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

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.

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.