. 2019 Jul;40(7):908-925.

doi: 10.1002/humu.23731. Epub 2019 Apr 24.

SLC35A2-CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals

Bobby G Ng¹, Paulina Sosicka¹, Satish Agadi², Mohammed Almannai², Carlos A Bacino^{2

3}, Rita Barone^{4

5}, Lorenzo D Botto⁶, Jennifer E Burton⁷, Colleen Carlston⁸, Brian Hon-Yin Chung⁹, Julie S Cohen¹⁰, David Coman^{11

12}, Katrina M Dipple^{13

14

15}, Naghmeh Dorrani¹⁶, William B Dobyns^{17

18}, Abdallah F Elias¹⁹, Leon Epstein²⁰, William A Gahl^{21

22}, Domenico Garozzo⁵, Trine Bjørg Hammer²³, Jaclyn Haven¹⁹, Delphine Héron²⁴, Matthew Herzog¹⁵, George E Hoganson⁷, Jesse M Hunter²⁵, Mahim Jain¹⁰, Jane Juusola²⁶, Shenela Lakhani²⁷, Hane Lee^{15

28}, Joy Lee^{29

30}, Katherine Lewis¹¹, Nicola Longo⁶, Charles Marques Lourenço³¹, Christopher C Y Mak⁹, Dianalee McKnight²⁶, Bryce A Mendelsohn³², Cyril Mignot²⁴, Ghayda Mirzaa^{17

18}, Wendy Mitchell^{33

34}, Hiltrud Muhle³⁵, Stanley F Nelson^{15

28

36}, Mariusz Olczak³⁷, Christina G S Palmer^{15

36

38}, Arthur Partikian³⁹, Marc C Patterson⁴⁰, Tyler M Pierson^{41

42

43}, Shane C Quinonez⁴⁴, Brigid M Regan⁴⁵, M Elizabeth Ross²⁷, Maria J Guillen Sacoto²⁶, Fernando Scaglia^{2

3

46}, Ingrid E Scheffer^{45

47}, Devorah Segal^{27

48}, Nilika Shah Singhal⁴⁹, Pasquale Striano⁵⁰, Luisa Sturiale⁵, Joseph D Symonds⁵¹, Sha Tang²⁵, Eric Vilain⁵², Mary Willis⁵³, Lynne A Wolfe^{21

22}, Hui Yang²⁶, Shoji Yano⁵⁴, Zöe Powis²⁵, Sharon F Suchy²⁵, Jill A Rosenfeld²⁶, Andrew C Edmondson², Stephanie Grunewald⁵⁵, Hudson H Freeze⁵⁶

Affiliations

¹ Human Genetics Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California.
² Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas.
³ Texas Children's Hospital, Houston, Texas.
⁴ Department of Clinical and Experimental Medicine, Child Neurology and Psychiatry, University of Catania, Catania, Italy.
⁵ CNR, Institute for Polymers, Composites and Biomaterials, Catania, Italy.
⁶ Departments of Pediatrics, Division of Medical Genetics, University of Utah, Salt Lake City, Utah.
⁷ Department of Pediatrics, University of Illinois College of Medicine, Peoria, Illinois.
⁸ Department of Pathology, University of Utah, Salt Lake City, Utah.
⁹ Department of Paediatrics & Adolescent Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
¹⁰ Division of Neurogenetics and Hugo W. Moser Research Institute, Kennedy Krieger Institute, Baltimore, Maryland.
¹¹ Department of Metabolic Medicine, Queensland Children's Hospital, Brisbane, Australia.
¹² Schools of Medicine, University of Queensland Brisbane, Griffith University Gold Coast, Brisbane, Australia.
¹³ Department of Pediatrics, University of Washington, Seattle, Washington.
¹⁴ Seattle Children's Hospital, Seattle, Washington.
¹⁵ Department of Human Genetics, UCLA, Los Angeles, California.
¹⁶ Department of Pediatrics, UCLA, Los Angeles, California.
¹⁷ Departments of Pediatrics, University of Washington, Seattle, Washington.
¹⁸ Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, Washington.
¹⁹ Department of Medical Genetics, Shodair Children's Hospital, Helena, Montana.
²⁰ Northwestern University Feinberg School of Medicine, Chicago, Illinois.
²¹ Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland.
²² Undiagnosed Diseases Program, Common Fund, National Institutes of Health, Bethesda, Maryland.
²³ Danish Epilepsy Center-Filadelfia, Dianalund, Denmark.
²⁴ APHP,Genetics Department, GH Pity Salpetriere, CRMR Intellectual Disabilities of Rare Causes, Sorbonne University, Paris, France.
²⁵ Ambry Genetics, Aliso Viejo, California.
²⁶ GeneDx, Gaithersburg, Maryland.
²⁷ Center for Neurogenetics Brain and Mind Research Institute Weill Cornell Medicine, New York, New York.
²⁸ Department of Pathology and Laboratory Medicine, UCLA, Los Angeles, California.
²⁹ Department of Metabolic Medicine, The Royal Children's Hospital, Melbourne, Parkville, Victoria, Australia.
³⁰ Department of Paediatrics, University of Melbourne, Parkville, Victoria, Australia.
³¹ Clinical Genetics and Neurogenetics, Centro Universitario Estacio de Ribeirao Preto, Ribeirao Preto, Brazil.
³² Department of Pediatrics, Division of Medical Genetics, University of California, San Francisco, San Francisco, California.
³³ Neurology Division Children's Hospital Los Angeles, Los Angeles, California.
³⁴ Department of Neurology, Keck School of Medicine, University of Southern California, Los Angeles, California.
³⁵ Department of Neuropediatrics, Christian-Albrechts-University of Kiel, Kiel, Germany.
³⁶ Department of Psychiatry & Biobehavioral Sciences, UCLA, Los Angeles, California.
³⁷ Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland.
³⁸ Institute for Society and Genetics, UCLA, Los Angeles, California.
³⁹ Departments of Pediatrics & Neurology, Keck School of Medicine of University of Southern California, Los Angeles, California.
⁴⁰ Division of Child and Adolescent Neurology, Mayo Clinic, Rochester, Minnesota.
⁴¹ Department of Pediatrics, Cedars-Sinai Medical Center, Los Angeles, California.
⁴² Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California.
⁴³ Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California.
⁴⁴ Division of Genetics, Department of Pediatrics, Metabolism and Genomic Medicine, University of Michigan, Ann Arbor, Michigan.
⁴⁵ The University of Melbourne, Austin Health, Melbourne, Australia.
⁴⁶ Joint BCM-CUHK Center of Medical Genetics, Prince of Wales Hospital, ShaTin, Hong Kong SAR.
⁴⁷ The University of Melbourne, Royal Children's Hospital, Florey Institute and Murdoch Children's Research Institute, Melbourne, Australia.
⁴⁸ Department of Pediatrics, Division of Child Neurology, Weill Cornell Medicine, New York, New York.
⁴⁹ Neurology & Pediatrics, University of California, San Francisco, California.
⁵⁰ Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, Pediatric Neurology and Muscular Diseases Unit, G. Gaslini Institute, University of Genoa, Genova, Italy.
⁵¹ Paediatric Neurosciences Research Group, Royal Hospital for Children, Queen Elizabeth University Hospitals, Glasgow, UK.
⁵² Center for Genetic Medicine Research, Children's National Medical Center, Columbia, Washington.
⁵³ Department of Pediatrics, Naval Medical Center, San Diego, California.
⁵⁴ Department of Pediatrics, Genetics Division, LAC+USC Medical Center, University of Southern California, Los Angeles, California.
⁵⁵ Department of Pediatrics, Division of Human Genetics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
⁵⁶ Metabolic Unit, Great Ormond Street Hospital NHS Trust, Institute for Child Health UCL, London, UK.

PMID: 30817854
PMCID: PMC6661012
DOI: 10.1002/humu.23731

SLC35A2-CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals

Bobby G Ng et al. Hum Mutat. 2019 Jul.

. 2019 Jul;40(7):908-925.

doi: 10.1002/humu.23731. Epub 2019 Apr 24.

Authors

Affiliations

¹ Human Genetics Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California.
² Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas.
³ Texas Children's Hospital, Houston, Texas.
⁴ Department of Clinical and Experimental Medicine, Child Neurology and Psychiatry, University of Catania, Catania, Italy.
⁵ CNR, Institute for Polymers, Composites and Biomaterials, Catania, Italy.
⁶ Departments of Pediatrics, Division of Medical Genetics, University of Utah, Salt Lake City, Utah.
⁷ Department of Pediatrics, University of Illinois College of Medicine, Peoria, Illinois.
⁸ Department of Pathology, University of Utah, Salt Lake City, Utah.
⁹ Department of Paediatrics & Adolescent Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
¹⁰ Division of Neurogenetics and Hugo W. Moser Research Institute, Kennedy Krieger Institute, Baltimore, Maryland.
¹¹ Department of Metabolic Medicine, Queensland Children's Hospital, Brisbane, Australia.
¹² Schools of Medicine, University of Queensland Brisbane, Griffith University Gold Coast, Brisbane, Australia.
¹³ Department of Pediatrics, University of Washington, Seattle, Washington.
¹⁴ Seattle Children's Hospital, Seattle, Washington.
¹⁵ Department of Human Genetics, UCLA, Los Angeles, California.
¹⁶ Department of Pediatrics, UCLA, Los Angeles, California.
¹⁷ Departments of Pediatrics, University of Washington, Seattle, Washington.
¹⁸ Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, Washington.
¹⁹ Department of Medical Genetics, Shodair Children's Hospital, Helena, Montana.
²⁰ Northwestern University Feinberg School of Medicine, Chicago, Illinois.
²¹ Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland.
²² Undiagnosed Diseases Program, Common Fund, National Institutes of Health, Bethesda, Maryland.
²³ Danish Epilepsy Center-Filadelfia, Dianalund, Denmark.
²⁴ APHP,Genetics Department, GH Pity Salpetriere, CRMR Intellectual Disabilities of Rare Causes, Sorbonne University, Paris, France.
²⁵ Ambry Genetics, Aliso Viejo, California.
²⁶ GeneDx, Gaithersburg, Maryland.
²⁷ Center for Neurogenetics Brain and Mind Research Institute Weill Cornell Medicine, New York, New York.
²⁸ Department of Pathology and Laboratory Medicine, UCLA, Los Angeles, California.
²⁹ Department of Metabolic Medicine, The Royal Children's Hospital, Melbourne, Parkville, Victoria, Australia.
³⁰ Department of Paediatrics, University of Melbourne, Parkville, Victoria, Australia.
³¹ Clinical Genetics and Neurogenetics, Centro Universitario Estacio de Ribeirao Preto, Ribeirao Preto, Brazil.
³² Department of Pediatrics, Division of Medical Genetics, University of California, San Francisco, San Francisco, California.
³³ Neurology Division Children's Hospital Los Angeles, Los Angeles, California.
³⁴ Department of Neurology, Keck School of Medicine, University of Southern California, Los Angeles, California.
³⁵ Department of Neuropediatrics, Christian-Albrechts-University of Kiel, Kiel, Germany.
³⁶ Department of Psychiatry & Biobehavioral Sciences, UCLA, Los Angeles, California.
³⁷ Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland.
³⁸ Institute for Society and Genetics, UCLA, Los Angeles, California.
³⁹ Departments of Pediatrics & Neurology, Keck School of Medicine of University of Southern California, Los Angeles, California.
⁴⁰ Division of Child and Adolescent Neurology, Mayo Clinic, Rochester, Minnesota.
⁴¹ Department of Pediatrics, Cedars-Sinai Medical Center, Los Angeles, California.
⁴² Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California.
⁴³ Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California.
⁴⁴ Division of Genetics, Department of Pediatrics, Metabolism and Genomic Medicine, University of Michigan, Ann Arbor, Michigan.
⁴⁵ The University of Melbourne, Austin Health, Melbourne, Australia.
⁴⁶ Joint BCM-CUHK Center of Medical Genetics, Prince of Wales Hospital, ShaTin, Hong Kong SAR.
⁴⁷ The University of Melbourne, Royal Children's Hospital, Florey Institute and Murdoch Children's Research Institute, Melbourne, Australia.
⁴⁸ Department of Pediatrics, Division of Child Neurology, Weill Cornell Medicine, New York, New York.
⁴⁹ Neurology & Pediatrics, University of California, San Francisco, California.
⁵⁰ Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, Pediatric Neurology and Muscular Diseases Unit, G. Gaslini Institute, University of Genoa, Genova, Italy.
⁵¹ Paediatric Neurosciences Research Group, Royal Hospital for Children, Queen Elizabeth University Hospitals, Glasgow, UK.
⁵² Center for Genetic Medicine Research, Children's National Medical Center, Columbia, Washington.
⁵³ Department of Pediatrics, Naval Medical Center, San Diego, California.
⁵⁴ Department of Pediatrics, Genetics Division, LAC+USC Medical Center, University of Southern California, Los Angeles, California.
⁵⁵ Department of Pediatrics, Division of Human Genetics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
⁵⁶ Metabolic Unit, Great Ormond Street Hospital NHS Trust, Institute for Child Health UCL, London, UK.

PMID: 30817854
PMCID: PMC6661012
DOI: 10.1002/humu.23731

Abstract

Pathogenic de novo variants in the X-linked gene SLC35A2 encoding the major Golgi-localized UDP-galactose transporter required for proper protein and lipid glycosylation cause a rare type of congenital disorder of glycosylation known as SLC35A2-congenital disorders of glycosylation (CDG; formerly CDG-IIm). To date, 29 unique de novo variants from 32 unrelated individuals have been described in the literature. The majority of affected individuals are primarily characterized by varying degrees of neurological impairments with or without skeletal abnormalities. Surprisingly, most affected individuals do not show abnormalities in serum transferrin N-glycosylation, a common biomarker for most types of CDG. Here we present data characterizing 30 individuals and add 26 new variants, the single largest study involving SLC35A2-CDG. The great majority of these individuals had normal transferrin glycosylation. In addition, expanding the molecular and clinical spectrum of this rare disorder, we developed a robust and reliable biochemical assay to assess SLC35A2-dependent UDP-galactose transport activity in primary fibroblasts. Finally, we show that transport activity is directly correlated to the ratio of wild-type to mutant alleles in fibroblasts from affected individuals.

Keywords: UDP-galactose; congenital disorders of glycosylation; glycoside; nucleotide sugar transporter.

PubMed Disclaimer

Conflict of interest statement

CONFLICT OF INTEREST STATEMENT

The Department of Molecular and Human Genetics at Baylor College of Medicine derives revenue from clinical genetic testing completed at Baylor Genetics Laboratories. Fernando Scaglia is involved in clinical trials supported by BioElectron, Reata Pharmaceuticals, and Stealth BioTherapeutics. Mary Willis declares views expressed herein are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the U.S. Government. Zöe Powis, Jesse M. Hunter, Sha Tang are employed by Ambry Genetics. Sharon F. Suchy, Jane Juusola, Dianalee McKnight, Maria Guillen Sacato and Hui Yang are employed by GeneDx, Inc., a wholly-owned subsidiary of OPKO Health, Inc. The other authors have no conflict.

Figures

**Figure 1.**
Schematic representation of the *SLC35A2* variants present in our 30 individuals. **(A)** Schematic representation showing exon location of all 30 *SLC35A2* variants identified in this study. (B) Schematic representation showing protein localization of all 30 SLC35A2 variants identified in this study. SLC35A2 topology was determined using 3D model generated with PHYRE2 server (Kelley, Mezulis, Yates, Wass & Sternberg, 2015).

**Figure 2.**
Clinical summary of 30 individuals with *de novo* variants in *SLC35A2*. Clinical information for each of the 30 individuals were provided by clinicians and summarized as a percentage of affected individuals.

**Figure 3.**
Optimization of the glycoside-based UDP-galactose transport assay. (A) GlcNAcβ−4-MU concentration, temperature and time of the assay were optimized. Glycoside concentration was tested using CHO (circles) and CHO-Lec8 (squares) cells. Temperature and time were optimized only on CHO lines. Transport efficiency at room temperature (diamonds) and 37 °C (triangles) was compared using increasing concentrations of GlcNAcβ−4-MU. Time curve (asterisk) was assayed at 37°C with 1 mM glycoside. (B) UDP-galactose transport was measured in CHO cells using optimized conditions. As a negative control CHO-Lec8 cells were employed. Specificity of the assay was determined by treating CHO cells with 0.025% Trition-X100 (CHO + TX100). The assay was performed in three biological repetitions. Error bars represent SD (C) Michaelis constant and the maximum UDP-galactose transport velocity were determined for CHO protein in optimized assay conditions. Each point represents a mean of 3 to 6 independent biological repetitions using different cell preparations and performed on different days. Error bars represents SEM. CPM – Counts Per Minute

**Figure 4.**
UDP-galactose transport assay in primary fibroblasts. UDP-[6-³H] galactose transport in permeabilized fibroblast cells with intact Golgi apparatus under optimized conditions. Each assay was performed in three biological repetitions and expressed as a percent of the control (* p < 0.05; ** p < 0.005; *** p<0.001).

**Figure 5.**
Determination of the wild-type to mutant allele ratios in fibroblasts (A) *SLC35A2* cDNA allele ratios in primary fibroblasts from control and SLC35A2-CDG subjects. Wild-type to mutant allele ratios were determined using a restriction enzyme-based assay. GM-00038, GM-03348 and GM-05381 cells were used as controls. Each assay was performed in duplicate in order to determine reproducibility of the assay. (B) UDP-galactose transporter activity was calculated based on allele ratio presented as a percentage of GM-00038 transport activity. To illustrate the activity of the respective mutant protein, the contribution of wild-type protein was subtracted.

**Figure 6.**
Western Blot analysis of SLC35A2 protein from primary fibroblast. (A) Western Blot analysis of SLC35A2 protein from a total of seven SLC35A2-CDG fibroblast lines as well as three controls (GM-00038, GM-03348 and GM-05381). Anti-α-tubulin antibody was used as a loading control. (B) Quantification was performed by normalizing SLC35A2 to α-tubulin from four independent blots using two biological replicates collected on separate days with error bars calculated as a StdDev.

**Figure 7.**
Immunofluorescence staining of primary fibroblasts. To assess glycosylation between control (GM-00038, GM-03348 and GM-05381) and subject fibroblasts (CDG-0187, CDG-0389, CDG-0416, CDG-0460, CDG-0468, CDG-0469 and CDG-1039) VVL lectin was used (green). This lectin specifically recognizes terminal GalNAc present in O-glycans, which is usually masked by galactose and blocks the lectin reactivity. UDP-galactose transporter was counterstained with anti-SLC35A2 antibody (red). Scale bar 20 μm.

**Figure 8.**
Characterization of CHO-Lec8 stable clones expressing SLC35A2. (A) UDP-[6-³H] galactose transport assay in parental CHO, CHO-Lec8 and CHO-Lec8 cells overexpressing respective variants of SLC35A2 protein. For better comparison, fibroblast results from control (GM-00038) and four affected individuals (CDG-0389, CDG-0416, CDG-0460, CDG-1039) were plotted. The transport for SLC35A2-CDG fibroblasts was expressed as the predicted activity of mutant transporter by subtracting the contribution of wild type protein, based on the calculated allele ratio. Each assay was performed in two biological repetitions. (B) Immunofluorescence staining of CHO-Lec8 cells overexpressing UDP-galactose transporter. Overexpressed HA-tagged SLC35A2 protein was detected with anti-HA antibody (green). VVL lectin staining was used to detect glycosylation changes between CHO-Lec8 cells which do or do not overexpress the transporter (red). Stable transfectants, but not clonal populations were analyzed. Scale bar 20 μm.

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References

1. Allen RC, Zoghbi HY, Moseley AB, Rosenblatt HM, & Belmont JW (1992). Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation. Am J Hum Genet, 51(6), 1229–1239. - PMC - PubMed
1. Bosch DG, Boonstra FN, de Leeuw N, Pfundt R, Nillesen WM, de Ligt J, … de Vries BB (2016). Novel genetic causes for cerebral visual impairment. Eur J Hum Genet, 24(5), 660–665. doi: 10.1038/ejhg.2015.186 - DOI - PMC - PubMed
1. Brandli AW, Hansson GC, Rodriguez-Boulan E, & Simons K (1988). A polarized epithelial cell mutant deficient in translocation of UDP-galactose into the Golgi complex. J Biol Chem, 263(31), 16283–16290. - PubMed
1. Brockhausen I, & Stanley P (2015). O-GalNAc Glycans In rd, Varki A, Cummings RD, Esko JD, Stanley P, Hart GW, Aebi M, Darvill AG, Kinoshita T, Packer NH, Prestegard JH, Schnaar RL, & Seeberger PH (Eds.), Essentials of Glycobiology (pp. 113–123). Cold Spring; Harbor (NY).
1. Bruneel A, Cholet S, Drouin-Garraud V, Jacquemont ML, Cano A, Megarbane A, … Fenaille F (2018). Complementarity of electrophoretic, mass spectrometric, and gene sequencing techniques for the diagnosis and characterization of congenital disorders of glycosylation. Electrophoresis. doi: 10.1002/elps.201800021 - DOI - PubMed

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SLC35A2-CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals

Affiliations

SLC35A2-CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

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Grants and funding

LinkOut - more resources

Full Text Sources

Medical

Molecular Biology Databases