doi:10.1093/brain/awab124 BRAIN 2021: 144; 2659–2669 | 2659 Biallelic PI4KA variants cause a novel neurodevelopmental syndrome with hypomyelinating leukodystrophy Edgard Verdura,1,2,† Agustı́ Rodrı́guez-Palmero,1,3,† Valentina Vélez-Santamaria,1,4 Laura Planas-Serra,1,2 Irene de la Calle,1 Miquel Raspall-Chaure,5,6 Agathe Roubertie,7,8 Mehdi Benkirane,9 Francesco Saettini,10 Lisa Pavinato,11 Giorgia Mandrile,12 Melanie O’Leary,13 Emily O’Heir,13 Estibaliz Barredo,14 Almudena Chacón,14 Vincent Michaud,15,16 Cyril Goizet,16,17 Montserrat Ruiz,1,2 Agatha Schlüter,1,2 Isabelle Rouvet,18Julia Sala-Coromina,5,6 Chiara Fossati,19 Maria Iascone,20 Francesco Canonico,21 Anna Marcé-Grau,5 Precilla de Souza,22 David R. Adams,22,23 Carlos Casasnovas,1,2,4 Heidi L. Rehm,13 Heather C. Mefford,24 Luis González Gutierrez-Solana,2,25 Alfredo Brusco,11,26 Michel Koenig,9 Alfons Macaya5,6 and Aurora Pujol1,2,27 † These authors contributed equally to this work. Phosphoinositides are lipids that play a critical role in processes such as cellular signalling, ion channel activity and membrane trafficking. When mutated, several genes that encode proteins that participate in the metabolism of these lipids give rise to neurological or developmental phenotypes. PI4KA is a phosphoinositide kinase that is highly expressed in the brain and is essential for life. Here we used whole exome or genome sequencing to identify 10 unrelated patients harbouring biallelic variants in PI4KA that caused a spectrum of conditions ranging from severe global neurodevelopmental delay with hypomye- lination and developmental brain abnormalities to pure spastic paraplegia. Some patients presented immunologic- al deficits or genito-urinary abnormalities. Functional analyses by western blotting and immunofluorescence showed decreased PI4KA levels in the patients’ fibroblasts. Immunofluorescence and targeted lipidomics indicated that PI4KA activity was diminished in fibroblasts and peripheral blood mononuclear cells. In conclusion, we report a novel severe metabolic disorder caused by PI4KA malfunction, highlighting the import- ance of phosphoinositide signalling in human brain development and the myelin sheath. 1 Neurometabolic Diseases Laboratory, Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, 08908, Barcelona, Catalonia, Spain 2 Centre for Biomedical Research in Network on Rare Diseases (CIBERER), Instituto de Salud Carlos III, 28029, Madrid, Spain 3 Pediatric Neurology Unit, Department of Pediatrics, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Catalonia, Spain 4 Neuromuscular Unit, Neurology Department, Hospital Universitari de Bellvitge, Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain 5 Neurology Research Group, Vall d’Hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain 6 Department of Paediatric Neurology, Vall d’Hebron University Hospital, Barcelona, Spain Received December 22, 2020. Revised February 10, 2021. Accepted February 15, 2021 C The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. V This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/ by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 2660 | BRAIN 2021: 144; 2659–2669 E. Verdura et al. 7 Département de Neuropédiatrie, Hôpital Gui de Chauliac Pôle Neurosciences Tête et Cou, Montpellier, France 8 INSERM U1051, Institut des Neurosciences de Montpellier, Montpellier, France 9 Laboratoire de Génétique de Maladies Rares EA7402, Institut Universitaire de Recherche Clinique, Université de Montpellier, CHU Montpellier, CEDEX 5, 34295 Montpellier, France 10 Paediatric Hematology Department, Fondazione MBBM, University of Milano Bicocca, Monza, Italy 11 Department of Medical Sciences, University of Torino, 10126 Torino, Italy 12 Thalassemia Centre and Medical Genetics Unit, San Luigi Gonzaga University Hospital, Orbassano, Italy 13 Center for Mendelian Genomics, Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA 14 Neuropediatric Department, Hospital Universitario Gregorio Maran ~ ón, Madrid, Spain 15 Molecular Genetics Laboratory, Bordeaux University Hospital, Bordeaux, Aquitaine, France 16 INSERM U1211, Rare Diseases Laboratory: Genetics and Metabolism, University of Bordeaux, Talence, Aquitaine, France 17 Reference Center for Rare Neurogenetic Diseases, Department of Medical Genetics, University Hospital Centre Bordeaux Pellegrin Hospital Group, Bordeaux, Aquitaine, France 18 Cellular Biotechnology Department and Biobank, Hospices Civils de Lyon, CHU de Lyon, Lyon, France 19 Department of Paediatrics, Fondazione MBBM, Monza, Italy 20 Molecular Genetics Laboratory, USSD LGM, Papa Giovanni XXIII Hospital, Bergamo, Italy 21 Department of Neuroradiology, University of Milan-Bicocca, San Gerardo Hospital, ASST di Monza, Monza, Italy 22 Office of the Clinical Director, National Human Genome Research Institute, NIH, Bethesda, MD, USA 23 Undiagnosed Diseases Program, The Common Fund, NIH, Bethesda, MD, USA 24 Division of Genetic Medicine, Department of Paediatrics, University of Washington, Seattle, WA 98195, USA 