. 2021 Oct;23(10):1873-1881.

doi: 10.1038/s41436-021-01215-9. Epub 2021 Jun 10.

PIGG variant pathogenicity assessment reveals characteristic features within 19 families

Camille Tremblay-Laganière¹, Reza Maroofian², Thi Tuyet Mai Nguyen¹, Ehsan Ghayoor Karimiani^{3

4}, Salman Kirmani⁵, Fizza Akbar⁵, Shahnaz Ibrahim⁵, Bushra Afroze⁵, Mohammad Doosti⁴, Farah Ashrafzadeh⁶, Meisam Babaei⁷, Stephanie Efthymiou², Marilena Christoforou², Tipu Sultan⁸, Roger L Ladda⁹, Heather M McLaughlin¹⁰, Rebecca Truty¹⁰, Sonal Mahida¹¹, Julie S Cohen^{11

12}, Kristin Baranano^{11

12}, Fatima Y Ismail^{12

13}, Millan S Patel¹⁴, Anna Lehman¹⁴, Andrew C Edmondson¹⁵, Amanda Nagy¹⁶, Melissa A Walker¹⁶, Saadet Mercimek-Andrews^{17

18}, Yuta Maki^{19

20}, Rani Sachdev^{21

22}, Rebecca Macintosh²¹, Elizabeth E Palmer^{21

22}, Grazia M S Mancini²³, Tahsin Stefan Barakat²³, Robert Steinfeld²⁴, Christina T Rüsch²⁴, Georg M Stettner²⁴, Matias Wagner^{25

26}, Saskia B Wortmann^{27

28}, Usha Kini²⁹, Angela F Brady³⁰, Karen L Stals³¹, Naila Ismayilova³², Sian Ellard^{31

33}, Danilo Bernardo³⁴, Kimberly Nugent³⁵, Scott D McLean³⁵, Stylianos E Antonarakis³⁶, Henry Houlden², Taroh Kinoshita^{37

38}, Philippe M Campeau³⁹, Yoshiko Murakami^{40

41}

Affiliations

¹ Department of Pediatrics, Centre Hospitalier Universitaire Sainte-Justine and University of Montreal, Montreal, QC, Canada.
² Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology and The National Hospital for Neurology and Neurosurgery, London, UK.
³ Genetics Research Centre, Molecular and Clinical Sciences Institute, St. George's Hospital, University of London, London, UK.
⁴ Next Generation Genetic Polyclinic, Mashhad, Iran.
⁵ Department of Pediatrics & Child Health, Aga Khan University, Karachi, Pakistan.
⁶ Department of Pediatric Neurology, Mashhad University of Medical Sciences, Mashhad, Iran.
⁷ Department of Pediatrics, North Khorasan University of Medical Sciences, Bojnurd, Iran.
⁸ Department of Pediatric Neurology, Institute of Child Health, The Children's Hospital Lahore, Lahore, Pakistan.
⁹ Department of Pediatrics, Milton S Hershey Medical Centre, Hershey, PA, USA.
¹⁰ Invitae Corporation, San Francisco, CA, USA.
¹¹ Division of Neurogenetics, Kennedy Krieger Institute, Baltimore, MD, USA.
¹² Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
¹³ Department of Pediatrics, United Arab Emirates University, Al Ain, UAE.
¹⁴ Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada.
¹⁵ Division of Human Genetics, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
¹⁶ Department of Neurology, Massachusetts General Hospital, Boston, MA, USA.
¹⁷ Division of Clinical and Metabolic Genetics, Department of Pediatrics, University of Toronto, The Hospital for Sick Children, Toronto, ON, Canada.
¹⁸ Department of Medical Genetics, Faculty of Medicine & Dentistry, University of Alberta, Stollery Children's Hospital, Alberta Health Services, Edmonton, AB, Canada.
¹⁹ Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.
²⁰ Project Research Center for Fundamental Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.
²¹ Sydney Children's Hospital, Centre for Clinical Genetics, Sydney Children's Hospital, High St, Randwick, UK.
²² School of Women's and Children's Health, University of New South Wales, High St, Randwick, UK.
²³ Department of Clinical Genetics, Erasmus MC University Medical Center, Rotterdam, The Netherlands, CA, Rotterdam, The Netherlands.
²⁴ Department of Pediatric Neurology, University Children's Hospital Zurich, University of Zurich, Zurich, Switzerland.
²⁵ Institute of Human Genetics, School of Medicine, Technical University Munich, Munich, Germany.
²⁶ Institute for Neurogenomics Helmholtz Zentrum München, Neuherberg, Germany.
²⁷ University Children's Hospital, Paracelsus Medical School, Salzburg, Austria.
²⁸ Amalias Children's Hospital, RadboudUMC, Nijmegen, the Netherlands.
²⁹ Oxford Centre for Genomic Medicine, Oxford University Hospitals NHS Trust, Oxford, UK.
³⁰ North West Thames Regional Genetics Service, London North West University Healthcare NHS Trust, Northwick Park Hospital, Harrow, UK.
³¹ Exeter Genomics Laboratory, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK.
³² Department of Paediatric Neurology, Chelsea and Westminster Hospital, London, UK.
³³ Institute of Biomedical and Clinical Science, College of Medicine and Health, University of Exeter, Exeter, UK.
³⁴ University of California San Francisco, Clinical Neurology, San Francisco, CA, UK.
³⁵ Department of Pediatrics, Baylor College of Medicine, The Children's Hospital of San Antonio, San Antonio, TX, USA.
³⁶ Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland.
³⁷ Yabumoto Department of Intractable Disease Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
³⁸ Department of Immunoglycobiology, World Premier International Immunology Frontier Research Center, Osaka University, Osaka, Japan.
³⁹ Department of Pediatrics, Centre Hospitalier Universitaire Sainte-Justine and University of Montreal, Montreal, QC, Canada. p.campeau@umontreal.ca.
⁴⁰ Yabumoto Department of Intractable Disease Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan. yoshiko@biken.osaka-u.ac.jp.
⁴¹ Department of Immunoglycobiology, World Premier International Immunology Frontier Research Center, Osaka University, Osaka, Japan. yoshiko@biken.osaka-u.ac.jp.

