Aug 2026· Thalassemia Reports· Vol 16, pp. 18· 0 citations· 39 references
TL;DR
A tiered, genotype-informed approach—combining HPLC/CE phenotyping, targeted molecular diagnostics, genetic modifier profiling, and periodic re-evaluation—optimizes diagnostic precision and guides individualized management across the thalassemia spectrum.
Abstract
Thalassemia represents the world’s most prevalent inherited hemoglobin disorder, affecting approximately 4.4 per 10,000 live births globally. Accurate genetic characterization is indispensable both for definitive diagnosis and for lifetime clinical monitoring. The past two decades have witnessed a paradigm shift from conventional protein-based assays toward comprehensive molecular techniques, including next-generation sequencing (NGS) and third-generation (long-read) sequencing, which in turn have enabled reproductive applications such as preimplantation genetic testing for monogenic disease (PGT-M) to identify unaffected embryos before implantation. (1) To systematically evaluate the molecular techniques available for confirming the diagnosis of alpha- and beta-thalassemia, including their diagnostic accuracy, indications, and limitations; (2) to examine how genotype–phenotype correlation and genetic modifier profiling inform clinical prognosis and therapeutic decision-making; and (3) to define evidence-based genetic monitoring parameters for longitudinal follow-up of patients receiving transfusions, iron chelation, and novel curative therapies including gene therapy. A comprehensive narrative review was conducted by systematically searching PubMed/MEDLINE for English-language peer-reviewed articles published between January 2000 and December 2024. Forty-three studies were ultimately included after applying predefined inclusion and exclusion criteria. Quality of included studies was assessed using SANRA (Scale for the Assessment of Narrative Review Articles). HPLC and capillary electrophoresis remain first-line phenotyping tools; DNA-based confirmation is mandatory for complete genotyping. Among known, previously characterized mutations, NGS-based targeted panels achieve > 95% detection sensitivity, but they require MLPA co-testing or long-read sequencing to detect structural variants such as large deletions. Genotype–phenotype prediction is substantially improved, though not rendered fully deterministic, by profiling three major modifier loci: XmnI (Gγ), BCL11A, and HBS1L-MYB. PGT-M using NGS achieves near-complete genotyping accuracy (>99%) with live birth rates of 40–60% per frozen embryo transfer cycle. For patients receiving curative gene therapy (exagamglogene autotemcel/Casgevy), molecular follow-up protocols spanning 15 years are now recommended. Cardiac T2* MRI remains the most reliable non-invasive tool for iron overload follow-up, superior to serum ferritin alone. A tiered, genotype-informed approach—combining HPLC/CE phenotyping, targeted molecular diagnostics, genetic modifier profiling, and periodic re-evaluation—optimizes diagnostic precision and guides individualized management across the thalassemia spectrum. Integration of PGT-M and long-read sequencing into standard care pathways, alongside robust gene therapy follow-up protocols, will define the next era of thalassemia genetics.
BACKGROUND
C-MoKa (Chromosome Conformation-based Karyotyping) is a novel 3D genome mapping platform, that enables simultaneous detection of structural variations (SVs), aneuploidies, copy number variations (CNVs), and uniparental disomy (UPD) in a single test. However, its performance in routine prenatal diagnosis is still unexplored. This study aimed to comprehensively evaluate the diagnostic performance and clinical utility of C-MoKa in prenatal diagnosis, and to assess its technical concordance with standard of care (SOC) testings in prenatal diagnosis.
METHODS
A two-phase study was designed. In phase 1, 56 retrospective participants with known chromosomal abnormalities (CAs) were recruited, and C-MoKa was performed on their cultured amniotic fluid (AF) samples. Concordance between C-MoKa and known CAs was analyzed. In phase 2, a prospective cohort of 208 participants for prenatal diagnosis were recruited. Samples underwent C-MoKa and karyotyping (KT), with parallel chromosomal microarray analysis (CMA) or improved whole-exome sequencing (iWES). In our study, supplementary copy number probes were enhanced in iWES detection, which could provide ~ 100 kb resolution across the genome, thus either CMA or iWES platform employed was used to assess the CNV detection. Diagnostic yields and concordance were evaluated, and the discordance of SVs and CNVs were further validated by fluorescence in situ hybridization (FISH) and CNV-seq, respectively.