25 Pediatric Neurology, Hospital Infantil Universitario Nin~ o Jesús, Madrid, Spain 26 Medical Genetics Unit, Città della Salute e della Scienza, University Hospital, 10126 Turin, Italy 27 Catalan Institution of Research and Advanced Studies (ICREA), Barcelona, Catalonia, Spain Correspondence to: Prof Aurora Pujol, MD, PhD Neurometabolic Diseases Laboratory, IDIBELL Hospital Duran i Reynals, Gran Via 199 08908 L’Hospitalet de Llobregat Barcelona, Spain E-mail: apujol@idibell.cat Keywords: PI4KA; phosphoinositol; inborn errors of metabolism; hypomyelinating leukodystrophy; spastic paraplegia Abbreviation: PI = phosphatidylinositol Introduction Notably, mutations in other genes that encode proteins that are involved in PI metabolism are associated with neurodevelopmental Phosphoinositide lipids at the plasma membrane, which are disorders. For instance, variants in the PIK3CA, PIK3R2, AKT3, and important determinants of membrane identity, play roles in cell FIG4 genes have been linked to the development of polymicrogyria, signalling, controlling cell shape and motility and thus also affect which can be associated or not with megalencephaly and capillary the interaction of cells with their environment.1 Phosphoinositide malformation,5,9–12 and PI4K2A variants have been associated with metabolism at the plasma membrane begins with the phosphoryl- intellectual disability and epilepsy.13 ation of phosphatidylinositol (PI) to PI(4)P, which is later further In this study, we identified 10 patients from unrelated families phosphorylated to PI(4,5)P2 and PI(3,4,5)P3. This first step is cata- carrying biallelic variants in PI4KA that caused a spectrum of con- lysed by phosphatidylinositol 4 kinase A (PI4KA, MIM *600286) and ditions ranging from severe global neurodevelopmental delay with is regulated by FAM126A and TTC7, which are subunits of the hypomyelination/delayed myelination and developmental brain same complex.2,3 Animal models in which PI4KA homologues abnormalities to pure spastic paraplegia. Analysis of protein are inactivated show profound abnormalities: downregulation of structure by 3D modelling and functional studies using western Pi4ka expression in zebrafish leads to multiple developmental blotting, immunofluorescence and targeted lipidomic studies of defects affecting the brain, heart, trunk and most prominently the the PI4KA pathway in patient cells were performed to confirm the loss of pectoral fins, while the Pi4ka orthologue knockout is lethal pathogenicity of the identified variants. in flies, mice and yeast.4–7 It is intriguing that pathogenic variants in FAM126A, a gene that encodes another partner of the PI4KA/TTC7/FAM126A protein com- Materials and methods plex, cause hypomyelinating leukodystrophy [hypomyelination and Genetic studies and variant assessment congenital cataracts(HCC); MIM #610532], with the main pathogenic mechanism of which is defective PI(4)P production in oligodendro- We identified PI4KA variants in probands by whole-exome or cytes, a process coregulated by the myelin basic protein (MBP).2,8 whole-genome sequencing in clinical diagnostic or research Novel leukodystrophy due to PI4KA loss BRAIN 2021: 144; 2659–2669 | 2661 settings. Candidate variants were validated by Sanger sequencing antibodies for 2 h at room temperature. Following incubation with and tested for co-segregation in all family members except for secondary antibodies for 1 h at room temperature, the proteins Patient 5’s mother (unavailable). The Genome Aggregation were detected with the ChemidocTM Touch Imaging System (Bio- Database (gnomAD v.2.1.1; https://gnomad.broadinstitute.org/; Rad). The bands were quantified with ImageLab (Bio-Rad). The pri- accessed 30 November 2020) was used to determine variant fre- mary antibodies that were used were anti-PI4KA (12411-1-AP quency in control populations. Variants were annotated with Proteintech) and anti-b-Actin (A2228, Sigma). The secondary anti- ANNOVAR. High-quality variants with an effect on the coding se- bodies that were used were polyclonal goat anti-mouse (P0447, quence or splice site regions with a frequency lower than 0.01 Dako Cytomation) and polyclonal goat anti-rabbit (P0448, Dako were retrieved from public databases (gnomAD and in-house data- Cytomation). The unmodified full-length blot is shown in bases). The functional impact of variants was analysed with vari- Supplementary Fig. 3. ous prediction tools, including PolyPhen-2, M-CAP, CADD, MutationTaster and LRT pred. Sequence alignment was performed Immunofluorescence using ClustalOmega (https://www.ebi.ac.uk/tools/msa/clustalo/; accessed 30 November 2020) with sequences extracted from NCBI A total of 150 000 cells were seeded on coverslips in wells contain- databases. The PI4KA complex (6bq1 template from the RCSB PDB ing 2 ml of 10% formalin and fixed in for 30 min at room tempera- database)3 was visualized with PyMOL version 2.2.3 (https://www. ture. To permeabilize and block the cells, the coverslips were pymol.org; accessed 30 November 