PMID: 34113002
PMCID: PMC9900493
DOI: 10.1038/s41436-021-01215-9

PIGG variant pathogenicity assessment reveals characteristic features within 19 families

Camille Tremblay-Laganière et al. Genet Med. 2021 Oct.

. 2021 Oct;23(10):1873-1881.

doi: 10.1038/s41436-021-01215-9. Epub 2021 Jun 10.

Authors

Affiliations

¹ Department of Pediatrics, Centre Hospitalier Universitaire Sainte-Justine and University of Montreal, Montreal, QC, Canada.
² Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology and The National Hospital for Neurology and Neurosurgery, London, UK.
³ Genetics Research Centre, Molecular and Clinical Sciences Institute, St. George's Hospital, University of London, London, UK.
⁴ Next Generation Genetic Polyclinic, Mashhad, Iran.
⁵ Department of Pediatrics & Child Health, Aga Khan University, Karachi, Pakistan.
⁶ Department of Pediatric Neurology, Mashhad University of Medical Sciences, Mashhad, Iran.
⁷ Department of Pediatrics, North Khorasan University of Medical Sciences, Bojnurd, Iran.
⁸ Department of Pediatric Neurology, Institute of Child Health, The Children's Hospital Lahore, Lahore, Pakistan.
⁹ Department of Pediatrics, Milton S Hershey Medical Centre, Hershey, PA, USA.
¹⁰ Invitae Corporation, San Francisco, CA, USA.
¹¹ Division of Neurogenetics, Kennedy Krieger Institute, Baltimore, MD, USA.
¹² Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
¹³ Department of Pediatrics, United Arab Emirates University, Al Ain, UAE.
¹⁴ Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada.
¹⁵ Division of Human Genetics, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
¹⁶ Department of Neurology, Massachusetts General Hospital, Boston, MA, USA.
¹⁷ Division of Clinical and Metabolic Genetics, Department of Pediatrics, University of Toronto, The Hospital for Sick Children, Toronto, ON, Canada.
¹⁸ Department of Medical Genetics, Faculty of Medicine & Dentistry, University of Alberta, Stollery Children's Hospital, Alberta Health Services, Edmonton, AB, Canada.
¹⁹ Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.
²⁰ Project Research Center for Fundamental Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.
²¹ Sydney Children's Hospital, Centre for Clinical Genetics, Sydney Children's Hospital, High St, Randwick, UK.
²² School of Women's and Children's Health, University of New South Wales, High St, Randwick, UK.
²³ Department of Clinical Genetics, Erasmus MC University Medical Center, Rotterdam, The Netherlands, CA, Rotterdam, The Netherlands.
²⁴ Department of Pediatric Neurology, University Children's Hospital Zurich, University of Zurich, Zurich, Switzerland.
²⁵ Institute of Human Genetics, School of Medicine, Technical University Munich, Munich, Germany.
²⁶ Institute for Neurogenomics Helmholtz Zentrum München, Neuherberg, Germany.
²⁷ University Children's Hospital, Paracelsus Medical School, Salzburg, Austria.
²⁸ Amalias Children's Hospital, RadboudUMC, Nijmegen, the Netherlands.
²⁹ Oxford Centre for Genomic Medicine, Oxford University Hospitals NHS Trust, Oxford, UK.
³⁰ North West Thames Regional Genetics Service, London North West University Healthcare NHS Trust, Northwick Park Hospital, Harrow, UK.