RESULTS
In the retrospective cohort with known CAs, C-MoKa achieved a 94.6% (53/56) diagnostic yield and 92.8% (52/56) concordance with KT + CNV. In the prospective cohort, its diagnostic yield was 21.2% (44/208), higher than KT (12.5%, 26/208) and CNV (18.3%, 38/208). The diagnostic yield (22.6%, 47/208) was achieved when CNV platform and C-MoKa were used together. C-MoKa exhibited concordance rates of 89.4% (186/208) with KT, 89.9% (187/208) with CNV, and 90.4% (188/208) with KT + CNV. Two SVs identified by C-MoKa but missed by KT were successfully determined by FISH, and five samples with additional CNVs identified by C-MoKa were consistently detected by CNV-seq.
CONCLUSIONS
Our findings demonstrate that C-MoKa is a highly effective and reliable method for prenatal diagnosis, exhibiting high concordance with SOC techniques. Compared to the conventional application of "KT + CNV" in prenatal setting, the combined use of CNV and C-MoKa appears to maximize the diagnostic yield.
Ying Zhou, Min Xie, L. Tian et al.· Journal of Translational Med...· 0 citations
Hypospadias is one of the most common birth defects in China and a key feature of differences in sex development (DSD), yet its genetic etiology remains largely unresolved. Current diagnostic approaches using DSD-targeted gene panels have a low rate of definitive diagnoses (5.5%), highlighting the need for more comprehensive genetic investigation. In this study, we performed next-generation sequencing (NGS) on the largest trio-based cohort of hypospadias to date, comprising 106 pediatric cases and their parents (92 trios). We achieved a definitive genetic diagnosis in 6.6% of patients, identifying pathogenic variants in canonical DSD genes such as AR, NR5A1 and WT1. By incorporating a broader spectrum of potentially clinically significant variants, we increased the overall genetic identification rate to 33.0% (35/106). Strikingly, trio analysis uncovered a significant burden of de novo loss-of-function (LoF) variants (2.2-fold enrichment, P=0.001), primarily driven by variants in genes associated with ciliopathies (10.35-fold, P=0.016), a previously underappreciated gene class in hypospadias. Furthermore, we identified and functionally validated two high-confidence risk genes, PRKCZ and HRNR, based on recurrent de novo variants. Functional assays confirmed that these variants disrupt key biological mechanisms, including cell proliferation, migration, and androgen signaling. Our large-scale trio approach substantially expands the genetic landscape of hypospadias, demonstrates the critical value of trio-based sequencing for improving diagnostic yield, and decisively implicates ciliary genes in its pathogenesis.
Yanqin You, Yingliu Luo, Honghui Zhou et al.· Science China Life Sciences· 0 citations
Abstract Objective: To evaluate real-world implementation of newborn genetic screening (NBGS) in terms of positivity rate, carrier frequency, and diagnostic accuracy for inherited metabolic disorders (IMDs), and to explore feasibility and challenges in regional clinical application. Methods: This study enrolled 1590 newborns (August 2023–November 2024) whose parents opted for NBGS. A targeted sequencing panel covering 465 genes for 596 diseases alongside traditional biochemical screening for 46 disorders were performed. Variants were classified per ACMG guidelines. Positive cases were recalled for confirmatory Sanger sequencing and auxiliary biochemical tests. We calculated uptake, carrier frequency, identified hotspot variants, and compared allele frequencies with gnomAD_EAS. Diagnostic performance was compared with biochemical screening. Results: Uptake was 10.41% (1590/15,272). Overall positivity was 7.74% (123/1590). Among these, 10 were positive for IMDs‑related genes, with 8 confirmed, giving a positive predictive value (PPV) of 80.00% for IMDs – significantly higher than biochemical screening’s 5.71%. We detected 2,354 variants, with carrier frequency 70.94%. Hotspot IMDs variants included MMACHC c.609G>A, c.658_660del, and MUT c.1286A>G, whose minor allele frequencies differed from gnomAD, indicating regional specificity. High carrier rates were also seen for lysosomal storage genes (GALC c.1901T>C, 53.57%; c.2041G>A, 32.14%). Conclusion: NBGS offers high PPV and specificity, reducing false positives and providing early molecular evidence for IMDs. Regional hotspot and MAF differences underscore the need for a local genetic database. Although the acceptance of NBGS is increasing, factors such as cost and varying levels of awareness among healthcare providers remain barriers to its broader implementation. This study provides preliminary data supporting the implementation of regional newborn genetic screening programs and informs secondary and tertiary prevention strategies.