2020). incubated for 20 min at room temperature in blocking buffer (1% BSA, 0.2% powdered milk, 2% NCS, 0.1 M glycine, and 0.1% TritonTM X-100). The cells were immunostained with primary antibodies for Patient recruitment 2 h at room temperature. Following incubation with secondary We obtained genotypic and phenotypic information from patients antibodies for 1 h at room temperature, the slides were mounted R with probable pathogenic PI4KA variants (after in silico criteria), using MowiolV. Confocal images were acquired using a Leica TCS from different hospitals identified with GeneMatcher.14 All SL laser scanning confocal spectral microscope (Leica patients were analysed by the neurologists and/or clinical geneti- Microsystems) and analysed with ImageJ (NIH, USA). The primary cists of their respective referral centre, who determined the para- antibodies that were used were anti-PI4KA (12411-1-AP, clinical analyses to be performed in each case. We reviewed Proteintech) and anti-PI(4)P (Z-P004, Echelon Biosciences Inc.). The R clinical information related to neurodevelopment, growth parame- secondary antibodies that were used were Alexa FluorV 555-conju- R ters, neurological manifestations, behaviour, dysmorphology, and gated goat anti-rabbit IgG (A-21428, Invitrogen) and Alexa FluorV the results of other exams. Hypomyelination was diagnosed when 488-conjugated goat anti-mouse IgG (A-11001, Invitrogen). neuroimaging evidenced mildly elevated hyperintensity of most cerebral white matter in T2-weighted images and mild hypointen- Lipidomics analysis sity, isointensity or mild hyperintensity relative to the cortex in Peripheral blood mononuclear cells (PBMCs) were isolated from T1-weighted images.15 Blood samples were obtained using stand- whole blood using standard methods. Quantification of phosphoi- ard methods. Written informed consent for genetic testing and nositide species was performed at ATK-Analytics Innovation and publication of the clinical information, including clinical pictures, Discovery using 200 lg of TCA-precipitated PBMCs as the starting was obtained from the parents or legal guardians of each patient material. Lipids were extracted and derivatized using TMS- according to the Declaration of Helsinki. Diazomethane prior to mass spectrometry, as described previous- The research project was approved by the Clinical Research ly.16,17 PI 17:0 and 20:4 and PI(4)P 17:0 and 20:4 (LM1502, LM1901, Ethics Committees of IDIBELL Institute (Patients 1 and 9, PR076/14), Avanti Polar Lipids) were used in parallel as internal standards. VHIR Institute [Patient 2, PR(AG)223/2017], CHU Bordeaux (Patient The results were analysed using MassLynx software. 4, Comité de Protection des Personnes Bordeaux—Outre Mer III), the Broad Institute (Patient 5, Partners IRB Protocol #:2016P001422), the University of Washington (Patient 6, #28853), Città della Salute Statistical analysis e della Scienza University Hospital (Patient 7, n. 0060884), the Statistical significance was assessed using Student’s t-test when Comitato Etico Brianza (Patient 8; Monza, Italy; PID-GENMET), the two groups were compared, and P 5 0.05 was considered IIGM Institute (Patient 10, Comité de Ética de la Investigación con significant. Medicamentos, CEIm) and the Montpellier Local Ethics Committee (Patient 3). Data availability Western blotting The authors confirm that the data supporting the findings of this study are available within the article and its Supplementary Human fibroblasts were homogenized in RIPA buffer (150 mM material. NaCl, 1% NonidetTM P40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0), sonicated for 1 min at 4 C, centrifuged for 10 min at 1000g mixed with 4 NuPAGE LDS Sample Buffer Results (Invitrogen) and heated for 5 min at 100 C. Protein concentrations Gene discovery and variant assessment were quantified using a BCA protein assay kit (Thermo Fisher Scientific). Twenty-five micrograms of each protein sample was In the framework of a research project aiming to end the diagnos- subjected to polyacrylamide gel electrophoresis for 30 min at 100 V tic odyssey in patients with neurogenetic diseases (URD-Cat, and for 1 h at 120 V in NuPAGE MOPS SDS Running Buffer Undiagnosed Rare Diseases Consortium of Catalonia), we per- (Invitrogen) supplemented with 5 mM sodium bisulfite (Ref. formed whole-exome sequencing and identified biallelic variants 243973, Sigma-Aldrich). The proteins were transferred to nitrocel- in PI4KA in two patients (Patients 1 and 2) affected with hypomye- lulose membranes. After blocking with 5% bovine serum albumin linating leukodystrophy. Patient 1 was a compound heterozygote (BSA, Sigma-Aldrich) in 0.05% TBS-Tween (TBS-T) for 1 h at room for a missense variant and a loss-of-function variant temperature, the membranes were incubated with primary (p.Glu1152Lys, p.Pro876SerfsTer36), while Patient 2 harboured a 2662 | BRAIN 2021: 144; 2659–2669 E. Verdura et al. homozygous missense variant (p.Gly1925Arg) located close to the study performed at 11 months of age showed