Splice-disruptive variants represent an underrecognized yet critical category of disease-causing mutations. While canonical splice site disruptions have long been associated with genetic disorders, it is now increasingly evident that synonymous, deep-intronic, and regulatory variants can also perturb splicing events and contribute to diseases. As genomic diagnostics shift from phenotype-first to genome-first paradigms, there is an urgent need for systematic strategies to identify and interpret such variants-including those residing in noncoding regions that escape detection by traditional annotation pipelines. This review provides an integrative overview of current in silico approaches for the annotation and interpretation of splice-disruptive variants. We outline the mechanistic diversity of splicing aberrations and discuss recent advances in computational prediction frameworks, including both deep learning-based models and motif-oriented tools. In parallel, we summarize experimental strategies that are used to validate predicted splicing effects and assess their pathogenic relevance. Focusing on clinically relevant contexts, we discuss how splicing-aware variant interpretation enhances diagnostic yield, informs the reclassification of variants of uncertain significance, and uncovers targets for therapeutic intervention. Finally, we consider the implications of such interpretation for RNA-targeted strategies, including antisense oligonucleotides, small-molecule modulators, and emerging RNA-editing platforms, particularly in neuromuscular and other splicing-driven disorders. Together, these insights underscore the expanding role of in silico splicing prediction in precision medicine, offering new diagnostic and therapeutic avenues for rare and undiagnosed genetic diseases.

SEC24D is a key component of the Coat Protein Complex II, which plays a critical role in the selective sorting and transport of cargo proteins from the endoplasmic reticulum. This function is particularly essential for the secretion of extracellular matrix proteins, including collagens. Biallelic pathogenic variants in SEC24D have been associated with Cole-Carpenter Syndrome 2, a rare skeletal dysplasia characterized by craniofacial abnormalities and recurrent fractures. We reported a 12-year-old male patient presenting with recurrent bone fractures, severe skeletal deformities, limb shortening, craniofacial dysmorphism and pseudoarthrosis, a feature not previously reported in this condition. Whole-exome sequencing identified a novel homozygous synonymous variant in SEC24D (c.2361C>T; p.Asn787=), located 16 bases upstream of the donor splice site of intron 18. Functional analyses revealed markedly reduced SEC24D expression and aberrant exon 18 skipping, supported by RNA-seq, qPCR, and Western blot. This case provided the first functional evidence for a synonymous variant in SEC24D causing disease via splicing disruption and expands both the phenotypic and genotypic spectrum of Cole-Carpenter Syndrome 2.

Sudden unexpected death in epilepsy (SUDEP) is one of the most frequent causes of death in patients with epilepsy, though the pathogenesis of SUDEP has not been well elucidated. Here, we report novel heterozygous KCND3 variants, p.V401L and p.V401M, identified in young patients with refractory epilepsy (RE) and neurodevelopmental disorders, and the functional properties of these variants. We aimed to investigate the electrophysiological changes in de novo KCND3 variants and analyse the pharmacological effects of quinidine on these variants. Chinese hamster ovary (CHO) cells were transiently co-transfected with wild-type (WT) and/or variant KCND3 and Kcnip2. Transient outward potassium currents (I) were recorded using the whole-cell patch-clamp method. The inhibitory effect of quinidine on I was evaluated. In electrophysiological analysis, CHO cells expressing the variant channels showed a significant increase in current density compared with those expressing WT channels. The I activation curves were shifted significantly to the left, and significantly slower inactivation time constants were observed in both variant channels. Recovery from inactivation of the variant channels was significantly slower than that of WT. Quinidine suppressed I in a concentration-dependent manner and accelerated the slow inactivation of variant channels. In conclusion, de novo KCND3 variants identified in patients with RE and neurodevelopmental disorders showed gain and loss of function effects on I. These patients may be at risk of developing early repolarization syndrome, leading to SUDEP. Increased I was suppressed by quinidine, suggesting that it may be an effective therapy for RE and possibly for preventing SUDEP.

