2yo p.G238D/ p.R436W – N/A, reported microcephaly
Pagnamenta et al., 2017
Yes (739–943)
– – – Submucosal cleft + – –
– –
GDD + Anal atresia
Yes (6mo) High DWI signal in globus pallidus and dorsal brainstem
8 M 21mo p.153S/ p.A452Gfs
Xue et al., 2016
A.M. Holtz, et al.
European Journal of Medical Genetics xxx (xxxx) xxxx
European Journal of Medical Genetics xxx (xxxx) xxxx
(NM_032634.4:c.1392delinsGA; p(Ile464Mfs*42); Fig. 2B). inherited in trans. Neither variant is present in the genome Aggregation Database (gnomAD; Karczewski et al., 2019). SIFT, PROVEAN, PolyPhen-2, and MutationTaster all predict a deleterious effect of the p.(Met451Arg) variant. This mutation is found downstream of the catalytic GPI ethanolamine phosphatase transferase domain at the N-terminal portion of the 2nd transmembrane domain and affects a highly conserved methionine residue (Fig. 2A–C). Missense mutations both within and outside of the GPI ethanolamine phosphatase transferase domain can cause reduced enzyme activity and compromise protein stability (Tanigawa et al., 2017). Thus, the p.(M451R) variant likely impairs enzyme activity, especially considering the high degree of phenotypic overlap between the patient described above and other children with bi-allelic PIGO variants. Interestingly, the severity of the phenotypic presentation correlates with the residual PIGO enzyme activity as defined in functional assays; however, this activity does not correlate with serum alkaline phosphatase levels or the levels of GPI-anchored proteins measured by flow cytometry (Tanigawa et al., 2017). The maternally inherited KRAS variant and paternally inherited MFN2 variant are unlikely to be contributing to the patient's presentation given the lack of features typical of Noonan syndrome and no parental history related to RASopathies or Charcot-Marie-Tooth Neuropathy type 2A. Patients typically present with one frameshift or nonsense variant, likely acting as a null allele, and one missense variant with variable residual enzyme function (Tanigawa et al., 2017). The lack of two null alleles in a patient suggests that residual enzyme activity is required for viability. Our patient certainly fits this trend; however, it is important to note that four recently described patients were found to possess either homozygous or compound heterozygous missense variants with relatively severe presentations (Morren et al., 2017; Pagnamenta et al., 2017; Zehavi et al., 2017). All four patients had severe developmental delay and 3/4 had seizure disorders (Morren et al., 2017; Zehavi et al., 2017). The patient in Pagnamenta et al. (p.Gly23Asp/p.Arg436Trp) did not have epilepsy as of their last evaluation at 2-years of age (Pagnamenta et al., 2017). Enzyme activity assays demonstrated that the PIGO p.Gly238Asp variant had no appreciable enzyme activity while the p.Arg436Trp had some residual function (Pagnamenta et al., 2017). Further functional characterization of PIGO missense variants will help to define this genotype-to-phenotype correlation. In conclusion, we describe a novel bi-allelic PIGO mutations presenting with overlapping and distinct features compared to other patients described with Mabry syndrome. The severity of the patient's genital and urinary tract malformations extends the phenotypic spectrum of inherited GPI deficiencies. The mechanisms by which mutations in GPI anchor biosynthesis affect tissue development and promote seizure activity remains largely unknown. Further animal models will be critical to better define the consequences of aberrant GPI-anchoring in disease pathogenesis and provide preclinical models to explore novel therapeutic approaches. Financial disclosure None of authors have financial relationships relevant to this article to disclose. Declaration of competing interest The authors declare no conflicts of interest. Acknowledgements We would like to acknowledge the family of the young girl described for their loving and compassionate advocacy for their child. We also thank them for asking us to write this manuscript to share her story and to help other children. We also acknowledge all of the physicians, nurses, and other staff at Boston Children's Hospital for their dedication
a
Mild (217–241) Mild
Mild
Yes (2816–5229)
Yes (5131) Yes (3079) Yes (2594) Yes (2816–5229) Hyperphosphatasia (U/L)
denotes the patient is deceased. GDD: global developmental delay, TEF/EA: trachea-esophageal fistula/esophageal atresia, ASD: atrial septal defect, PA: pulmonary artery.