axonal sensory neur- PI4KA catalytic site, suggesting possible consanguinity. Through opathy. Six patients had cerebellar ataxia, and five presented a GeneMatcher,14 we recruited eight additional patients from inde- movement disorder, including tremor in two patients, dystonia in pendent families carrying biallelic variants in PI4KA with compat- two patients, choreoathetosis in one patient and stereotypies in ible phenotypes (Figs 1 and 2). Accordingly, the pLI value of PI4KA another patient. There were no formal diagnoses of behavioural was 0.00031, and the pRec value was 1, indicating that biallelic disorders, but two patients showed hyperexcitable behaviour. Six deleterious variants in this gene are pathogenic.18 All identified patients developed epileptic seizures, which appeared in the neo- variants were ultra-rare with minor allele frequencies (MAF) natal period in four patients and were very frequent initially but 50.00001 in the Genome Aggregation Database (gnomAD) became less frequent later. Patient 6 remained without seizures (Supplementary Table 1). Eight patients were compound heterozy- after stopping anti-epileptic treatment at 18 months of age. gous, while two [Patient 2, p.(Gly1925Arg) and Patient 8, Seizures were generalized, focal and myoclonic, and in Patient 1, p.(Asp1854Asn)] were homozygous. Twelve of the identified var- episodes suggestive of infantile spasms were described at iants were missense variants, one was an in-frame deletion of one 3.5 months of age. In Patients 1–3, seizures usually appeared to be amino acid, and the remaining four variants were predicted to re- associated with fever or microbial infections and could evolve into sult in loss-of-function. All missense/in-frame variants affected status epilepticus. The EEG results of Patients 1 and 2 showed conserved amino acids and were predicted to be deleterious by multifocal epileptiform discharges, with paracentral predomin- various prediction tools (Table 1 and Supplementary Fig. 1). PI4KA ance being observed in Patient 1, which improved over time, and contains an a-solenoid domain, a dimerization domain, and a ‘cra- slow background activity. In Patient 4, frontotemporal spike-wave dle’ domain (which contains three contact surfaces with TTC7, discharges were reported, and in Patient 8, bilateral frontal anoma- with which PI4KA interacts directly), as well as a catalytic domain lies evolved into marked abnormalities of background activity and at the C-terminus that is responsible for phosphorylation activity frequent diffuse spike and spike-wave discharges; however, in the and is anchored at the cell membrane.2,3 Interestingly, 7 of 13 con- other three patients, interictal EEG was normal. served missense/in-frame variants were clustered near the active Other clinical features included nystagmus in four patients, site of PI4KA in the catalytic and ‘cradle’ domains (Fig. 1C and strabismus in two patients, and myopia and bilateral iris and ret- Supplementary Fig. 2). All patients harbouring one loss-of-function inal coloboma in one patient. Sensorineural hearing loss was variant were compound heterozygotes with a missense or in- reported in one patient. Four patients had feeding difficulties in frame variant, with no patients harbouring two loss-of-function early childhood, and two required nasogastric tube feeding during variants. Patients 4 and 9 shared one of the truncating variants the first weeks of life. Patients 2, 3, 5 and 8 had immunological (p.Thr2053SerfsTer4) but displayed discordant phenotypes. Patient problems, including hypogammaglobulinaemia in three patients 9, who carried an in-frame variant near the catalytic domain of the and lymphopaenia and autoimmune neutropaenia in one patient second allele had a milder presentation, showing spastic parapar- each. It is worth noting that symptoms of bowel dysfunction (vom- esis with an age of onset of 17. Patient 4 harboured a missense iting, diarrhoea, constipation or gastroesophageal reflux disease) variant in the a-solenoid domain and presented with severe hypo- were reported in Patients 3, 5, 7 and 8. Finally, Patients 2, 6 and 8 myelinating leukodystrophy with onset at 1 year of age. presented abnormalities of the genito-urinary system, such as cryptorchidism, renal cysts and duplication of the collecting Clinical features system. We studied 10 patients harbouring biallelic, ultrarare and probably Group 2 deleterious variants after in silico predictions in PI4KA. Table 1 out- lines the main clinical features of the patients, and the Patients with predominant spastic paraparesis (Patients 9 and 10). Supplementary material includes the patients’ clinical summaries These patients presented a milder phenotype characterized by and demographics. Weight, length and cranial circumference were progressive spastic paraparesis with onset at 2 and 17 years and all normal at birth, while at the last examination (median age pes cavus. One showed mild intellectual disability, and the other 7 years; 3–42 years), three patients (Patients 1, 2 and 6) had low patient had normal cognition. Patient 9 received a diagnosis of weight, four (Patients 1, 3, 6 and 8) had short height and four Crohn’s disease with a stenosing-inflammatory pattern and cor- (Patients 2, 3, 6 and 8) had a head circumference less than two ticosteroid-dependent course at 21 years of age. standard deviations (SD) below the mean. The median age of dis- ease onset was 2 years (birth to 17 years). Brain MRI findings Based on neurological involvement, patients in this cohort fall The MRI images of all the patients are shown in Fig. 2. Group 1 into two main groups. patients showed a pattern of marked, diffuse supratentorial and infratentorial hypomyelination associated with white matter atro- Group 1 phy and a thin corpus callosum (Patients 1–4), incomplete/delayed Patients with developmental encephalopathy with hypomyelinat- myelination (Patients 5–7) and bilateral perisylvian polymicrogyria ing leukodystrophy/delayed myelination and structural brain (Patient 8). The corpus callosum was dysplastic in Patients 3, 5 and anomalies (Patients 1–8). These patients presented global develop- 6. In Patients 1, 4 and 6, there was brainstem and cerebellar hypo- mental delay in the first 12 months of life; in five of these patients, plasia/atrophy, which remained unchanged up to age 2 in Patient clinical manifestations started in the neonatal period, mainly seiz- 1. In addition, there was a component of cerebellar atrophy that ures and/or severe hypotonia. At the last examination (3–19 years progressed in successive controls in Patients 3, 4 and 7 (inferior of age), all of the patients presented moderate-to-severe intellec- lobe). The severity of myelin involvement correlated with clinical tual disability, except for Patient 5, in whom intellectual disability manifestations, which were more severe in Patients 1–3. In con- was mild. Four patients were non-verbal, and four had not trast, cerebral neuroimaging in the clinically milder patients achieved ambulation, with Patient 3 being non-ambulatory even at (Patients 9 and 10) was normal, except for an arachnoid cyst of the the age of 13 years. Axial hypotonia with limb spasticity and pyram- posterior fossa in Patient 9, which was considered an unrelated idal signs were present in all patients except Patient 2, in whom finding. However, in these patients (and in Patients 1–3), cranial severe global hypotonia predominated and a neurophysiological MRI revealed cervical spinal cord atrophy. Novel leukodystrophy due to PI4KA loss BRAIN 2021: 144; 2659–2669 | 2663 Figure 1 PI4KA variant features. (A) Family trees. Square = male; circle = female; filled symbols = affected individuals; open symbols = unaffected carriers; WT = wild-type allele. (B) Structure of PI4KA protein and the mutations identified in this study. Top: Missense/in-frame variants. Bottom: Loss-of-function variants. Light blue = a-solenoid domain; cyan = dimerization domain; dark blue = ‘cradle’ domain; dark purple = catalytic domain. (C) 3D representation of the PI4KA catalytic domain. Blue = PI4KA; green = TTC7B; pink = A1, PI4KA inhibitor occupying the ATP-binding space in the catalytic domain. The red balls represent the location of the missense/in-frame variants found in our cohort. Note the clustering of missense/in- frame variants near the catalytic site of PI4KA. 2664 | BRAIN 2021: 144; 2659–2669 E. Verdura et al. Figure 2 MRI features of patients harbouring biallelic PI4KA variants. Left: Axial T1-weighted sequences. Middle: Axial T2-weighted sequences. Right: Sagittal T1-weighted sequences. Patients 1 and 2 exhibited diffuse hypomyelination, global white matter atrophy with posterior predominance, a thin corpus callosum and colpocephaly. Patient 1 also presented with brainstem and cerebellar hypoplasia. Patients 3 and 4 had diffuse hypomyeli- nation with a thin corpus callosum and progressive cerebellar atrophy. Patient 4 also had brainstem atrophy. Patients 5–7 exhibited delayed myelin- ation with mild ventriculomegaly. Patients 5 and 6 showed a dysplastic corpus callosum. Patient 6 had brainstem and cerebellar atrophy and Patient 7 exhibited atrophy of the cerebellar inferior lobe. Patient 8 showed bilateral perisylvian polymicrogyria. Patients 9 and 10 showed cervical spinal cord atrophy, and arachnoid cyst of the posterior fossa was observed in Patient 9, the images of whom were otherwise normal. PI4KA activity in vitro and in vivo performed lipidomics analysis to quantify global phosphoinositide (PI, PIP and PIP2) levels in Patients 1, 2, 9 and 10. All of the patients We were able to obtain skin biopsies and/or blood samples from showed a significantly decreased PIP/PI ratio compared to age- Patients 1,3, 4, 9 and 10, and performed functional analysis of their matched control subjects, indicating decreased PI4KA activity in variants. Western blot analysis showed that the expression of the these patients (Fig. 3E). PI4KA protein was strongly decreased in the fibroblasts of Patients 1, 3, 4, 9 and 10 compared