Background Fragile X syndrome (FXS) is the most frequent cause of inherited X-linked intellectual disability. Conventional FXS genetic testing methods mainly focus on FMR1 CGG expansions and fail to identify AGG interruptions, rare intragenic variants, and large gene deletions. Methods A long-range PCR and long-read sequencing-based assay termed comprehensive analysis of FXS (CAFXS) was developed and evaluated in Coriell and clinical samples by comparing to Southern blot analysis and triplet repeat–primed PCR (TP–PCR). Results CAFXS accurately detected the number of CGG repeats in the range of 93 to at least 940 with mass fraction of 0.5% to 1% in the background of normal alleles, which was 2–4-fold analytically more sensitive than TP–PCR. All categories of mutations detected by control methods, including full mutations in 30 samples, were identified by CAFXS for all 62 clinical samples. CAFXS accurately determined AGG interruptions in all 133 alleles identified, even in mosaic alleles. CAFXS successfully identified 2 rare intragenic variants including the c.879A > C variant in exon 9 and a 697-bp microdeletion flanking upstream of CGG repeats, which disrupted primer annealing in TP–PCR assay. In addition, CAFXS directly determined the breakpoints of a 237.1-kb deletion and a 774.0-kb deletion encompassing the entire FMR1 gene in 2 samples. Conclusions Long-read sequencing-based CAFXS represents a comprehensive assay for identifying FMR1 CGG expansions, AGG interruptions, rare intragenic variants, and large gene deletions, which greatly improves the genetic screening and diagnosis for FXS.
Background The aim is to evaluate the clinical utility of a long-read sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) in prenatal diagnosis of thalassemia. Methods A total of 278 fetuses from at-risk pregnancies identified in thalassemia carrier screening by PCR-based methods were recruited from 9 hospitals, and PCR-based methods were employed for prenatal diagnosis. CATSA was performed retrospectively and blindly for all 278 fetuses. Results Among the 278 fetuses, 263 (94.6%) had concordant results and 15 (5.4%) had discordant results between the 2 methods. Of the 15 fetuses, 4 had discordant thalassemia variants within the PCR detection range and 11 had additional variants identified by CATSA. Independent PCR and Sanger sequencing confirmed the CATSA results. In total, CATSA and PCR-based methods correctly detected 206 and 191 fetuses with variants, respectively. Thus, CATSA yielded a 7.9% (15 of 191) increment as compared with PCR-based methods. CATSA also corrected the predicted phenotype in 8 fetuses. Specifically, a PCR-based method showed one fetus had homozygous HBB c.52A > T variants, while CATSA determined the variant was heterozygous, which corrected the predicted phenotype from β-thalassemia major to trait, potentially impacting the pregnancy outcome. CATSA additionally identified α-globin triplicates in 2 fetuses with the heterozygous HBB c.316-197C > T variant, which corrected the predicted phenotype from β-thalassemia trait to intermedia and changed the disease prognosis. Conclusions CATSA represents a more comprehensive and accurate approach that potentially enables more informed genetic counseling and improved clinical outcomes compared to PCR-based methods.
In prenatal diagnosis, chromosomal aberrations, such as aneuploidies and copy number variation (CNV), are one of the important reasons for ultrasound structural abnormalities and products of conceptions (POC). CNVs are pervasive in human genome and account for a large fraction of the population diversity in humans (Girirajan, Campbell, & Eichler, 2011). Many CNVs located in specific genome regions also have clinical significance or have strong associations with well-characterized genomic disorders, such as 22q11 deletion syndrome (Velocardiofacial/DiGeorge syndrome; Faas et al.,
Hemophilia A (HA) is the most frequently occurring X-linked bleeding disorder caused by heterogeneous variants in the F8 gene, one of the largest genes known. Conventional molecular analysis of F8 requires a combination of assays, usually including long range (LR)-PCR or inverse PCR for inversions, Sanger sequencing or next-generation sequencing for single-nucleotide variants (SNVs) and indels, and multiplex ligation-dependent probe amplification for large deletions or duplications. This study aimed to develop a LR-PCR and long-reads sequencing (LRS)-based assay termed comprehensive analysis of hemophilia A (CAHEA) for full characterization of F8 variants. The performance of CAHEA was evaluated in 272 samples from 131 HA pedigrees with a wide spectrum of F8 variants by comparing to conventional molecular assays. CAHEA identified F8 variants in all the 131 pedigrees, including 35 intron 22-related gene rearrangements, three intron 1 inversion (Inv1), 85 SNVs and indels, one large insertion and seven large deletions. Compared to the conventional methods combined altogether, CAHEA assay demonstrated 100% sensitivity and specificity for identifying various type of F8 variants and had the advantages of directly determining the break regions/points of large inversions, insertions and deletions, which enabled analyzing the mechanisms of recombination at the junction sites and pathogenicity of the variants. CAHEA represents a comprehensive assay towards full characterization of F8 variants including intron 22 and intron 1 inversions, SNVs/indels, large insertions and deletions, greatly improving the genetic screening and diagnosis for HA.
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