³¹ Exeter Genomics Laboratory, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK.
³² Department of Paediatric Neurology, Chelsea and Westminster Hospital, London, UK.
³³ Institute of Biomedical and Clinical Science, College of Medicine and Health, University of Exeter, Exeter, UK.
³⁴ University of California San Francisco, Clinical Neurology, San Francisco, CA, UK.
³⁵ Department of Pediatrics, Baylor College of Medicine, The Children's Hospital of San Antonio, San Antonio, TX, USA.
³⁶ Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland.
³⁷ Yabumoto Department of Intractable Disease Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
³⁸ Department of Immunoglycobiology, World Premier International Immunology Frontier Research Center, Osaka University, Osaka, Japan.
³⁹ Department of Pediatrics, Centre Hospitalier Universitaire Sainte-Justine and University of Montreal, Montreal, QC, Canada. p.campeau@umontreal.ca.
⁴⁰ Yabumoto Department of Intractable Disease Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan. yoshiko@biken.osaka-u.ac.jp.
⁴¹ Department of Immunoglycobiology, World Premier International Immunology Frontier Research Center, Osaka University, Osaka, Japan. yoshiko@biken.osaka-u.ac.jp.

PMID: 34113002
PMCID: PMC9900493
DOI: 10.1038/s41436-021-01215-9

Abstract

Purpose: Phosphatidylinositol Glycan Anchor Biosynthesis, class G (PIGG) is an ethanolamine phosphate transferase catalyzing the modification of glycosylphosphatidylinositol (GPI). GPI serves as an anchor on the cell membrane for surface proteins called GPI-anchored proteins (GPI-APs). Pathogenic variants in genes involved in the biosynthesis of GPI cause inherited GPI deficiency (IGD), which still needs to be further characterized.

Methods: We describe 22 individuals from 19 unrelated families with biallelic variants in PIGG. We analyzed GPI-AP surface levels on granulocytes and fibroblasts for three and two individuals, respectively. We demonstrated enzymatic activity defects for PIGG variants in vitro in a PIGG/PIGO double knockout system.

Results: Phenotypic analysis of reported individuals reveals shared PIGG deficiency-associated features. All tested GPI-APs were unchanged on granulocytes whereas CD73 level in fibroblasts was decreased. In addition to classic IGD symptoms such as hypotonia, intellectual disability/developmental delay (ID/DD), and seizures, individuals with PIGG variants of null or severely decreased activity showed cerebellar atrophy, various neurological manifestations, and mitochondrial dysfunction, a feature increasingly recognized in IGDs. Individuals with mildly decreased activity showed autism spectrum disorder.

Conclusion: This in vitro system is a useful method to validate the pathogenicity of variants in PIGG and to study PIGG physiological functions.

PubMed Disclaimer

Conflict of interest statement

COMPETING INTERESTS

The authors declare no competing interests.

Figures

**Fig. 1. Pedigrees and genotypes of families with biallelic *PIGG* variants.**
Filled squares correspond to affected males and filled circles correspond to affected females. Open squares or circles with a center dot correspond to male or female unaffected carriers.

