CLINICAL RESEARCH www.jasn.org Germline-Derived Gain-of-Function Variants of Gsa-Coding GNAS Gene Identified in Nephrogenic Syndrome of Inappropriate Antidiuresis Mami Miyado,1 Maki Fukami,1 Shuji Takada,2 Miho Terao,2 Kazuhiko Nakabayashi,3 Kenichiro Hata,3 Yoichi Matsubara,4 Yoko Tanaka ,5 Goro Sasaki,5 Keisuke Nagasaki ,6 Masaaki Shiina,7 Kazuhiro Ogata,7 Youhei Masunaga,8 Hirotomo Saitsu,9 and Tsutomu Ogata 1,8 Departments of 1Molecular Endocrinology, 2Systems BioMedicine, 3Maternal-Fetal Biology and 4Head Office, National Research Institute for Child Health and Development, Tokyo, Japan; 5Department of Pediatrics, Tokyo Dental College, Ichikawa General Hospital, Ichikawa, Japan; 6Department of Homeostatic Regulation and Development, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan; 7Department of Biochemistry, Yokohama City University Graduate School of Medicine, Yokohama, Japan; and Departments of 8Pediatrics and 9Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Japan ABSTRACT Background The stimulatory G-protein a-subunit encoded by GNAS exons 1–13 (GNAS-Gsa)mediates signal transduction of multiple G protein–coupled receptors, including arginine vasopressin receptor 2 (AVPR2). Various germline-derived loss-of-function GNAS-Gsa variants of maternal and paternal origin have been found in pseudohypoparathyroidism type Ia and pseudopseudohypoparathyroidism, respec- tively. Specific somatic gain-of-function GNAS-Gsa variants have been detected in McCune–Albright syndrome and may result in phosphate wasting. However, no germline-derived gain-of-function variant has been identified, implying that such a variant causes embryonic lethality. Methods We performed whole-exome sequencing in two families with dominantly inherited nephrogenic syndrome of inappropriate antidiuresis (NSIAD) as a salient phenotype after excluding a gain-of-function variant of AVPR2 and functional studies for identified variants. Results Whole-exome sequencing revealed two GNAS-Gsa candidate variants for NSIAD: GNAS-Gsa p.(F68_G70del) in one family and GNAS-Gsa p.(M255V) in one family. Both variants were absent from public and in-house databases. Of genes with rare variants, GNAS-Gsa alone was involved in AVPR2 sig- naling and shared by the families. Protein structural analyses revealed a gain-of-function–compatible con- formational property for p.M255V-Gsa, although such assessment was not possible for p.F68_G70del-Gsa. Both variants had gain-of-function effects that were significantly milder than those of McCune–Albright syndrome–specific somatic Gsa variants. Model mice for p.F68_G70del-Gsa showed normal survivability and NSIAD-compatible phenotype, whereas those for p.M255V-Gsa exhibited severe failure to thrive. Conclusions This study shows that germline-derived gain-of-function rare variants of GNAS-Gsa exist and cause NSIAD as a novel Gsa-mediated genetic disease. It is likely that AVPR2 signaling is most sensitive to GNAS-Gsa’s gain-of-function effects. J Am Soc Nephrol 30: 877–889, 2019. doi: https://doi.org/10.1681/ASN.2018121268 CLINICAL RESEARCH Received December 27, 2018. Accepted February 2, 2019. Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan, or Prof. Tsutomu Ogata, Department of Pediatrics, Hamamatsu M.M. and M.F. contributed equally to this work. University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan. Email: [email protected] or Published online ahead of print. Publication date available at [email protected] www.jasn.org. Copyright © 2019 by the American Society of Nephrology Correspondence: Dr. Maki Fukami, Department of Molecular Endocrinology, National Research Institute for Child Health and J Am Soc Nephrol 30: 877–889, 2019 ISSN : 1046-6673/3005-877 877 CLINICAL RESEARCH www.jasn.org The GNAS complex locus generates multiple gene products, Significance Statement including the stimulatory G-protein a-subunit encoded by GNAS exons 1–13 (GNAS-Gsa).1,2 GNAS-Gsa is biallelically The stimulatory G-protein a-subunit GNAS-Gsa mediates signal expressed in most tissues, including the renal collecting transduction of multiple G protein–coupled receptors, including fi duct, and it is predominantly expressed from the maternal arginine vasopressin receptor 2 (AVPR2). Speci c gain-of-function variants in AVPR2 are known causes of nephrogenic syndrome allele in a few tissues, such as the renal proximal tubule, of inappropriate antidiuresis (NSIAD), an arginine vasopressin– – thyroid, gonad, and pituitary.1 5 G-protein a-subunit (Gsa) independent antidiuresis. In two families with NSIAD, after ex- mediates signal transductions of multiple G protein–coupled cluding AVPR2 gain-of-function variants, the authors identified two receptors, including parathyroid hormone 1 receptor (PTH1R), novel germline-derived variants of GNAS-Gsa. They also showed GNAS thyroid-stimulating hormone receptor (TSHR), follicle- that both of the -Gsa variants had gain-of-function effects that were milder than those of specific somatic GNAS-Gsa variants stimulating hormone receptor (FSHR), luteinizing hormone/ reported in McCune–Albright syndrome, a condition that may re- choriogonadotropin receptor (LHCGR), growth hormone– sult in renal phosphate wasting. The results refute the widely be- releasing hormone receptor (GHRHR), and arginine vasopres- lieved concept that a germline-derived GNAS-Gsa gain-of-function sin receptor 2 (AVPR2).6 To date, various germline-derived variant is absent because of embryonic lethality and reveal the loss-of-function (LOF) variants of GNAS-Gsa of maternal and genetic heterogeneity in NSIAD. paternal origin have been found in pseudohypoparathyroidism type Ia (PHP-Ia) and pseudopseudohypoparathyroidism Oligonucleotides (PPHP), respectively,1,2,6,7 and specific somatic gain-of-function Oligonucleotides used in this study are shown in Supplemental (GOF) variants of GNAS-Gsa, such as p.R201C and p.R201H, Table 1. have been detected in McCune–Albright syndrome (MAS) and endocrine tumors.1,6,8 However, no germline-derived GOF var- Molecular Diagnostic Studies iant of GNAS-Gsa has been identified, implying that such a We performed Sanger sequencing, genome-wide array com- variant causes embryonic lethality.9 parative genomic hybridization (aCGH), and whole-exome Nephrogenic syndrome of inappropriate antidiuresis sequencing (WES) using leukocyte genomic DNA samples. (NSIAD) is a rare disorder caused by impaired renal capacity Sanger sequencing was carried out for all of the coding exons to excrete a free water load into the urine under undetectable or and their splice sites of AVPR2 and for variant-positive GNAS- very low plasma arginine vasopressin (AVP) values.10 Although Gsa exons on the ABI 3130xl Genetic Analyzer (Thermo this condition is associated with blood hypo-osmolality and Fisher Scientific). To confirm heterozygous variants, sequenc- hyponatremia as well as inappropriately elevated urine osmo- ing was performed for subcloned wild-type (WT) and variant lality and sodium concentration as observed in the syndrome alleles. of inappropriate antidiuretic hormone secretion (SIADH),11 Genome-wide aCGH was performed with a catalog human NSIAD and SIADH show a sharp contrast in the plasma array (131M format; ID G4447A) according to the manufac- AVP/ADH value. Consistent withsuchanAVP-independent turer’s instructions (Agilent Technologies). Obtained data antidiuresis, NSIAD has been shown to be caused by specific were analyzed using the default settings of the Agilent Geno- GOF variants of AV PR2, such as p.R137C, p.R137L, and mic Workbench 7.0. Copy number alterations were regarded p.F229V.10,12 as normal variants if they had been registered in Database of Here, we report relatively mild germline-derived GOF var- Genomic Variants (http://dgv.tcag.ca/dgv/app/home) or Clin- iants of GNAS-Gsa identified in two families with NSIAD as a Var (http://www.ncbi.nlm.nih.gov/clinvar/). salient phenotype. This study demonstrates the presence of WES was carried out using SureSelect Human All Exon V6 germline-derived GOF variants of GNAS-Gsa and establishes (Agilent Technologies). Captured libraries were sequenced by a novel Gsa-mediated genetic disease. NextSeq 500 (Illumina) with 150-bp paired end reads. Reads were aligned to the reference genome (Human GRCh37/hg19; http://genome.ucsc.edu/) using BWA-MEM (Version 0.7.12) METHODS with default parameters. Duplicated reads were removed by Picard (Version 2.9.2), and local realignment and base quality Ethical Approval recalibration were performed by GATK Version 3.7. Variants Human studies were approved by the Institutional Review were identified with the GATK HaplotypeCaller, and those Board Committees at Hamamatsu University School of Med- with minor allele frequencies of ,0.005 in all of the following icine (91–002) and National Research Institute for Child public databases (the whole-genome and exome data for the Health and Development (519), and they were performed East Asian population in the Genome Aggregation Database after obtaining written informed consent. Mouse studies [gnomAD_genome_EAS andgnomAD_exome_EAS],13 the were approved by the Animal Care and Use Committee of Human Genetic Variation Database,14 allele frequency data National Research Institute for Child Health and Develop- of 2049 Japanese individuals,15 and in-house database) were ment (A2014–001), and they were conducted in accordance selected as rare variants. Final variants were annotated with with the approved protocols. Annovar.16 In silico pathogenicity