Detection of skewed X-chromosome inactivation in Fragile X syndrome and X chromosome aneuploidy using quantitative melt analysis.

Methylation of the fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary positioned fragile X related epigenetic element 2 (FREE2), reveals skewed X-chromosome inactivation (XCI) in fragile X syndrome full mutation (FM: CGG > 200) females. XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs). In this study, 10 FREE2 CpG sites were targeted using methylation specific quantitative melt analysis (MS-QMA), including 3 sites that could not be analysed with previously used EpiTYPER system. The method was applied for detection of skewed XCI in FM females and in different types of SCA. We tested venous blood and saliva DNA collected from 107 controls (CGG < 40), and 148 FM and 90 SCA individuals. MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals. MS-QMA output also showed significant correlation with the EpiTYPER reference method in FM males and females (P < 0.0001) and SCAs (P < 0.05). In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility

Editorial for the Fragile X Syndrome Genetics Special Issue: May 2023

Fragile X syndrome (FXS) is the leading single-gene cause of inherited intellectual disability and autism [...

Special Issue: Genetics of Prader–Willi Syndrome

This Special Issue includes 15 peer-reviewed articles for publication by experts in Prader–Willi syndrome (PWS) and their reflective area of interest impacting this rare disorder [...

Exploring autism symptoms in an Australian cohort of patients with Prader-Willi and Angelman syndromes

Abstract Background Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurodevelopmental disorders that are caused by abnormal expression of imprinted genes in the 15q11-13 region. Dysregulation of genes located in this region has been proposed as a susceptibility factor for autism spectrum disorder (ASD) in both disorders. Methods This study aimed to explore symptoms of ASD in 25 PWS and 19 AS individuals aged between 1 and 39 years via objective assessment. Participants completed the Autism Diagnostic Observation Schedule-2nd Edition (ADOS-2) and a developmentally or age-appropriate intellectual functioning assessment. All participants had their genetic diagnosis confirmed using DNA methylation analysis and microarray testing of copy number changes within the 15q11-13 region. Results Participants with PWS had significantly higher overall and social affect calibrated severity scores (CSS) on the ADOS-2 compared to AS participants (p = .0055 and .0015, respectively), but the two groups did not differ significantly on CSS for the repetitive and restricted behaviour domain. Conclusions PWS cases presented with greater symptoms associated with ASD compared to individuals with AS. Mental health issues associated with PWS may contribute to elevated symptoms of ASD, particularly in adolescents and adults with PWS

Clinical and Molecular Differences between 4-Year-Old Monozygous Male Twins Mosaic for Normal, Premutation and Fragile X Full Mutation Alleles

This study describes monozygotic (MZ) male twins with fragile X syndrome (FXS), mosaic for normal size (NS: <44 CGGs), premutation (PM: 55–199 CGG) and full mutation (FM alleles ≥ 200) alleles, with autism. At 4 years of age chromosomal microarray confirmed monozygosity with both twins showing an XY sex complement. Normal size (30 CGG), PM (99 CGG) and FM (388–1632 CGGs) alleles were detected in Twin 1 (T1) by standard polymerase chain reaction (PCR) and Southern blot testing, while only PM (99 CGG) and FM (672–1025) alleles were identified in Twin 2 (T2). At ~5 years, T2 had greater intellectual impairments with a full scale IQ (FSIQ) of 55 and verbal IQ (VIQ) of 59, compared to FSIQ of 62 and VIQ of 78 for T1. This was consistent with the quantitative FMR1 methylation testing, revealing 10% higher methylation at 80% for T2; suggesting that less active unmethylated alleles were present in T2 as compared to T1. AmplideX methylation PCR also identified partial methylation, including an unmethylated NS allele in T2, undetected by standard testing. In conclusion, this report demonstrates significant differences in intellectual functioning between the MZ twins mosaic for NS, PM and FM alleles with partial FMR1 promoter methylation

β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies

Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55-199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called β-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10-11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships