Loss-of-Function Variants in PPP1R12A: From Isolated Sex Reversal to Holoprosencephaly Spectrum and Urogenital Malformations

doi:10.1016/j.ajhg.2019.12.004

Case Reports

. 2020 Jan 2;106(1):121-128.

doi: 10.1016/j.ajhg.2019.12.004. Epub 2019 Dec 26.

Loss-of-Function Variants in PPP1R12A: From Isolated Sex Reversal to Holoprosencephaly Spectrum and Urogenital Malformations

Joel J Hughes¹, Ebba Alkhunaizi², Paul Kruszka³, Louise C Pyle⁴, Dorothy K Grange⁵, Seth I Berger⁶, Katelyn K Payne⁷, Diane Masser-Frye⁸, Tommy Hu¹, Michelle R Christie⁹, Nancy J Clegg⁹, Joshua L Everson¹⁰, Ariel F Martinez¹, Laurence E Walsh⁷, Emma Bedoukian⁴, Marilyn C Jones⁸, Catharine Jean Harris¹¹, Korbinian M Riedhammer¹², Daniela Choukair¹³, Patricia Y Fechner¹⁴, Meilan M Rutter¹⁵, Sophia B Hufnagel¹⁶, Maian Roifman², Gad B Kletter¹⁷, Emmanuele Delot¹⁸, Eric Vilain¹⁸, Robert J Lipinski¹⁰, Chad M Vezina¹⁰, Maximilian Muenke¹, David Chitayat²

Affiliations

¹ Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
² The Prenatal Diagnosis and Medical Genetics Program, Department of Obstetrics and Gynecology, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, M5G 1X5, Canada; Division of Clinical and Metabolic Genetics, Department of Pediatrics, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, M5G 1X8, Canada.
³ Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: paul.kruszka@nih.gov.
⁴ Division of Human Genetics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
⁵ Department of Pediatrics, Division of Genetics and Genomic Medicine, Washington University School of Medicine, St. Louis, MO, 63110, USA.
⁶ Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA; Center for Genetic Medicine Research, Children's National Hospital, Washington, DC 20010, USA; Department of Genomics and Precision Medicine, George Washington University, Washington, DC 20037, USA.
⁷ Division of Child Neurology, Riley Hospital for Children, Indianapolis, Indiana, 46202, USA.
⁸ Department of Pediatrics, Division of Genetics, University of California San Diego-Rady Children's Hospital, San Diego, CA 92123, USA.
⁹ Texas Scottish Rite Hospital for Children, Dallas, TX 75219, USA.
¹⁰ Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA; Molecular and Environmental Toxicology Center, University of Wisconsin-Madison, Madison, WI 53706, USA.
¹¹ Department of Pediatric Genetics, University of Missouri Medical Center, Columbia, MO 65212, USA.
¹² Institute of Human Genetics, Klinikum rechts der Isar, Technical University of Munich, Munich, 4JQ2+9Q, Germany; Department of Nephrology, Klinikum rechts der Isar, Technical University of Munich, Munich, 4JQ2+9Q, Germany.
¹³ Division of Paediatric Endocrinology and Diabetology, University Children's Hospital, 69120 Heidelberg, Germany.
¹⁴ Division of Pediatric Endocrinology, Seattle Children's Hospital, University of Washington, Seattle, WA 98105, USA.
¹⁵ Division of Endocrinology, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.
¹⁶ Rare Disease Institute, Children's National Hospital, Washington, DC 20010, USA.
¹⁷ Pediatric Endocrinology, Mary Bridge Children's Hospital, Tacoma, WA 98404, USA.
¹⁸ Center for Genetic Medicine Research, Children's National Hospital, Washington, DC 20010, USA; Department of Genomics and Precision Medicine, George Washington University, Washington, DC 20037, USA.

PMID: 31883643
PMCID: PMC7042489
DOI: 10.1016/j.ajhg.2019.12.004

Case Reports

Loss-of-Function Variants in PPP1R12A: From Isolated Sex Reversal to Holoprosencephaly Spectrum and Urogenital Malformations

Joel J Hughes et al. Am J Hum Genet. 2020.

. 2020 Jan 2;106(1):121-128.

doi: 10.1016/j.ajhg.2019.12.004. Epub 2019 Dec 26.

