Intellectual disability (ID), a neurodevelopmental disorder imposing significant psychological and economic burdens on families and society, has a global prevalence of 1–3% (Bustos and Findlay, 2023; Nair et al., 2022). X-linked intellectual disability (XLID) accounts for approximately 5–15% of all ID cases (Bustos and Findlay, 2023). Fragile X syndrome (FXS, OMIM 300624), the leading cause of XLID, represents ~50% of XLID cases and is the most common monogenic cause of inherited ID (Fernández et al., 2015). FXS follows an X-linked semidominant inheritance pattern, driven by abnormal expansion and methylation of CGG trinucleotide repeats in the 5′ untranslated region (5’UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene (Hagerman and Hagerman, 2021). This abnormality disturbs the expression of fragile X messenger ribonucleoprotein (FMRP), encoded by FMR1, resulting in various disorders of FXS, including neural development (Rodriguez et al., 2020; Sirois et al., 2024).

FXS is classified into four types based on the FMR1 CGG repeat length: normal (≤44 repeats), intermediate (45–54 repeats), pre-mutation (55–200 repeats), and full-mutation (>200 repeats) (Spector et al., 2021). Individuals with full-mutation exhibit marked phenotypic heterogeneity: more than 99% of males and ~50% of females manifest with FXS, with females having milder symptoms (Clinical Genetics Group of Medical Geneticist Branch of Chinese Medical Doctor Association et al., 2022). Among pre-mutation carriers, 40–75% of males and 16–20% of females develop fragile X-associated tremor/ataxia syndrome (FXTAS) (Hagerman and Hagerman, 2016), while 20% of females are also at risk for fragile X-associated primary ovarian insufficiency (FXPOI) (Jacquemont et al., 2007; Shelly et al., 2021). Female pre-mutation carriers have the CGG repeat expansion risk, which results in transmitting full-mutation to offspring; this risk rate is as high as 100% when the CGG repetition number is more than 140 (Nolin et al., 2003). Thus, preconception and prenatal FXS screening exhibit valuable clinical significance in offering reproductive decisions to individuals with abnormal CGG repeats of FMR1 and reducing FXS incidence.

Here, we identified that the three ID individuals in this study were suffering from FMR1 full-mutation through multiple approaches—whole-exome sequencing (WES), triplet-repeat primed PCR (TP-PCR) with capillary electrophoresis (CE), and long-range PCR (LR-PCR) with long-read sequencing (LRS) (Liang et al., 2022). In addition, four pre-mutation carriers, including the counselee (III-2), were also detected. Furthermore, we discussed insights into the genetic diagnosis and counseling of FXS based on this pedigree.

FXS, the leading cause of XLID, arises predominantly from CGG repeat expansions in the 5’UTR of FMR1 (Monaghan et al., 2013; Spector et al., 2021). Pre-mutation carriers of FMR1 often exhibit no clinical symptoms during reproductive years, yet carry a risk of transmitting full-mutation to offspring (Monaghan et al., 2013). In the pedigree here, female pre-mutation carriers in the II generation showed no FXS-associated manifestations, yet three full-mutation cases with ID were identified in the III generation. This highlights the critical role of FXS pre-mutation carrier screening in informing reproductive counseling, thereby reducing FXS incidence and enhancing the proportion of healthy offspring. Through comprehensive family history collection and multiple genetic testing, FXS was identified as the genetic etiology of ID in the investigated pedigree. Furthermore, insights into FXS genetic diagnosis and counseling strategies are discussed here.

FXS is the most common inherited cause of ID (Ciaccio et al., 2017). Its phenotypic spectrum is highly complex, comprising not only neurodevelopmental abnormalities underlying ID but also multi-system impairments (Ciaccio et al., 2017). In full-mutation individuals, ID is the typical phenotype, or is accompanied by autism and attention-deficit/hyperactivity disorder (Spector et al., 2021). With advancing age, pre-mutation carriers of both sexes are at risk for FXTAS, while females face additional risk of FXPOI (Hagerman and Hagerman, 2016; Jacquemont et al., 2007; Shelly et al., 2021). The additional X chromosome in females typically results in milder phenotypes of both full-mutation individuals and pre-mutation carriers compared to males (Spector et al., 2021). In our study, full-mutation female III-6 manifested with milder ID compared to males III-1 and III-8, also likely attributed to a shorter CGG repeat length (283 vs. 427 and 319). Notably, an asymptomatic full-mutation female with 280 CGG triplets has been reported (Hung et al., 2019). The high phenotypic heterogeneity of FXS complicates genetic counseling by limiting specific evidence for rapid genetic mapping. Given its relatively high prevalence, preconception and prenatal screening for FXS are crucial for early detection and intervention.

