Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder affecting women of reproductive age, yet the molecular mechanisms influencing its pathophysiology remain poorly defined. A comprehensive prospective case-control study was conducted to elucidate the follicular fluid (FF) hormone and metabolite profile in women with PCOS and its implications for oocyte maturation and fertilization. The study involved 40 age- and body mass index (BMI)-matched women undergoing in vitro fertilization (IVF), including 20 diagnosed with PCOS and 20 controls with infertility due to tubal or male factors. A distinctive hormone profile in the FF of women with PCOS was identified, characterized by significantly higher anti-Müllerian hormone (AMH) levels (24.90±17.61 vs. 16.68±17.67 pmol/L, p=0.0039) and lower progesterone (8253±4748 vs. 25362±10862 ng/mL, p<0.0001) and estradiol levels (388.23±210.58 vs. 651.48±390.79 ng/mL, p=0.0208) compared to normoovulatory controls. Moreover, a metabolite fingerprint associated with glycolytic and mitochondrial dysfunction was observed, as evidenced by lower lactate (4575.44±1507.76 vs. 5595.34±1073.32 μmol/L, p=0.0182) and formate (64.51±16.06 vs. 75.81±16.63 μmol/L, p=0.0351) levels and higher citrate levels (136.93±52.53 vs. 109.15±24.17 μmol/L, p=0.0409) in the FF of women with PCOS. These findings suggest that the molecular profile of the FF in women with PCOS is related to granulosa cell glycolytic and mitochondrial dysfunction, which can have a negative impact on oocyte fertilization potential. The study provides an integrative analysis of the FF hormone and metabolite profile in women with PCOS, offering insights into the molecular mechanisms underlying the reproductive dysfunctions associated with this condition.

Autosomal Recessive Spinocerebellar Ataxia 20, SCAR20, is a rare condition characterized by intellectual disability, lack of speech, ataxia, coarse facies and macrocephaly, caused by SNX14 variants. While all cases described are due to homozygous variants that generally result in loss of protein, so far there are no other cases of reported compound heterozygous variants. Here we describe the first non-consanguineous SCAR20 family, the second Portuguese, with two siblings presenting similar clinical features caused by compound heterozygous SNX14 variants: NM_001350532.1:c.1195C>T, p.(Arg399^*) combined with a novel complex genomic rearrangement. Quantitative PCR (Q-PCR), long-range PCR and sequencing was used to elucidate the region and mechanisms involved in the latter: two deletions, an inversion and an AG insertion: NM_001350532.1:c.[612+3028_698-2759del;698-2758_698-516inv;698-515_1171+1366delinsAG]. In silico analyses of these variants are in agreement with causality, enabling a genotype-phenotype correlation in both patients. Clinical phenotype includes dystonia and stereotypies never associated with SCAR20. Overall, this study allowed to extend the knowledge of the phenotypic and mutational spectrum of SCAR20, and to validate the role of Sorting nexin-14 in a well-defined neurodevelopmental syndrome, which can lead to cognitive impairment. We also highlight the value of an accurate clinical evaluation and deep phenotyping to disclose the molecular defect underlying highly heterogeneous condition such as intellectual disability.

Background: Premutations in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene, defined as between 55 and 200 CGGs, have been implicated in fragile X-associated primary ovarian insufficiency (FXPOI). Only 20% of female premutation carriers develop early ovulatory dysfunction, the reason for this incomplete penetrance is unknown. This study validated the mathematical model in premutation alleles, after assigning each allele a score representing allelic complexity. Subsequently, allelic scores were used to investigate the impact of allele complexity on age at amenorrhea for 58 premutation cases (116 alleles) previously published. Methods: The allelic score was determined using a formula previously described by our group. The impact of each allelic score on age at amenorrhea was analyzed using Pearson’s test and a contour plot generated to visualize the effect. Results: Correlation of allelic score revealed two distinct complexity behaviors in premutation alleles. No significant correlation was observed between the allelic score of premutation alleles and age at amenorrhea. The same lack of significant correlation was observed regarding normal-sized alleles, despite a nearly significant trend. Conclusions: Our results suggest that the use of allelic scores combination have the potential to explain female infertility, namely the development of FXPOI, or ovarian dysfunction, despite the lack of correlation with age at amenorrhea. Such a finding is of great clinical significance for early identification of females at risk of ovulatory dysfunction, enhancement of fertility preservation techniques, and increasing the probability for a successful pregnancy in females with premutations. Additional investigation is necessary to validate this hypothesis.

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.

The polymorphic trinucleotide repetitive region in the FMR1 gene 5′UTR contains AGG interspersions, particularly in normal-sized alleles (CGG < 45). In this range repetitive stretches are typically interrupted once or twice, although alleles without or with three or more AGG interspersions can also be observed. AGG interspersions together with the total length of the repetitive region confer stability and hinder expansion to pathogenic ranges: either premutation (55 < CGG < 200) or full mutation (CGG > 200). The AGG interspersions have long been identified as one of the most important features of FMR1 repeat stability, being particularly important to determine expansion risk estimates in female premutation carriers. We sought to compute the combined AGG interspersion numbers and patterns, aiming to define FMR1 repetitive tract complexity combinations. A mathematical model, the first to compute this cumulative effect, was developed and validated using data from 131 young and healthy females. Plotting of their allelic complexity enabled the identification of two statistically distinct groups – equivalent and dissimilar allelic combinations. The outcome, a numerical parameter designated allelic score, depicts the repeat substructure of each allele, measuring the allelic complexity of the FMR1 gene including the AGGs burden, thus allowing new behavioral scrutiny of normal-sized alleles in females.

Over 100 X-linked intellectual disability genes have been identified, with triplet repeat expansions at the FMR1 (FRAXA) and AFF2 (FRAXE) genes being the causative agent in two of them. The absence of FRAXE pathognomonic features hampers early recognition, delaying testing and molecular confirmation. Hence, our laboratory uses a multiplex PCR-based strategy to genotype both FRAXA and FRAXE. However, AFF2 expansions are missed giving rise to an uninformative result in around 20% of female samples. To rule out undetected expansions and confirm homozygosity Southern blot analysis is performed being labour- and resource-intensive. The aim of this study is to develop a timely and economic triplet-primed amplification (TP-PCR) screening strategy to size the AFF2 GCC repeat and accurately assess homozygosity as well as pinpoint multiplex-PCR false negatives in female samples. In order to achieve this, validation was performed in a cohort of 500 females with a previous uninformative FRAXE PCR result. Interestingly, the presence of a T > C SNP (rs868949662), contiguous to the GCC repetitive tract, allows triplet primer binding in two additional repeats, increasing the discrimination power of the TP-PCR assay in heterozygous and homozygous samples. Twelve alleles outside the normal range were recognized: eight intermediate and four premutated, which seems relevant considering the rarity of the AFF2 expansions. All genotypes are concordant with that obtained by Southern blotting, confirming this as a strict, reproducible and low-cost homozygosity screening strategy that enables the identification of small expanded alleles missed by the routine multiplex-PCR due to allele dropout. Overall, this assay is capable of spotting multiplex-PCR false negatives besides identifying alleles up to > 80 GCC repeats. Furthermore, the occurrence of intermediate repeat sizes with unexpected frequency, introduces new areas of clinical research in this cohort in understanding these less explored AFF2 repeat sizes and newly associated phenotypes.