Weiss B et al. (2023), Identification of novel genes including NAV2 as...

XB-ART-60493

Front Endocrinol (Lausanne) 2023 Jan 01;14:1258313. doi: 10.3389/fendo.2023.1258313.

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Identification of novel genes including NAV2 associated with isolated tall stature.

Weiss B , Ott T , Vick P , Lui JC , Roeth R , Vogel S , Waldmüller S , Hoffmann S , Baron J , Wit JM , Rappold GA .

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Very tall people attract much attention and represent a clinically and genetically heterogenous group of individuals. Identifying the genetic etiology can provide important insights into the molecular mechanisms regulating linear growth. We studied a three-generation pedigree with five isolated (non-syndromic) tall members and one individual with normal stature by whole exome sequencing; the tallest man had a height of 211 cm. Six heterozygous gene variants predicted as damaging were shared among the four genetically related tall individuals and not present in a family member with normal height. To gain insight into the putative role of these candidate genes in bone growth, we assessed the transcriptome of murine growth plate by microarray and RNA Seq. Two (Ift140, Nav2) of the six genes were well-expressed in the growth plate. Nav2 (p-value 1.91E-62) as well as Ift140 (p-value of 2.98E-06) showed significant downregulation of gene expression between the proliferative and hypertrophic zone, suggesting that these genes may be involved in the regulation of chondrocyte proliferation and/or hypertrophic differentiation. IFT140, NAV2 and SCAF11 have also significantly associated with height in GWAS studies. Pathway and network analysis indicated functional connections between IFT140, NAV2 and SCAF11 and previously associated (tall) stature genes. Knockout of the all-trans retinoic acid responsive gene, neuron navigator 2 NAV2, in Xenopus supports its functional role as a growth promotor. Collectively, our data expand the spectrum of genes with a putative role in tall stature phenotypes and, among other genes, highlight NAV2 as an interesting gene to this phenotype.

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Species referenced: Xenopus laevis
Genes referenced: cngb1 esr1 fgfr3 ift140 nav2 npr2 scaf11 tcf7 ywhae
gRNAs referenced: nav2 gRNA2 nav2 sgRNA1

???displayArticle.omims??? ACHONDROPLASIA; ACH [+]
Phenotypes: Xla Wt + nav2 CRISPR (Fig. 3 AB) [+]

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	Figure 1 Pedigree and height data. (A) Pedigree of family: Black symbols indicate individuals with tall stature. Individual I.2 is considered as borderline tall stature (grey symbol). White symbols indicate normal stature. Exome sequencing was carried out on DNA from individuals I.1, II.1, II.4 and III.1. DNA was also available from I.2, II.2 and III.2 but not for exome sequencing; no DNA was available from individual II.3 (declined). (B) Data on height and year of birth of Family A Height data were adjusted for secular trend and shrinking. SDS, Standard deviation score.
	Figure 2 Ingenuity (IPA) Network analysis. The newly identified genes were used to show functional connections in context of known protein networks, based on data from the literature. No connections to known proteins could be established for CNGB1.
	Figure 3 CRISPR/Cas-mediated knockout of nav2 causes enhanced embryonic growth. (A, B) Tadpole total body length measurements of three independent experiments. All experiments revealed a slightly increased average body size in nav2 sgRNA-injected specimens as compared to control littermates. (A) Three single experiments with sgRNA1 that specifically targets an exon orthologous to the exon harboring the human NAV2 missense mutation p.R2201C (exp.1, n=38 animals, stage 45; exp.2, n=102 animals, stage 44; exp.3, n=18 animals, stage 45); (B) Combined data of the three experiments in (A) after normalization to the mean of their controls revealed a significant increase of relative body sizes after sgRNA1 injections (p-value=0.023). Boxplot whiskers (error bars) are 1.5 * IQR +Q3 (upper), or 1.5 * IQR -Q1 (lower). co, control; exp., experiment; sgRNA, single-guide RNA; mm, millimeter.
	Supplementary Figure 1 \| Ingenuity (IPA) Network analysis. Known genes from the literature (n=86; in green colour) underlying tall stature were analysed using the IPA network analysis. The newly identified genes were added to predict functional connections in the context of known protein networks.
	Supplementary Figure 2 \| nav2 sgRNA2 causes wild type appearance in about half of the animals and organ defects and reduced embryonic growth in the other half. (AC) Overall appearance of representative stage 45 control specimen (A, A), or nav2 sgRNA2-injected specimen (B, B), illustrating organ malformations with edema formation in about 50% of nav2 sgRNA2 injections. Tadpoles are shown in lateral (A, B) and ventral view (A, B). (C, D) Quantification of total body length of tadpoles with organ defects (grey fraction in C), showing reduced body length when compared to control specimens (A, C). (E) Quantification of total body length of tadpoles without organ defects (dark blue fraction in C) of the two individual experiments with sgRNA2 (exp.1, n=53 animals, stage 45; exp.2, n=31 animals, stage 45) demonstrating a mild but not statistically significant increase in body size. co, control; sgRNA, single-guide RNA; mm, millimeter; OD, organ defects; st., stage; wta, wild type appearance.

References [+] :

Accogli, Loss of Neuron Navigator 2 Impairs Brain and Cerebellar Development. 2023, Pubmed