In this study, the counselee III-2 sought preconception counseling due to her brother (proband III-1) presenting with ID. Initially, only partial pedigree data covering II-1, II-2, III-1, III-2, III-3, and III-4 were provided, with a request for WES. However, WES failed to identify pathogenic variants consistent with the family’s phenotype. Recontacting the counselee for extended pedigree analysis revealed three individuals with ID in the family. Genetic segregation analysis showed that the phenotypic inheritance pattern did not align with classical autosomal or X-linked dominant/recessive models (Figure 3A). Considering FXS is the most common inherited cause of ID and follows an X-linked semidominant inheritance pattern, TP-PCR/CE analysis was performed on proband III-1’s DNA sample, confirming a FMR1 full-mutation. This case revealed the limitations of WES in FXS diagnosis and emphasizes the indispensable role of comprehensive pedigree data collection in genetic analysis (Figure 3A).

Current FXS diagnostic modalities primarily include Southern blot analysis, TP-PCR/CE, and CAFXS (Spector et al., 2021). Short-read sequencing technologies like WES are inadequate for FXS diagnosis (Spector et al., 2021). The GC-rich CGG repeat tract poses challenges for amplification and accurate mapping, often leading to undetected expansions over 200 repeats, missed mosaicism, and inability to define AGG interruption patterns—key factors in assessing mutation stability (Spector et al., 2021). This explains the failure of WES to identify the genetic etiology in this ID-affected pedigree. The screening value of the above methods was limited by their high costs and prolonged workflows. While TP-PCR/CE and nanopore sequencing shortened testing duration, CAFXS provided the highest diagnostic accuracy, albeit with higher costs and longer turnaround times (Xia et al., 2025). Development of cost-effective, high-accuracy diagnostic methods for FXS could potentially revolutionize current testing paradigms.

To achieve the precise assessment of the FMR1 gene, CAFXS was employed in this pedigree analysis. CAFXS also confirmed the diagnosis of III-1 as a full-mutation individual. While prior studies indicate a near 100% risk of full-mutation transmission when female pre-mutation carriers have ≥140 CGG repeats (Nolin et al., 2003), counselee III-2 was identified as a pre-mutation carrier with 71 repeats, fewer than her mother II-2 (152 repeats). Despite this intergenerational reduction in CGG repeat length, pre-mutation carriers II-2, II-4, and II-6 each transmitted full-mutation to their offspring. These findings demonstrate that bidirectional CGG repeat dynamics (expansion and reduction) occur during genetic transmission, complicating predictive modeling for offspring risk. Advancing mechanistic insights into FXS could unlock novel strategies to refine transmission risk prediction and reduction.

The primary pathogenesis of FXS arises from dynamic expansions of CGG trinucleotide repeats in the FMR1 5’UTR (Hagerman and Hagerman, 2021) (Figure 3B). In full-mutation individuals (CGG repeats ≥200), local DNA methylation silences FMR1, driving FMRP deficiency and FXS (Hagerman and Hagerman, 2021; Malachowski et al., 2023) (Figure 3B). The CGG repeat length further influences FXS pathogenesis by modulating inter-chromosomal interactions to regulate long synaptic genes expression (Malachowski et al., 2023) (Figure 3B).

Pre-mutation carriers (55 < CGG repeats <200) exhibit ≥5-fold FMR1 mRNA elevation, driving degenerative disorders like FXTAS and FXPOI via RNA toxicity or repeat-associated non-AUG (RAN) translation (Hagerman and Hagerman, 2021). Expanded CGG repeats undergo RAN translation to produce FMRpolyG, a uORF that inhibits FMRP synthesis (Sellier et al., 2017) (Figure 3B). FMRpolyG interacts with LAP2b to disrupt nuclear lamina structure, induce neuronal cell death, and form toxic aggregates linked to motor deficits and shortened lifespan in transgenic mice, thereby driving FXTAS pathogenesis (Rodriguez et al., 2020; Sirois et al., 2024).

