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. 2022 Dec;24(12):2501-2515.
doi: 10.1016/j.gim.2022.08.025. Epub 2022 Sep 30.

The p190 RhoGAPs, ARHGAP35, and ARHGAP5 are implicated in GnRH neuronal development: Evidence from patients with idiopathic hypogonadotropic hypogonadism, zebrafish, and in vitro GAP activity assay

Margaret F Lippincott  1 , Wanxue Xu  2 , Abigail A Smith  3 , Xinyu Miao  4 , Agathe Lafont  5 , Omar Shennib  6 , Gordon J Farley  2 , Riwa Sabbagh  2 , Angela Delaney  7 , Maria Stamou  2 , Lacey Plummer  2 , Kathryn Salnikov  2 , Neoklis A Georgopoulos  8 , Veronica Mericq  9 , Richard Quinton  10 , Frederic Tran Mau-Them  11 , Sophie Nambot  12 , Asma Hamad  13 , Helen Brittain  13 , Rebecca S Tooze  14 , Eduardo Calpena  14 , Andrew O M Wilkie  14 , Marjolaine Willems  15 , William F Crowley  16 , Ravikumar Balasubramanian  2 , Nathalie Lamarche-Vane  4 , Erica E Davis  3 , Stephanie B Seminara  2
Affiliations

The p190 RhoGAPs, ARHGAP35, and ARHGAP5 are implicated in GnRH neuronal development: Evidence from patients with idiopathic hypogonadotropic hypogonadism, zebrafish, and in vitro GAP activity assay

Margaret F Lippincott et al. Genet Med. 2022 Dec.

Abstract

Purpose: The study aimed to identify novel genes for idiopathic hypogonadotropic hypogonadism (IHH).

Methods: A cohort of 1387 probands with IHH underwent exome sequencing and de novo, familial, and cohort-wide investigations. Functional studies were performed on 2 p190 Rho GTPase-activating proteins (p190 RhoGAP), ARHGAP35 and ARHGAP5, which involved in vivo modeling in larval zebrafish and an in vitro p190A-GAP activity assay.

Results: Rare protein-truncating variants (PTVs; n = 5) and missense variants in the RhoGAP domain (n = 7) in ARHGAP35 were identified in IHH cases (rare variant enrichment: PTV [unadjusted P = 3.1E-06] and missense [adjusted P = 4.9E-03] vs controls). Zebrafish modeling using gnrh3:egfp phenotype assessment showed that mutant larvae with deficient arhgap35a, the predominant ARHGAP35 paralog in the zebrafish brain, display decreased GnRH3-GFP+ neuronal area, a readout for IHH. In vitro GAP activity studies showed that 1 rare missense variant [ARHGAP35 p.(Arg1284Trp)] had decreased GAP activity. Rare PTVs (n = 2) also were discovered in ARHGAP5, a paralog of ARHGAP35; however, arhgap5 zebrafish mutants did not display significant GnRH3-GFP+ abnormalities.

Conclusion: This study identified ARHGAP35 as a new autosomal dominant genetic driver for IHH and ARHGAP5 as a candidate gene for IHH. These observations suggest a novel role for the p190 RhoGAP proteins in GnRH neuronal development and integrity.

Keywords: Developmental disorder; Idiopathic hypogonadotropic hypogonadism; Intellectual disability; Puberty; Rho GTPase–activating protein.

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Conflict of interest statement

Conflict of Interest The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.. Pedigrees with rare ARHGAP35 variants.
a. Loss of function variants; enrichment compared to controls, unadjusted p= 3.1E-06; b. RhoGAP domain missense variants; enrichment compared to controls, adjusted p=4.9E-3. c. GeneMatcher loss of function variants. Parents are shown when their phenotype is known. Abbreviations: “+”, wild-type allele; “M”, variant allele; nIHH, normosmic idiopathic hypogonadotropic hypogonadism; KS, Kallmann syndrome; CDP, constitutional delay of growth and puberty
Figure 2.
Figure 2.. Rare variants identified in ARHGAP35.
a. Schematic of ARHGAP35 protein (GenBankID: NP_004482.4) and position of loss of function variants and RhoGAP domain missense variants (phenotypes: black – idiopathic hypogonadotropic hypogonadism (IHH), green – developmental disorder (DD) previously published, green underlined DD this report, red - neonatal hypogonadism and DD). FF, phenyalanine domains (blue); pG1, pseudo-GTPase domain 1 (salmon); pG2, pseudoGTPase domain 2 (salmon); PBD, phospholipid binding domain (gray); RhoGAP, Rho GTPase-activating proteins domain (yellow); P, proline rich domain (gray); amino acid position is labeled at the bottom. b. Conservation of missense variants identified in IHH in the RhoGAP domain across multiple vertebrate species.
Figure 3.
Figure 3.. Characterization of GnRH3-GFP neuronal patterning in arhgap35a, arhgap35b, and arhgap5 mutant zebrafish.
a, c, e. Representative dorsal images acquired from 5 dpf larvae to evaluate gnrh3:gfp signal. Anterior, left; posterior, right. Dotted lines indicate eyes and the most anterior region of the head. Scale bar, 100μm. b, d, f. Quantification of GnRH3-GFP+ signal in 5 dpf siblings compared across genotypes obtained from in-crosses of heterozygous mutant adults. Abbreviations: au, arbitrary units; WT, wild type; Het, heterozygous; Hom, homozygous; ns, not significant; significant differences detected with a one-way ANOVA; *p<0.05, **p<0.01; error bars indicate standard deviation.
Figure 4:
Figure 4:. RhoGAP modeling of ARHGAP35 missense variants in vitro.
In vitro GAP activity assay towards RhoA in the absence (intrinsic) or presence of p190A-GAP wild type (WT) or mutant proteins. Arg1284Trp substitution is a loss-of-function variant in p190A GAP domain. Data are presented as mean ± S.E.M of three independent experiments (n=3). **, p < 0.01; ***, p < 0.001; unpaired student’s t test.
Figure 5:
Figure 5:. Kallmann Syndrome Pedigrees with rare ARHGAP5 variants.
a. Pedigrees of probands with previously unreported loss of function variants. ”+”, wild-type allele; “M”, variant allele. b. Schematic of human ARHGAP5 protein (GenBank ID: NP_001025226.1) and position of variants identified in IHH cases. FF, phenyalanine domains (blue); pG1, pseudo-GTPase domain 1 (salmon); pG2, pseudoGTPase domain 2 (salmon); K, lysine rich domain (gray); RhoGAP, Rho GTPase-activating proteins domain (yellow); P, proline rich domain (gray); amino acid position is labeled at the bottom.
See this image and copyright information in PMC

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