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. 2022 Jul 14:10:888923.
doi: 10.3389/fped.2022.888923. eCollection 2022.

FGD1 Variant Associated With Aarskog-Scott Syndrome

Yilin Zhu  1 , Qingqing Chen  1 , Haiyan Lin  2 , Huifei Lu  1 , Yangbin Qu  1 , Qingfeng Yan  1   3   4 , Chunlin Wang  1
Affiliations

FGD1 Variant Associated With Aarskog-Scott Syndrome

Yilin Zhu et al. Front Pediatr. .

Abstract

Background: Aarskog-Scott syndrome, a rare X-linked genetic disorder, is identified by combined clinical manifestations of short stature, facial, skeletal, and genital anomalies. Annually, two or three new cases are diagnosed with Aarskog-Scott syndrome, which is associated with FGD1 variants. However, there is no specific treatment for Aarskog-Scott syndrome due to its unclear mechanism.

Methods: Clinical data were collected when the patient first visited the hospital. Trio whole-exome sequencing and Sanger sequencing were performed for the genetic cause of disease. To evaluate the pathogenicity of the variants in vitro, stable cell lines were constructed using lentivirus infection in 143B cell. Furthermore, Western blot was used to verify the expression of signaling pathway-related proteins, and the transcription levels of osteogenic-related genes were verified by luciferase reporter gene assay.

Results: A 7-year-old boy was manifested with facial abnormalities, intellectual disability, and short stature (-3.98 SDS) while the growth hormone level of stimulation test was normal. Trio whole-exome sequencing and Sanger sequencing identified a variant (c.1270A>G, p.Asn424Asp) in FGD1 gene. The Asn424 residue was highly conserved and the hydrogen bond in the FGD1 variant protein has changed, which led to decrease in the interaction with CDC42 protein. In vitro study showed that the Asn424Asp variant significantly decreased the transcription levels of OCN, COL1A1, and ALP activity, and it activated the phosphorylation of JNK1.

Conclusion: Molecular biological mechanisms between abnormal expression of FGD1and Aarskog-Scott syndrome remain poorly understood. In our study, c.1270A>G variant of FGD1 resulted in Aarskog-Scott syndrome, and the analysis of pathogenicity supports the deleterious effect of the variant. Furthermore, we demonstrated the weakened affinity of the mutant FGD1 and CDC42. Decreased expression of osteogenic-related gene and abnormal activation of JNK1 were also shown in this work.

Keywords: Aarskog-Scott syndrome; CDC42; FGD1; JNK1; short stature.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Clinical characteristics of the proband. (A) Image of the proband. Facial abnormalities included triangular face, long philtrum, low-set ears, and short nose. (B) Hand X-ray of the patient. (C) The pedigree analysis and pedigree symbols. The squares and circles refer to males and females, respectively, and the arrow indicates the proband. A filled symbol represents a person affected with AAS, and the center of the black spot circle indicates carrier status. (D) Sanger sequencing chromatogram demonstrates mutation from affected individuals and heterozygous variant from unaffected family members.
Figure 2
Figure 2
Molecular analysis. (A) A schematic diagram showing the location of the mutation site. (B) Interspecific conservation analysis of the p.Asn424Asp mutation in FGD1, highlighted by the box. (C) Three-dimensional structure model of the local structural changes between wildtype and mutant FGD1 protein, as predicted by Dynamut. Red arrows represent the difference in hydrogen between the wildtype and mutant.
Figure 3
Figure 3
The interaction of the variant of FGD1 and CDC42 has been decreased, which might be contributing to the increase of p-JNK1. (A) Schematic diagram of interaction between FGD1 protein and CDC42 protein by NanoLuc® Binary Technology. (B) To facilitate the comparison, luciferase values were normalized to Control values (100%), histogram and bar chart represent the relative value of FGD1-CDC42 binding capacity. (C) Western blotting analysis of Flag-FGD1, p-JNK1, and T-JNK1 in 143B cell lines. (D,E) Quantified analysis of Western blotting of Flag-FGD1, p-JNK1, and T-JNK1 in 143B cell lines. **p < 0.01, and ***p < 0.001.
Figure 4
Figure 4
Comparison of the osteogenic differentiations between wildtype 143B and mutant 143B. (A) ALP activity determined by ALP activity assay. (B) ALP activity was measured every other day for 10 days since stable cell lines cultured with osteogenic medium. (C) The transcription of OCN was evaluated by dual luciferase reporter gene assay. (D) The transcription of COL1A1 were evaluated by dual luciferase reporter gene assay. ***p < 0.001.
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