FMRpolyG also contributes to FXPOI pathogenesis by disrupting granulosa cell mitosis, via reduced mTOR phosphorylation and nuclear architecture perturbation, thereby accelerating antral follicle atresia, diminishing anti-Müllerian hormone (AMH) production, and activating primordial follicles prematurely (Rose and Brown, 2020; Shelly et al., 2021). Furthermore, expanded CGG-repeat RNA forms stable intranuclear foci that sequester RNA-binding proteins (e.g., FUS, PA2G4, TRA2β), disrupting their regulation, and driving granulosa cell death independently of FMRpolyG expression (Rosario et al., 2022) (Figure 3B). FXPOI risk peaks at 59–99 CGG repeats, declining at 100–200 (Pastore et al., 2019). Pre-mutation carriers require higher doses of FSH for ovarian stimulation but exhibit similar ovarian response and pregnancy rates compared to full-mutation individuals (Ranisavljevic et al., 2020). FXPOI patients with FMR1 pre-mutations exhibit a mean menopause onset 5 years earlier than non-carriers (Shelly et al., 2021). The allelic score integrating CGG length and AGG patterns fails to independently predict amenorrhea age in FMR1 pre-mutation carriers (Rodrigues et al., 2024). Thus, predictive assessment of FXPOI risk allows for timely recommendations on cryopreserving reproductive materials (e.g., embryos, oocytes, ovarian tissue), providing substantial fertility preservation benefits to female FXS pre-mutation carriers intending to delay pregnancy (La Marca and Mastellari, 2021). However, the unclear mechanism of CGG repeat instability hinders the prediction of FXPOI risk in pre-mutation carriers or full-mutation transmission to offspring. Additionally, limited FXPOI knowledge among women’s healthcare providers may delay FMR1 testing for POI patients, underscoring the need for enhanced education on FXPOI-FXS associations and integrated genetic counseling to enable timely screening and risk assessment (Singleton et al., 2025). Large-scale cohort studies are therefore critical for establishing clinical patterns and providing evidence for genetic counseling and reproductive guidance.

In this pedigree, both pre-mutation (II-2, II-4, and II-6) and full-mutation (III-6) females achieved successful natural pregnancies. The primary concern of counselee III-2 (a pre-mutation carrier female) centered on the risk of ID in offspring. While higher maternal CGG repeat lengths correlate with increased offspring full-mutation risk, approaching 100% for female pre-mutation carriers with CGG repeats≥140 (Nolin et al., 2003). Even more, maternal AGG interruptions in the FMR1 gene mitigate the risk of offspring developing FXS (Yrigollen et al., 2012). However, the inherent unpredictability of dynamic CGG repeat expansions is strikingly evident. Maternal carrier II-2 (152 CGG repeats) gave birth to four children: one with full-mutation (III-1, 427 repeats), one with reduced pre-mutation (III-2, 71 repeats), and two with normal genotype (III-3 and III-4), indicating unpredictable expansion dynamics in this pedigree. Notably, III-2’s CGG repeat length (71) decreased from the maternal 152, complicating the risk prediction. The full-mutation individual III-6 (283 repeats) here exhibits mild ID, and prior reports describe asymptomatic females with 280 CGG repeats. These findings highlight the limitations of using repeat length alone to predict full-mutation risk or ID severity in III-2’s offspring. To mitigate full-mutation transmission risk for counselee III-2, available strategies include prenatal diagnosis after natural conception or preimplantation genetic testing for assisted reproductive technologies (Cohen et al., 2021; Hung et al., 2019; Persico et al., 2023; Sonigo et al., 2021).

FXS underlies ID in this pedigree, highlighting the critical clinical importance of screening ID families with atypical inheritance patterns. Such screening aids in preventing diagnostic oversights related to FXS and decreases the incidence of the syndrome by offering genetic and reproductive counseling to individuals who are pre-mutation carriers.