to those of age-matched controls (Fig. 3B). This result was supported by immunofluorescence images (Fig. 3C and D). To evaluate the activity of PI4KA, we used an anti- Discussion body against the head group of its reaction product, PI(4)P, as pre- We describe here a novel neurological syndrome caused by bial- viously described.2 Immunofluorescence revealed lower levels of lelic mutations in the PI4KA gene. The clinical spectrum ranges PI(4)P in our patients’ fibroblasts (Fig. 3C and D). Finally, we also from a neurodevelopmental disorder of neonatal onset associated Table 1 Main clinical features of the 10 patients with biallelic PI4KA variants General Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 information Gender Female Male Female Female Male Male Female Male Male Male Ethnicity Caucasian Caucasian Caucasian Caucasian Caucasian Asian/Caucasian Caucasian Turkish Caucasian Latin American Age of onset Newborn Newborn 6 months 1 year Newborn Newborn 4 months Newborn 17 years 2 years Age at exam 4 years 3 years 13 years 19 years 10 years 6 years 11 years 5 years 40 years 18 years Variant cDNA c.2624dupC c.5773G4C c.3884A4G c.3592G4A c.1852C4T c.5116+1G4A c.1414A4C c.5560G4A c.5459_5461 c.4666G4A c.3454G4A c.5773G4C c.6122T4C c.6156_6159delGACA c.4990G4A c.5960A4G c.355C4T c.5560G4A delAAG c.5159C4T c.6156_6159delGACA Novel leukodystrophy due to PI4KA loss Protein p.(Pro876 p.(Gly1925Arg) p.(His1295Arg) p.(Ala1198Thr) p.(Arg618Ter) p.? p.(Ser472Arg) p.(Asp1854Asn) p(.Glu1820del) p.(Val1556Met) SerfsTer36) p.(Gly1925Arg) p.(Met2041Thr) p.(Thr2053 SerfsTer4) p.(Asp1664Asn) p.(Asn1987Ser) p.(Arg119Trp) p.(Asp1854Asn) p.(Thr2053SerfsTer4) p.(Thr1720Ile) p.(Glu1152Lys) Examination Weight (SD) 11.4 kg (–2.6) 10.5 kg (–2.4) 39 kg (–1) 49 kg (–1) 26.8 kg (1.23) 15.8 kg (–2.3) 40 kg (–0.4) 14.5 kg (–1.4) 64 kg (–0.9) Not available Height (SD) 92 cm (–3.8) Not available 141 cm (–2.7) 169 cm ( + 0.8) 133.6 cm (0.77) 105.8 cm (–2) 148 cm (–0.6) 100 cm (–2) 171 cm (–1) Not available HC (SD) 48.5 cm (–1.4) 46.2 cm (–3.3) 52 cm (–2) Not available 50 cm (0.5) 47 cm (–3.8) 52.3 cm (–1.9) 47 cm (–3.5) Not available 56 cm (–0.3) Motor signs Spastic Hypotonia Spastic paraparesis Spastic Spastic Spastic Spastic Spastic Spastic Spastic tetraparesis tetraparesis paraparesis tetraparesis paraparesis tetraparesis paraparesis paraparesis Ataxia + – + + + + + – – – Epilepsy + + (IS) ++ + + – + – ++ – – Additional Dystonia, Axonal sensory Irritability (first Tremor, Hand flapping Stereotypic Dystonia features choreoathetosis, neuropathy, months), tremor, nystagmus when excited movements, hyperexcitability, nystagmus, startle very smiley, ocular nystagmus nystagmus dyspraxia Development Gross motor Severely delayed Severely delayed Severy delayed Delayed Delayed Delayed Delayed Severely No delay Delayed delayed Walking Not achieved Not achieved Not achieved Delayed 2.8 years 4.5 years 5years Not achieved Normal 15 months Language None None Delayed Single words Delayed None Delayed None Normal Normal Intellectual Severe Severe Moderate Severe Moderate Severe Severe Severe Normal Mild disability MRI description Diffuse Diffuse Diffuse Diffuse Delayed Delayed Delayed Bilateral Arachnoid cyst Cervical spinal hypomyelination, hypomyelination, hypomyelination, hypomyelination, myelination, myelination, myelination, perisylvian of the posterior cord atrophy WM atrophy, WM atrophy, dysplastic thin CC, thin CC, cerebral, dysplastic CC, dysplastic CC, external polymicrogyria fossa, cervical ventriculomegaly, ventriculomegaly, cerebellar atrophy brainstem and cerebral atrophy, brainstem and hydrocephalus, spinal cord thin CC, brainstem thin CC with calcifications cerebellar atrophy ventriculomegaly cerebellar atrophy cerebellar atrophy and cerebellar atrophy hypoplasia + = present; – = absent; + + = severe; CC = corpus callosum; HC = head circumference; ID = intellectual disability; IS = infantile spasms; WM = white matter. BRAIN 2021: 144; 2659–2669 | 2665 2666 | BRAIN 2021: 144; 2659–2669 E. Verdura et al. Figure 3 PI4KA activity evaluation. (A) Schematic representation of P4KA pathway. (B) Western blot of PI4KA protein and quantification. Patients’ fibroblasts (n = 5) and controls (CTL, n = 5). (C) Immunofluorescence of PI4KA protein and the head group of the lipid PI(4)P and (D) its quantification on a minimum of 100 cells. Patient’s fibroblasts (n = 5) and controls (CTL, n = 4). (E) Targeted lipidomics against phosphatidylinositol (PI), PIP and PIP2, in human PBMCs from control (CTL) children (n = 5), control adults (n = 5); and PI4KA deficient patients (n = 4). Data presented as mean ± SD. *P 5 0.05, **P 5 0.01, ***P 5 0.001 (two-tailed Student’s t-test). Box plot centre line corresponds to the median, lower and upper limits to the first and third quar- tiles (25th and 75th percentiles), respectively, and whiskers to 1.5  the interquartile range. with severe hypomyelinating leukodystrophy with pontocerebellar presentations, such as juvenile or adult-onset spastic paraplegias. hypoplasia (or even polymicrogyria in one case) to spastic parapar- TUBB4A-related leukodystrophy, X-linked adrenoleukodystrophy, esis beginning in adolescence. This cohort illustrates how muta- Pelizaeus-Merzbacher disease and Alexander disease are paradigmat- tions in genes causing childhood leukodystrophies at the most ic for this well-described phenomenon.19–24 Although in most cases severe end of the clinical spectrum, can also give rise to milder recessive mutations in this gene will manifest as hypomyelinating Novel leukodystrophy due to PI4KA loss BRAIN 2021: 144; 2659–2669 | 2667 leukodystrophy, we propose the term ‘PI4KA-spectrum’ to describe complete loss-of-function, and a certain degree of residual PI4KA the different neurological conditions associated with this gene. activity is retained. Despite a clear enrichment of missense var- Pyramidal tract involvement with increased limb muscle tone iants close to PI4KA’s active site hence supporting the pathogenic predominates in most of the patients in this cohort. Remarkably, role of these variants, we did not find a patient with two loss-of- Patient 2 shows severe global hypotonia with areflexia and abnor- function alleles, suggesting that the missense variants identified mal nerve conduction studies, indicating peripheral nervous sys- in this cohort may be hypomorphic. Patients 1, 4, 5 and 9 har- tem involvement, which is in line with the neuropathy described boured loss-of-function variants (out-of-frame indels or stop var- in a mouse model with Pi4ka inactivation restricted to Schwann iants) that were always in trans with missense variants located far cells.25 In comparison with patients with mutations in FAM126A from the active site [p.(Glu1152Lys), p.(Asp1664Asn), (HCC), another member of the PI4KA/TTC7/FAM126A complex,2 p.(Ala1198Thr)] or small in-frame mutations [Patient 9, four of our patients had an earlier clinical onset in the neonatal p.(Glu1820del)], suggesting that the impairment of PI4KA activity period and were neurologically more severely affected. resulting from these variants may have been less severe than that Furthermore, epilepsy appears to be more common than in HCC caused by other variants. Two patients harbouring the patients,26 and immunological or gastrointestinal symptoms have p.(Thr2053SerfsTer4) variant presented different phenotypes, indi- not been reported in patients with HCC. On the other hand, it is cating that p.(Ala1198Thr) (Patient 4) might be more deleterious striking that none of the patients in the present study developed than p.(Glu1820del) (Patient 9); however, phenotype-genotype cor- cataracts, which suggests that their presence may be directly due relation in a small cohort of patients poses a challenge. to malfunction of FAM126A. Peripheral neuropathy is an invariable Although the clinical manifestations presented by our cohort of feature of HCC, whereas it was only observed in one of the patients patients were prominently neurological, five patients presented reported here. Regarding neuroimaging, Patient 4 exhibited a with an immune disorder and/or a history of recurrent infections. hypomyelinating pattern with a more conspicuous periventricular On the other hand, one patient suffered from Crohn’s disease, T2 hyperintensity, similar to the pattern described in patients with whereas more non-specific manifestations, such as vomiting, diar- FAM126A mutations.26 In contrast, the polymicrogyria and brain- rhoea, constipation or gastroesophageal reflux disease, were pre- stem and cerebellar hypoplasia reported in our cohort have not sent in four patients. It is tempting to speculate that some of these been described in FAM126A patients. extraneurological manifestations could be caused by disruption Further differential diagnosis should be made with other dis- of the interactions between PI4KA and TTC7A, defects that have eases associated with hypomyelination, mainly PLP1 disorders, been associated with immunodeficiency and gastrointestinal and specifically disorders that present with cerebellar atrophy or manifestations.32 hypoplasia, such as Pol III-related disorders, 18q syndrome, The pathogenic effects of PI4KA mutations remain poorly TUBB4A- and RARS-associated hypomyelination and hereditary understood. PI4KA and PI(4)P levels were significantly diminished spastic paraplegia (both pure and complex forms). Disorders that in the fibroblasts of five patients. PI(4,5)P2, a product of PI(4)P phos- cause pontocerebellar hypoplasia and myelination delay should phorylation, is a substrate for the PI3K-AKT-mTOR pathway, also be taken into account.27 The combination of severe develop- which is critical for the myelination process.2,8 PIP levels were mental delay, motor impairment and early-onset seizures is a fea- reduced in the PBMCs of four patients, and specifically PI(4)P was ture of developmental and epileptic encephalopathies (DEEs), and diminished as shown in fibroblasts of five patients, suggesting patients with these diseases often show posterior fossa anomalies, that