Authors

Affiliations

¹ Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
² The Prenatal Diagnosis and Medical Genetics Program, Department of Obstetrics and Gynecology, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, M5G 1X5, Canada; Division of Clinical and Metabolic Genetics, Department of Pediatrics, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, M5G 1X8, Canada.
³ Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: paul.kruszka@nih.gov.
⁴ Division of Human Genetics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
⁵ Department of Pediatrics, Division of Genetics and Genomic Medicine, Washington University School of Medicine, St. Louis, MO, 63110, USA.
⁶ Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA; Center for Genetic Medicine Research, Children's National Hospital, Washington, DC 20010, USA; Department of Genomics and Precision Medicine, George Washington University, Washington, DC 20037, USA.
⁷ Division of Child Neurology, Riley Hospital for Children, Indianapolis, Indiana, 46202, USA.
⁸ Department of Pediatrics, Division of Genetics, University of California San Diego-Rady Children's Hospital, San Diego, CA 92123, USA.
⁹ Texas Scottish Rite Hospital for Children, Dallas, TX 75219, USA.
¹⁰ Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA; Molecular and Environmental Toxicology Center, University of Wisconsin-Madison, Madison, WI 53706, USA.
¹¹ Department of Pediatric Genetics, University of Missouri Medical Center, Columbia, MO 65212, USA.
¹² Institute of Human Genetics, Klinikum rechts der Isar, Technical University of Munich, Munich, 4JQ2+9Q, Germany; Department of Nephrology, Klinikum rechts der Isar, Technical University of Munich, Munich, 4JQ2+9Q, Germany.
¹³ Division of Paediatric Endocrinology and Diabetology, University Children's Hospital, 69120 Heidelberg, Germany.
¹⁴ Division of Pediatric Endocrinology, Seattle Children's Hospital, University of Washington, Seattle, WA 98105, USA.
¹⁵ Division of Endocrinology, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.
¹⁶ Rare Disease Institute, Children's National Hospital, Washington, DC 20010, USA.
¹⁷ Pediatric Endocrinology, Mary Bridge Children's Hospital, Tacoma, WA 98404, USA.
¹⁸ Center for Genetic Medicine Research, Children's National Hospital, Washington, DC 20010, USA; Department of Genomics and Precision Medicine, George Washington University, Washington, DC 20037, USA.

PMID: 31883643
PMCID: PMC7042489
DOI: 10.1016/j.ajhg.2019.12.004

Abstract

In two independent ongoing next-generation sequencing projects for individuals with holoprosencephaly and individuals with disorders of sex development, and through international research collaboration, we identified twelve individuals with de novo loss-of-function (LoF) variants in protein phosphatase 1, regulatory subunit 12a (PPP1R12A), an important developmental gene involved in cell migration, adhesion, and morphogenesis. This gene has not been previously reported in association with human disease, and it has intolerance to LoF as illustrated by a very low observed-to-expected ratio of LoF variants in gnomAD. Of the twelve individuals, midline brain malformations were found in five, urogenital anomalies in nine, and a combination of both phenotypes in two. Other congenital anomalies identified included omphalocele, jejunal, and ileal atresia with aberrant mesenteric blood supply, and syndactyly. Six individuals had stop gain variants, five had a deletion or duplication resulting in a frameshift, and one had a canonical splice acceptor site loss. Murine and human in situ hybridization and immunostaining revealed PPP1R12A expression in the prosencephalic neural folds and protein localization in the lower urinary tract at critical periods for forebrain division and urogenital development. Based on these clinical and molecular findings, we propose the association of PPP1R12A pathogenic variants with a congenital malformations syndrome affecting the embryogenesis of the brain and genitourinary systems and including disorders of sex development.

Keywords: MYPT1; PPP1R12A; disorders of sex development; embryogenesis; encephalocele; facial dysmorphism; forebrain; holoprosencephaly; hypospadias; omphalocele.

Published by Elsevier Inc.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Figure 1**
PPP1R12A with Variant Annotations and Highlighted Regions Per UniProt, domains include a Lysine-Valine-Lysine-Phenylalanine (KVKF) motif (red), multiple ankyrin repeats (yellow), rho-associated coiled-coil-containing protein kinase 1 (ROCK1) and rho-associated coiled-coil-containing protein kinase 2 (ROCK2) interaction site (orange), and a leucine zipper domain which binds a cGMP-dependent protein kinase 1. Stop gain and frameshift variants are notated in black with the splice-site variant in red at the approximate site predicted to result in a premature termination codon and nonsense-mediated decay. Diagram was created using Domain Graph version 2.0.

**Figure 2**
Brain: Mouse *in situ* Hybridization of the Prosencephalic Neural Folds Gestational day 8.25 mouse embryos were stained via *in situ* hybridization in order to determine gene expression patterns. A ventral view is shown for whole mounts. Transverse sections through the prosencephalic neural folds (at the level of the dashed line in schematic) were stained in order to visualize gene expression in specific cellular compartments. *Ppp1r12a* localized to the head mesenchyme and is absent from extra-embryonic membranes. nf—neural folds, h—heart, ne—neuroectoderm, hm—head mesenchyme, eem—extra-embryonic membranes. Scale bar = 100 μm.