Jialin Mu, Meng Sun, Yulin Li et al.· Annals medicus· 0 citations
In this narrative review, we outline the historical progression of, and modern advancements in, prenatal diagnosis for monogenic diseases. Initially dependent on invasive sampling procedures, prenatal diagnosis has evolved through the integration of high-throughput molecular techniques, such as next-generation sequencing. These techniques now allow for the comprehensive detection of pathogenic variants across the genome. A major breakthrough occurred with the discovery of cell-free fetal DNA (cfDNA) in maternal plasma, originating from the placenta. This discovery enabled the development of non-invasive prenatal testing (NIPT), which is now widely used. However, non-invasive prenatal diagnosis (NIPD) for monogenic diseases took longer to develop, mainly due to the need for highly sensitive sequencing methods and sophisticated statistical analysis to determine maternal transmission. Currently, only a few countries have implemented NIPD in clinical settings. The multitude of available technologies and protocols can complicate the information provided during antenatal consultations. However, mastery of the knowledge and ethical issues surrounding NIPD will ensure optimal service and better care for pregnancies at risk of monogenic disease transmission.
Camille Verebi, J. Nectoux, Thierry Bienvenu· Archives of Medical Research· 0 citations
Simple Summary This study evaluated ultra-deep error-corrected next-generation sequencing (NGS) in 134 patients with myeloproliferative neoplasms (MPNs) to determine its diagnostic and prognostic value. Ultra-deep sequencing detected many low-variant-allele-frequency (VAF < 5%) mutations that conventional methods could miss, including 25% of CALR, 43% of MPL, and 82% of TP53 mutations. In contrast, most JAK2 mutations had higher VAFs and were reliably detected by standard testing. During leukemic transformation, all TP53 mutations showed VAFs > 5%, suggesting clonal expansion during disease progression. Lower JAK2 p.V617F VAFs were associated with patients who remained untreated, indicating potential clinical relevance for treatment decisions. Additional mutations such as ASXL1 and SRSF2 were observed in patients with disease progression. Overall, the findings suggest that panel-based ultra-deep NGS improves mutation detection, enhances diagnostic sensitivity, and may provide valuable prognostic information for MPN management, although larger prospective studies are needed to confirm these results.
Namsoo Kim, Yehyun Kang, Hye Won Kook et al.· Cancers· 0 citations
Phenylketonuria (PKU) is a common inherited metabolic disorder in newborns. Early diagnosis and timely intervention are essential to prevent neurodevelopmental impairment. Traditional newborn screening primarily relies on measuring blood phenylalanine (Phe) levels, which can lead to both false-positive and false-negative results. Long-read sequencing (LRS) offers high coverage and long-fragment resolution, enabling comprehensive detection of
PAH
gene variants and potentially improving PKU screening and diagnosis.
We developed a comprehensive
PAH
gene sequencing technology (CAPAH) based on the HiFi LRS platform, and applied it to the second-tier screening for PKU in newborns. First, CAPAH was validated for consistency with traditional NGS, MLPA, and Sanger sequencing in 20 samples with known genotypes. Subsequently, a retrospective analysis of 200 newborns with positive or borderline primary screening results was performed to assess the impact of CAPAH on variant detection, genotype-phenotype associations, and second-tier screening performance.
CAPAH achieved complete consistency (100%) with traditional methods in the 20 validation samples. It enabled direct determination of cis-trans configurations and precise localization of large deletion breakpoints. Among 200 newborns, CAPAH identified 103 newborns with biallelic variants, 47 carriers, and 50 negative individuals, reducing the recall rate from primary screening by 48.5% (97/200). Among 131 newborns with negative or excluded PKU based on rescreening blood Phe levels, CAPAH detected 40 newborns carrying compound heterozygous variants, preventing approximately 20% of potential missed detections. In the 103 newborns with biallelic variants, CAPAH identified 64
PAH
variants, including 47 missense, eight splice-site, four nonsense, and five deletion variants. Genotype-phenotype analysis revealed that the proportion of severe biallelic PKU-related variants (classic and mild PKU) was significantly higher in the rescreen-positive group than in the rescreen-negative group (25.00% vs. 14.29%). Homozygosity for c.158G > A or its combination with PKU- or mild hyperphenylalaninemia (MHP)-related variants was associated with blood Phe levels within the MHP range (2–6 mg/dL). When c.158G > A was combined with PKU-related variants, rescreening Phe levels were significantly elevated (
p
< 0.01).
CAPAH enables comprehensive detection of the
PAH
gene, including complex variants and cis–trans configurations, thereby improving the accuracy of second-tier PKU screening in newborns and reducing unnecessary recalls. This approach provides an efficient and practical molecular diagnostic tool for newborn screening of inherited metabolic disease.
Shuyuan Xue, Ziyi Feng, Jingying Zhu et al.· Orphanet Journal of Rare Dis...· 0 citations