Yes (418–624)
+ + N/A
– – Hypertelorism, upslanting palpebral fissures, broad nasal bridge, blue sclera, elongated lashes Mild (260–490) + – Tented upper lip
+ – Tented upper lip
+ – Thick eye brows, epicanthal folds, high-arched palate
+ + – + + Tented upper lip + + Upslanting palpebral fissures, anteverted nares, micrognathia + + Upslanting palpebral fissures, anteverted nares, micrognathia
– – –
– – –
–
Submucosal cleft
– Cleft lip and palate Cleft palate
Craniofacial anomalies Distal digit hypoplasia Nail hypoplasia Facial dysmorphisms
– Malformed – Malformed Hearing loss Ear anomalies
Source
Table 1 (continued)
–
+ – – – – – + Malformed + Large dysmorphic ear
+ Large dysmorphic ear Cleft palate
– –
Morren et al., 2017 Tanigawa et al., 2017
Zehavi et al., 2017
– Low-set
Pagnamenta et al., 2017
A.M. Holtz, et al.
5
European Journal of Medical Genetics xxx (xxxx) xxxx
A.M. Holtz, et al.
Fig. 2. Mapping of known PIGO variants. Diagram of PIGO protein showing all known missense (A) and nonsense/frameshift (B) variants described in the literature. Orange denotes transmembrane domains and blue represents the GPI ethanolamine phosphatase transferase 3 domain. The variants described in this report are denoted in red. (C) Multi-species alignment of M451 showing conservation of this methionine residue. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
and exceptional care. The variants described in this report were identified as part of the SouthSeq project (U01HG007301) and sequencing was performed at the HudsonAlpha Institute for Biotechnology in Huntsville, AL.
Schleicher, M., Soto, J., Tibbetts, K., Tolonen, C., Wade, G., Talkowski, M.E., Neale, B.M., Daly, M.J., Macarthur, D.G., 2019. Variation across 141,456 Human Exomes and Genomes Reveals the Spectrum of Loss-Of-Function Intolerance across Human Protein-Coding Genes. bioRxiv 531210. Krawitz, P.M., Murakami, Y., Hecht, J., Krüger, U., Holder, S.E., Mortier, G.R., Delle Chiaie, B., De Baere, E., Thompson, M.D., Roscioli, T., Kielbasa, S., Kinoshita, T., Mundlos, S., Robinson, P.N., Horn, D., 2012. Mutations in PIGO, a member of the GPI-anchor-synthesis pathway, cause hyperphosphatasia with mental retardation. Am. J. Hum. Genet. 91, 146–151. https://doi.org/10.1016/j.ajhg.2012.05.004. Kuki, I., Takahashi, Y., Okazaki, S., Kawawaki, H., Ehara, E., Inoue, N., Kinoshita, T., Murakami, Y., 2013. Vitamin B6-responsive epilepsy due to inherited GPI deficiency. Neurology 81, 1467–1469. https://doi.org/10.1212/WNL.0b013e3182a8411a. Manea, E., 2018. A step closer in defining glycosylphosphatidylinositol anchored proteins role in health and glycosylation disorders. Mol. Genet. Metab. Rep. 16, 67–75. https://doi.org/10.1016/j.ymgmr.2018.07.006. Morren, M.-A., Jaeken, J., Visser, G., Salles, I., Van Geet, C., Simeoni, I., Turro, E., Freson, K., 2017. PIGO deficiency: palmoplantar keratoderma and novel mutations. Orphanet J. Rare Dis. 12 (1 12), 101. https://doi.org/10.1186/s13023-017-0654-9. 2017. Nakamura, K., Osaka, H., Murakami, Y., Anzai, R., Nishiyama, K., Kodera, H., Nakashima, M., Tsurusaki, Y., Miyake, N., Kinoshita, T., Matsumoto, N., Saitsu, H., 2014. PIGO mutations in intractable epilepsy and severe developmental delay with mild elevation of alkaline phosphatase levels. Epilepsia 55, e13–e17. https://doi.org/10.1111/epi. 12508. Pagnamenta, A.T., Murakami, Y., Taylor, J.M., Anzilotti, C., Howard, M.F., Miller, V., Johnson, D.S., Tadros, S., Mansour, S., Temple, I.K., Firth, R., Rosser, E., Harrison, R.E., Kerr, B., Popitsch, N., Kinoshita, T., Taylor, J.C., Kini, U., 2017. Analysis of exome data for 4293 trios suggests GPI-anchor biogenesis defects are a rare cause of developmental disorders. Eur. J. Hum. Genet. 