**Figure 3**
Urogenital: Mouse and Human Immunostaining of the Genitourinary Tract (A–B) Tissue sections from mouse genitourinary tracts at gestation day (GD) 13. (C–D) Mouse genitourinary tracts at GD13.75. (E–F) Human genitourinary tracts at week 10 were immunostained in order to detect protein localization patterns. PPP1R12A was detected in genital tubercle epithelium (ectoderm derived), bladder, and urethral epithelium (endoderm derived), and a subset or urogenital sinus mesenchymal cells (arrowhead) bladder detrusor smooth muscle. (E′) PPP1R12A localized to epithelial cell nuclei of human urethra (lower image, inset). (F′, arrowhead) PPP1R12B detected in developing human detrusor smooth muscle (B, D, F, F′) but not in developing mouse or human bladder or urogenital sinus epithelial cells. Abbreviations are B—bladder, GT—genital tubercle, U—urethra.

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References

1. Grassie M.E., Moffat L.D., Walsh M.P., MacDonald J.A. The myosin phosphatase targeting protein (MYPT) family: a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type 1δ. Arch. Biochem. Biophys. 2011;510:147–159. - PubMed
1. Kiss A., Erdődi F., Lontay B. Myosin phosphatase: Unexpected functions of a long-known enzyme. Biochim. Biophys. Acta Mol. Cell Res. 2019;1866:2–15. - PubMed
1. Ito M., Nakano T., Erdodi F., Hartshorne D.J. Myosin phosphatase: structure, regulation and function. Mol. Cell. Biochem. 2004;259:197–209. - PubMed
1. Shichi D., Arimura T., Ishikawa T., Kimura A. Heart-specific small subunit of myosin light chain phosphatase activates rho-associated kinase and regulates phosphorylation of myosin phosphatase target subunit 1. J. Biol. Chem. 2010;285:33680–33690. - PMC - PubMed
1. Shimizu H., Ito M., Miyahara M., Ichikawa K., Okubo S., Konishi T., Naka M., Tanaka T., Hirano K., Hartshorne D.J. Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase. J. Biol. Chem. 1994;269:30407–30411. - PubMed

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[1] Grassie M.E., Moffat L.D., Walsh M.P., MacDonald J.A. The myosin phosphatase targeting protein (MYPT) family: a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type 1δ. Arch. Biochem. Biophys. 2011;510:147–159. - PubMed

[2] Grassie M.E., Moffat L.D., Walsh M.P., MacDonald J.A. The myosin phosphatase targeting protein (MYPT) family: a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type 1δ. Arch. Biochem. Biophys. 2011;510:147–159. - PubMed

[3] Kiss A., Erdődi F., Lontay B. Myosin phosphatase: Unexpected functions of a long-known enzyme. Biochim. Biophys. Acta Mol. Cell Res. 2019;1866:2–15. - PubMed

[4] Kiss A., Erdődi F., Lontay B. Myosin phosphatase: Unexpected functions of a long-known enzyme. Biochim. Biophys. Acta Mol. Cell Res. 2019;1866:2–15. - PubMed

[5] Ito M., Nakano T., Erdodi F., Hartshorne D.J. Myosin phosphatase: structure, regulation and function. Mol. Cell. Biochem. 2004;259:197–209. - PubMed

[6] Ito M., Nakano T., Erdodi F., Hartshorne D.J. Myosin phosphatase: structure, regulation and function. Mol. Cell. Biochem. 2004;259:197–209. - PubMed

[7] Shichi D., Arimura T., Ishikawa T., Kimura A. Heart-specific small subunit of myosin light chain phosphatase activates rho-associated kinase and regulates phosphorylation of myosin phosphatase target subunit 1. J. Biol. Chem. 2010;285:33680–33690. - PMC - PubMed

[8] Shichi D., Arimura T., Ishikawa T., Kimura A. Heart-specific small subunit of myosin light chain phosphatase activates rho-associated kinase and regulates phosphorylation of myosin phosphatase target subunit 1. J. Biol. Chem. 2010;285:33680–33690. - PMC - PubMed

[9] Shimizu H., Ito M., Miyahara M., Ichikawa K., Okubo S., Konishi T., Naka M., Tanaka T., Hirano K., Hartshorne D.J. Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase. J. Biol. Chem. 1994;269:30407–30411. - PubMed

[10] Shimizu H., Ito M., Miyahara M., Ichikawa K., Okubo S., Konishi T., Naka M., Tanaka T., Hirano K., Hartshorne D.J. Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase. J. Biol. Chem. 1994;269:30407–30411. - PubMed

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Loss-of-Function Variants in PPP1R12A: From Isolated Sex Reversal to Holoprosencephaly Spectrum and Urogenital Malformations

Affiliations

Loss-of-Function Variants in PPP1R12A: From Isolated Sex Reversal to Holoprosencephaly Spectrum and Urogenital Malformations

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Abstract

Conflict of interest statement

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