**Fig. 2. Clinical findings.**
(a) Photographs from individual 1 showing facial dysmorphism. This individual present with mildly coarse face, strabismus, long philtrum, and shallow nose tip. (b) Magnetic resonance image (MRI) from affected individuals showing cerebellar atrophy. Individual 8A: T1 weighted sagittal image shows minimal increase in the interfoliate distance of cerebellum superiorly at the age of 3 years. Individual 8B: T1 weighted sagittal image shows atrophy of the anterior lobe of the cerebellum (superior vermian atrophy) at the age of 2 years. Individual 13B: sagittal, coronal, and transverse MRI images at 12 years old showing cerebellar vermian atrophy predominantly affecting the superior vermis. Cerebellar atrophy was found in individuals 2, 3, 8A, 8B, 11, 12, 13A, 13B, and 19.

**Fig. 3. PIGG protein structure and location of variants.**
(a) Figure generated using ProteinPaint. Variants from affected individuals are in blue and matched according to each family’s genotype (F01 to F19). Transmembrane and luminal domains are from UniProt. Already published pathogenic variants are in green.^,, ClinVar likely pathogenic and pathogenic variants are in yellow and red respectively. (b) Figure generated using Protter. Transmembrane domains are from UniProt and numbered 1 to 13. Missense variants are in pink and the two nonsense variants resulting in a truncated protein are in blue (p.Tyr934Ter and p.Tyr957Ter). (c) Conservation of affected residues and C-term region across vertebrates. Multiple alignment from UCSC Genome Browser.

**Fig. 4. Functional analysis of *PIGG* variants.**
(a) Fluorescence-activated cell sorting (FACS) from granulocytes of affected individuals. GPI-anchored proteins (GPI-AP) surface levels (FLAER, CD16, CD55, and CD59) on granulocytes for individuals 8A (red), 8B (green), and 13A (green) compared to their unaffected heterozygous mother (purple), father (blue), and unrelated controls (black). (b) FACS from fibroblasts of affected individuals. GPI-AP surface levels (FLAER, CD73, and CD109) on fibroblasts from individuals 16 and 17 (green) compared to control (black). (c) Functional assay using strong promoter-driven PIGG variants. HEK293 *PIGO/PIGG* DKO cells were transfected with strong promoter-driven (pME) wild-type or mutant *PIGG* complementary DNA (cDNA). Two days later, expression of DAF was analyzed by FACS. Most of the variants could not rescue the expression at all except F13, showing partial rescue. Blue percentages indicates % of rescue compared to wild-type *PIGG*. PIGG variant activity was shown by the percentage of geometric mean of DAF fluorescent intensity of each PIGG variant transfectant against that of the PIGG wild-type transfectant. (d) Functional assay using the weak promoter to increase sensitivity for variant dysfunction. HEK293 *PIGO/PIGG* DKO cells were transfected with strong promoter-driven (pME) or a weak promoter-driven (pTK) wild-type or mutant *PIGG* cDNA. Two days later, expression of DAF was analyzed by FACS except for F17 (pTK), showing expression of CD59. All variants could rescue similar level to wild-type *PIGG* when using strong promoter (100% of rescue). Driven by weak promoter, variants showed various level of restoration of GPI-APs. PIGG variant activity was shown by the percentage of geometric mean of DAF fluorescent intensity of each PIGG variant transfectant against that of the PIGG wild-type transfectant.

See this image and copyright information in PMC

References

1. Zhao JJ et al. Reduced cell surface levels of GPI-linked markers in a new casewith PIGG loss of function. Hum. Mutat 38, 1394–1401 (2017). - PMC - PubMed
1. Murakami Y. et al. Mutations in PIGB cause an inherited GPI biosynthesis defectwith an axonal neuropathy and metabolic abnormality in severe cases. Am. J. Hum. Genet 105, 384–394 (2019). - PMC - PubMed
1. Makrythanasis P. et al. Pathogenic variants in PIGG cause intellectual disabilitywith seizures and hypotonia. Am. J. Hum. Genet 98, 615–626 (2016). - PMC - PubMed
1. Murakami Y. et al. Mechanism for release of alkaline phosphatase caused byglycosylphosphatidylinositol deficiency in patients with hyperphosphatasia mental retardation syndrome. J. Biol. Chem 287, 6318–6325 (2012). - PMC - PubMed
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PIGG variant pathogenicity assessment reveals characteristic features within 19 families

Affiliations

PIGG variant pathogenicity assessment reveals characteristic features within 19 families

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

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Grants and funding

LinkOut - more resources

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Medical

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