their use as biomarkers for diagnosis and prognosis may be cerebral dysgenesis or delayed myelination. Hence, this group of indicated. Moreover, and given the important role of PI(4)P in the diseases should also be considered.28 Finally, other disorders asso- transport of phosphatidylserine, a phospholipid abundantly found ciated with neuronal migration and polymicrogyria should be in myelin, to the plasma membrane,33,34 PI4KA malfunction may included in the differential diagnosis.29,30 lead to disturbances in the brain cell membrane lipidome, result- Previously, mutations in PI4KA were reported in three foetuses ing in aberrant myelination. Concordantly, PI4KA inhibition has from a single family who showed bilateral polymicrogyria with been shown to significantly reduce phosphatidylserine levels in hypoplasia/dysplasia of the cerebellum, olivary and dentate nu- cultured cells by inhibiting its transport to the plasma membrane, cleus abnormalities, joint contractures and overlapping fingers.31 a phenomenon also observed in Lenz-Majewski syndrome, a de- All three foetuses were compound heterozygous for two loss-of- velopmental condition that involves similar abnormalities in function variants: a premature stop variant [p.(Arg796Ter)] and a PI(4)P/phosphatidylserine metabolism.35 Furthermore, inositol missense variant located in the active site that abrogated kinase phospholipids are also important in processes such as actin activity [p.(Asp1854Asn)]. Therefore, the phenotypes presented by remodelling and oligodendrocyte membrane polarization, which these foetuses may represent the most severe end of the pheno- are essential for myelination. This process is similar to the actin typic spectrum of diseases associated with PI4KA mutations since polymerization-depolymerization sequence required for correct no abnormalities were detected during pregnancy in our cases. cell migration during brain development25 and may underlie both This suggests that our patients may retain higher residual PI4KA the myelination and neurodevelopmental defects we describe activity than previously reported patients.31 In support of this hy- (polymicrogyria and brainstem-cerebellar hypoplasia). Interesting- pothesis, Pi4ka mouse models harbouring two complete loss-of- ly, mice with knockout of Pi4k2a, another kinase involved in PI(4)P function alleles are embryonic lethal,5 while pi4ka knockdown formation from PI, develop late-onset features resembling heredi- zebrafish, which retain some Pi4ka activity, show multiple devel- tary spastic paraplegia, and the inhibition of PI4K activity disrupts opmental abnormalities.4 Interestingly, the only patient in our the retrograde axonal transport of neurotrophins.36 The alteration study who shared a variant with the family reported by of axonal transport is one of the main processes involved in her- Pagnamenta et al.31 [p.(Asp1854Asn)] also developed polymicrogy- editary spastic paraplegia37 and is also dependent on oligodendro- ria, suggesting that this variant may be specifically associated cyte function.38 with this developmental brain anomaly. Furthermore, the fact that Given that lipid metabolism defects appear to play an import- this variant in homozygosity leads to a milder phenotype than the ant role in the pathophysiology of PI4KA-associated disorders and foetuses in Pagnamenta et al.,31 who had the p.Asp1854Asn variant other leukodystrophies and hereditary spastic paraplegias, thera- in compound heterozygous state with a stop codon variant, peutic avenues for these patients could involve normalization of strongly suggests that this missense variant would not cause a lipidic disturbances impacting myelination.39,40 Alternatively, 2668 | BRAIN 2021: 144; 2659–2669 E. Verdura et al. upregulation of PI4KA expression or treatment with drugs that the NIMH (MH111661). D.R.A. and A.P. are members of the specifically treat secondary deficiencies in interacting proteins Undiagnosed Disease Network International (UDNI). (TTC7B and FAM126A) may prove helpful in alleviating symptoms in the most severe patients in the future.41,42 Competing interests In summary, we describe a novel inherited error of metabolism caused by PI4KA malfunction resulting in a broad phenotypic spec- The authors report no competing interests. trum ranging from severe global neurodevelopmental delay asso- ciated with hypomyelinating leukodystrophy and/or brain Supplementary material developmental anomalies such as pontocerebellar hypoplasia/at- Supplementary material is available at Brain online. rophy (or even polymicrogyria) in the most severe forms to spastic paraplegia in milder cases. The presence of immunological defi- cits, gastrointestinal manifestations or genito-urinary abnormal- References ities may be helpful for the diagnosis of these patients. 1. Burke JE. Structural basis for regulation of phosphoinositide kinases and their involvement in human disease. Mol Cell. 2018; Acknowledgements 71(5):653–673. 2. 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