Fragile X syndrome (FXS), an X-linked disorder, is the most common cause of intellectual disability. Repeats of CGG trinucleotides 5 to 40 in the 5′-UTR of the FMR1 gene exist in the normal population; however, over 200 repeats will lead to the development of the disease. FMR1 gene amplification, methylation, and transcriptional silencing lead to a loss of FMRP function. In addition, CGG repeats between 55 and 199 also present with the disease, with the most common symptoms being fragile X-related tremor and ataxia syndrome (FXTAS) in men and fragile X primary ovarian hypoplasia (FXPOI) in women. The global mutation rate of FXS patients is 1:5,000 for males and 1:4,000–1:8,000 for females (Santoro et al., 2012; Saldarriaga et al., 2014). Due to incomplete penetrance of the gene, 100% of males with full mutation showed FXS, while females with full mutation have an incidence of approximately one-third due to random inactivation of one X chromosome. Given the high carrier rate of FXS, CGG amplification experiments are often required for patients with intellectual disability. In recent years, with the advent of next-generation sequencing technology, an increased number of mutations within the FMR1 gene have been discovered. In this study, a novel frameshift mutation was identified in a family with a clinical phenotype suspected of FXS. We recommend that whole exome sequencing (WES) should be the primary consideration in the case of negative CGG amplification tests.

Fragile X syndrome (FXS) is the most common genetic cause of intellectual disability and is more common in men as an X-linked dominant genetic disorder. Its clinical features include social impairment, autism, delayed language development, neurological dysfunction (seizures and sleep abnormalities), and characteristic appearance (large protruding ears, long faces, highly arched palates, hyperextended finger joints) (Berry-Kravis et al., 2010; Santoro et al., 2012). Although FXS is usually caused by the amplification of the CGG repeat of FMR1, a small number of FMR1 gene mutations leading to FXS have been reported to date (Quartier et al., 2017; Sitzmann et al., 2018; Carroll et al., 2020). These mutations include deletions, splicing, missense, and nonsense mutations.

The FMR1 gene maps to Xp27.3, has a gene length of 39 kb, and consists of 17 exons and 16 introns. The FMR1 gene directs the synthesis of FMRP, which is an RNA-binding protein. Dysregulation of the translation/transport/stability of these mRNAs in the absence of FMRP has a cascade effect on many pathways, resulting in a final phenotype of FXS (Maurin et al., 2014). This protein is found in many tissues, including the brain, testes, and ovaries. In the brain, it is thought to be able to regulate specific gene expression during neurodevelopment and affect the plasticity of neural cell synapses, resulting in the inability of dendrites to develop to mature size and shape (Bassell, 2011; Deng and Klyachko, 2021; Prieto et al., 2021).

In this study, based on the proband’s clinical presentation and pedigree analysis, the diagnosis was speculated to be FXS, which was found to be negative after performing routine CGG repeat amplification tests. Subsequent WES of this family revealed the presence of c. 585_586del in exon 7 of the FMR1 gene, which may form a truncated ion form of FMRP. Quartier et al. (2017) reported that deletion of exon 17 of FMR1 leads to a truncated FMRP and a decreased expression level of this truncated form of FMRP in the patients. Therefore, we propose that c. 585_586del in exon 7 in this study may also lead to a decreased expression level of the truncated form of FMRP and contribute to the phenotypes (Quartier et al., 2017).

Male carriers of mutations in the FMR1 gene are generally FXS patients, while female carriers have high clinical heterogeneity, and phenotypes from normal intelligence to severe intellectual disability may exist, which are more likely to be associated with learning disabilities, socio-emotional, and mental health problems (Hagerman et al., 2017; Reches, 2019). During the development of female embryos, most genes on one X chromosome in each cell are randomly inactivated, resulting in approximately half of the cells in the body expressing genes from each X chromosome. The theoretical ratio of two types of cells is 50:50. The type of X chromosome inactivation in the female population (the ratio of X inactivated cells in the father to X inactivated cells in the mother) follows the normal distribution, and a small number of normal women will deviate from 50:50. Usually, a ratio greater than or equal to 90:10 is used as the criterion for determining non-random inactivation of X; however, 80:20 is also used as a criterion for non-random inactivation (Robinson et al., 2001; Uehara et al., 2001). It is believed that only a few embryonic progenitor cells will continue to form the brain; therefore, it is believed that the ratio of affected X-activated cells to silenced cells significantly affects the expression level of FMRP in the developing central nervous system, and the difference in this ratio may lead to significant changes in the phenotype of FXS women. In fact, the phenotype is commonly milder in females than in males (Loesch et al., 2004; Bartholomay et al., 2019).

The males of this family have some clinical features, such as intellectual disability, prominent forehead, flaring ears, and long face. The mother and grandmother of the proband only showed excessive extension of long face and fingers, which may be related to the skewness of the female X chromosome. In this study, the X chromosome inactivation test was performed on the mother of the proband, and the result showed that the ratio of X chromosome from the father to X chromosome from the mother was approximately 85:15, which was non-random inactivation. The normal expression of the FMR1 gene was greater than the abnormal expression, which can be used to explain the slight clinical phenotype of female carriers.

At present, conventional detection methods for FXS mainly use PCR and capillary technology to detect the presence of CGG trinucleotide amplification in the 5′UTR of the FMR1 gene. The application of chromosome microarray technology to detect FMR1 gene deletion (full, partial, or mosaicism) has also been reported (Coffee et al., 2009; Gonçalves et al., 2016; Ciaccio et al., 2017). WES is recommended for FMR1 point mutation detection in cases with negative CGG tests or FMR1 gene deletion when FXS syndrome is suspected. According to HGMD (professional 2023.2), 89 pathogenic mutations (45 gross deletions, 16 repeat variations, 7 gross insertions, 5 splicing, 4 missense, 1 nonsense, 3 regulatory, 4 small deletions, 3 insertions/indels, and 1 complex rearrangement) have been reported. Among them, at least 17 pathogenic mutations (5 splicing, 4 missense, 1 nonsense, 4 small deletions, and 3 insertions/indels), accounting for 19.1% of all the reported pathogenic mutations, could be identified by WES. The proband in this study had a negative CGG expansion result; however, WES identified a frameshift mutation, which was verified by Sanger sequencing. This frameshift mutation disrupts the reading frame of the FMR1 gene, resulting in a premature stop codon after 8 aberrant amino acids and forming a truncated protein. Therefore, the proband and his two uncles developed an FXS phenotype. Population databases showing FMR1 gene variant c.585_586del are very rare and it has not been reported in the gnomAD, ExAC, HGMD, or ClinVar database or in relevant literature.

In summary, a c.585_586del frameshift mutation in the FMR1 gene was detected in the proband of this family by WES, which was the cause of FXS in the proband. This study provides a theoretical basis for genetic counseling and fertility guidance in this family. Moreover, the discovery of a new variant also expands the mutation spectrum of this gene. We also emphasize the importance of using WES as a clinical tool. The discovery of FMR1 gene mutations may lead to a better understanding of the function of FMRP, especially at the synapse, and an understanding of the pathways underlying these alterations is essential to develop many targeted therapies for FXS. Understanding the relationship between specific mutations and the different effects of FMRP on pre-synaptic and/or post-synaptic function will be interesting and should be further studied in clinical cases and animal models (Hagerman et al., 2014; Jacquemont et al., 2014; Sitzmann et al., 2018; Prieto et al., 2021).