25, 669–679. https://doi.org/10.1016/ j.ajhg.2016.03.024. Tanigawa, J., Mimatsu, H., Mizuno, S., Okamoto, N., Fukushi, D., Tominaga, K., Kidokoro, H., Muramatsu, Y., Nishi, E., Nakamura, S., Motooka, D., Nomura, N., Hayasaka, K., Niihori, T., Aoki, Y., Nabatame, S., Hayakawa, M., Natsume, J., Ozono, K., Kinoshita, T., Wakamatsu, N., Murakami, Y., 2017. Phenotype-genotype correlations of PIGO deficiency with variable phenotypes from infantile lethality to mild learning difficulties. Hum. Mutat. 38, 805–815. https://doi.org/10.1002/humu. 23219. Waymire, K.G., Mahuren, J.D., Jaje, J.M., Guilarte, T.R., Coburn, S.P., MacGregor, G.R., 1995. Mice lacking tissue non-specific alkaline phosphatase die from seizures due to defective metabolism of vitamin B-6. Nat. Genet. 11, 45–51. https://doi.org/10. 1038/ng0995-45. Xue, J., Li, H., Zhang, Y., Yang, Z., 2016. Clinical and genetic analysis of two Chinese
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ejmg.2019.103802. References Bellai-Dussault, K., Nguyen, T.T.M., Baratang, N.V., Jimenez-Cruz, D.A., Campeau, P.M., 2018. Clinical variability in inherited glycosylphosphatidylinositol deficiency disorders. Clin. Genet. https://doi.org/10.1111/cge.13425. Fleming, L., Lemmon, M., Beck, N., Johnson, M., Mu, W., Murdock, D., Bodurtha, J., Hoover-Fong, J., Cohn, R., Bosemani, T., Barañano, K., Hamosh, A., 2015. Genotypephenotype correlation of congenital anomalies in multiple congenital anomalies hypotonia seizures syndrome (MCAHS1)/PIGN-related epilepsy. Am. J. Med. Genet. 170, 77–86. https://doi.org/10.1056/NEJMoa1306555. Fujihara, Y., Ikawa, M., 2016. GPI-AP release in cellular, developmental, and reproductive biology. J. Lipid Res. 57, 538–545. https://doi.org/10.1073/pnas. 0710608105. Horn, D., Wieczorek, D., Metcalfe, K., Baric, I., Paležac, L., Ćuk, M., Petković Ramadža, D., Krüger, U., Demuth, S., Heinritz, W., Linden, T., Koenig, J., Robinson, P.N., Krawitz, P., 2013. Delineation of PIGV mutation spectrum and associated phenotypes in hyperphosphatasia with mental retardation syndrome. Eur. J. Hum. Genet. 22, 762–767. https://doi.org/10.1002/ajmg.a.34102. Karczewski, K.J., Francioli, L.C., Tiao, G., Cummings, B.B., Alföldi, J., Wang, Q., Collins, R.L., Laricchia, K.M., Ganna, A., Birnbaum, D.P., Gauthier, L.D., Brand, H., Solomonson, M., Watts, N.A., Rhodes, D., Singer-Berk, M., England, E.M., Seaby, E.G., Kosmicki, J.A., Walters, R.K., Tashman, K., Farjoun, Y., Banks, E., Poterba, T., Wang, A., Seed, C., Whiffin, N., Chong, J.X., Samocha, K.E., Pierce-Hoffman, E., Zappala, Z., O'Donnell-Luria, A.H., Minikel, E.V., Weisburd, B., Lek, M., Ware, J.S., Vittal, C., Armean, I.M., Bergelson, L., Cibulskis, K., Connolly, K.M., Covarrubias, M., Donnelly, S., Ferriera, S., Gabriel, S., Gentry, J., Gupta, N., Jeandet, T., Kaplan, D., Llanwarne, C., Munshi, R., Novod, S., Petrillo, N., Roazen, D., Ruano-Rubio, V., Saltzman, A.,
6
European Journal of Medical Genetics xxx (xxxx) xxxx
A.M. Holtz, et al. infants with Mabry syndrome. Brain Dev. 38, 807–818. https://doi.org/10.1016/j. braindev.2016.04.008. Zehavi, Y., Renesse, von, A., Daniel-Spiegel, E., Sapir, Y., Zalman, L., Chervinsky, I., Schuelke, M., Straussberg, R., Spiegel, R., 2017. A homozygous PIGO mutation associated with severe infantile epileptic encephalopathy and corpus callosum
hypoplasia, but normal alkaline phosphatase levels. Metab. Brain Dis. 32, 2131–2137. https://doi.org/10.1016/j.braindev.2016.04.008. Zurzolo, C., Simons, K., 2016. Glycosylphosphatidylinositol-anchored proteins: membrane organization and transport. Biochim. Biophys. Acta 1858, 632–639. https:// doi.org/10.1016/j.bbamem.2015.12.018.
7