X-linked intellectual disability is a common cause of inherited cognitive deficit affecting mostly males. There are several genetic causes implicated in this condition, which has hampered the establishment of an accurate diagnosis. We developed a multiplex-PCR assay for the mutational hotspot regions of the FMR1, AFF2 and ARX genes.

We describe a female infant with Fragile-X syndrome, with a fully expanded FMR1 allele and preferential inactivation of the homologous X-chromosome carrying a de novo deletion. This unusual and rare case demonstrates the importance of a detailed genomic approach, the absence of which could be misguiding, and calls for reflection on the current clinical and diagnostic workup for developmental disabilities. We present a female infant, referred for genetic testing due to psychomotor developmental delay without specific dysmorphic features or relevant family history. FMR1 mutation screening revealed a methylated full mutation and a normal but inactive FMR1 allele, which led to further investigation. Complete skewing of X-chromosome inactivation towards the paternally-inherited normal-sized FMR1 allele was found. No pathogenic variants were identified in the XIST promoter. Microarray analysis revealed a 439 kb deletion at Xq28, in a region known to be associated with extreme skewing of X-chromosome inactivation. Overall results enable us to conclude that the developmental delay is the cumulative result of a methylated FMR1 full mutation on the active X-chromosome and the inactivation of the other homologue carrying the de novo 439 kb deletion. Our findings should be taken into consideration in future guidelines for the diagnostic workup on the diagnosis of intellectual disabilities, particularly in female infant cases.

X-chromosome inactivation (XCI) is a developmental process to compensate the imbalance in the dosage of X-chromosomal genes in females. A skewing of the XCI pattern may suggest a carrier status for an X-linked disease or explain the presence of a severe phenotype. In these cases, it is important to determine the XCI pattern, conventionally using the gold standard Human Androgen-Receptor Assay (HUMARA), based on the analysis of the methylation status at a polymorphic CAG region in the first exon of the human androgen receptor gene (AR). The aim of this study was to evaluate whether the methylation status of the fragile mental retardation protein translational regulator gene (FMR1) can provide an XCI pattern similar to that obtained by HUMARA. A set of 48 female carriers of FMR1 gene normal-sized alleles was examined using two assays: HUMARA and a FMR1 methylation PCR (mPCR). Ranges were defined to establish the XCI pattern using the methylation pattern of the FMR1 gene by mPCR. Overall, a 77% concordance of the XCI patterns was obtained between the two assays, which led us to propose a set of key points and a stepwise analysis towards obtaining an accurate result for the XCI pattern and to minimize the underlying pitfalls.

In a patient with Usher syndrome and atypical muscle complaints, we have identified two separate variants in MYO7A and NEB genes by exome sequencing. The homozygous variants in these two recessive genes could explain the full phenotype of our patient.

We investigated whether FMR1 allelic complexity—integrating CGG repeat length with the number and pattern of AGG interspersions—can be used as a predictor of ovarian reserve and in vitro fertilization (IVF) success. This cohort study included 124 females with infertility attributed to female factors undergoing intracytoplasmic sperm injection (ICSI). The total CGG repeat lengths and AGG interspersion patterns of the FMR1 gene were determined by conventional polymerase chain reaction (PCR) and triplet-primed PCR. The allelic complexity (allelic score) was calculated using a previously described formula by combining the allelic scores, allowing for the stratification of samples into equivalent and dissimilar groups. No statistically significant differences were observed in ovarian reserve markers or overall IVF outcomes between the two groups. However, within the dissimilar group, the allelic score of allele 1 was significantly correlated with the number of both injected metaphase II and two-pronuclei oocytes. These findings suggest that FMR1 allelic complexity may contribute to predicting IVF success, particularly in females classified in the dissimilar group, who appear more susceptible to IVF failure than those in the equivalent group. Further research into the predictive utility of FMR1 could provide valuable insights for fertility assessment and enhance assisted reproductive technologies.