Inborn errors of metabolism (IEMs) lead to early-onset neurodegenerative disorders often caused by mitochondrial dysfunction. In this study, we identified a homozygous frameshift mutation (c.283dupG; p.Cys65LeufsTer13) in SLC27A3, identified through exome sequencing in a 2-month-old female proband presenting with developmental regression, hypotonia, seizure, feeding difficulty, and bilateral putaminal lesions on brain magnetic resonance imaging (MRI). The mutation results in a truncated, non-functional protein and complete loss of SLC27A3 expression in proband-derived fibroblasts. Results show the absence of SLC27A3 and aberrant mitochondrial morphology with clumped networks. Metabolic profiling showed elevated acyl-carnitine levels in the cytosol of proband cells, indicative of disrupted fatty acid oxidation. Additionally, mitochondrial respiratory chain activity was significantly reduced, and flow cytometry revealed increased cell death in mutant cells compared to controls. Protein-protein interaction analysis revealed SLC27A3 networks linked to fatty acid metabolism, ER-associated degradation (ERAD), and ion transport. GO enrichment demonstrated strong associations with transporter activity, protein homeostasis, and ER-mitochondrial membrane networks. Regional expression profiling showed high SLC27A3 transcript levels in the basal ganglia, correlating with the observed neuropathology. These findings position SLC27A3 as a critical lipid transporter involved in neuronal energy metabolism and proteostasis, and implicate its loss in mitochondrial encephalopathy. This study expands the genotypic and phenotypic spectrum of metabolic neurodevelopmental disorders and highlights the importance of fatty acid transport proteins in mitochondrial health and brain development. Our findings propose SLC27A3 as a novel candidate gene for early-onset mitochondrial disorders.

MECP2 duplication syndrome results from duplication of the MECP2 gene, encoding methyl-CpG-binding protein 2. Structural variations in this region can be detected by short-read next-generation sequencing, but resolving its precise genomic architecture remains challenging because of the involvement of complex and highly repetitive sequences. This study investigated the hidden structural variations using optical genome mapping and targeted long-read nanopore sequencing. We identified 14 breakpoints within the Xq28 regions encompassing MECP2 in four individuals from four families with MECP2 duplication syndrome. Combining the above methods enabled us to identify all the precise breakpoints, except for two inversions embedded within highly repetitive sequences. This also represents the most precise delineation to date of complex structural variants in MECP2 duplication syndrome. Notably, leveraging long nanopore reads (> 75 kb) allowed us to span low-copy repeat regions, including the approximately 72 kb J-group low-copy repeat which was difficult to be resolved, as well as GC-rich segments and dense clusters of short interspersed nuclear elements such as Alu, thus enhancing breakpoint-detection accuracy. We also detected previously underreported rare and complex rearrangement patterns. These findings highlight the power of integrating long-read sequencing with optical genome mapping for the delineation of complex genomic architectures, thus enhancing our understanding of the genomic structure underlying MECP2 duplication syndrome.

Congenital myopathies are a group of genetically heterogeneous neuromuscular disorders characterized by early-onset hypotonia and muscle weakness. While many congenital myopathies have historically been attributed to structural defects in muscle fibers, accumulating evidence reveals that dysfunction of satellite cells-the resident stem cells essential for muscle growth and regeneration-can also cause congenital myopathy. In this review, we focus on four genes critical for satellite cell biology: PAX7, MYOD1, MEGF10, and MYMK, and discuss how pathogenic variants in these genes contribute to muscle pathology. Mutations in PAX7, a transcription factor essential for satellite cell specification and maintenance, have been identified in patients with progressive congenital myopathy and scoliosis. MYOD1 variants affect the transcriptional regulation of myogenic differentiation and have been reported in individuals with congenital muscle hypoplasia. Loss-of-function variants in MEGF10, which mediates satellite cell proliferation, result in early-onset myopathy characterized by severe weakness and areflexia. Mutations in MYMK, essential for myoblast fusion, lead to congenital myopathy with facial and axial weakness. Together, these studies illustrate that distinct steps in satellite cell function-including specification, commitment, proliferation, and fusion-are critical for normal muscle development and maintenance. Recognizing that genetic defects affecting any of these processes can lead to congenital myopathies, redefining the disease spectrum beyond purely structural muscle disorders. Expanding our understanding of satellite cell biology will be key to elucidating the full spectrum of congenital myopathies and identifying targeted therapeutic strategies.

Whole-genome sequence information currently available for large-scale sequencing studies is biased toward European descent populations. Such bias causes difficulties in identifying disease-associated genetic variations in non-European populations, including the Japanese. Here, to comprehensively identify genetic variants, we sequenced 3135 individuals representing the genetic diversity of the Japanese population. Of the 44,757,785 identified variants, 31.0% exhibiting a minor allele frequency of <1% were novel. Using these variants, we constructed a reference haplotype and graph-structured reference sequence to facilitate accurate imputation and variant characterization. Our findings suggest that integrating genetic variations from ethnically diverse populations into the prevailing catalogs is essential to achieve precision medicine for all populations.

Tenascin-R (TNR) is an extracellular matrix glycoprotein that is essential for the formation of perineuronal nets in the central nervous system and is critical for neurite outgrowth, synaptic plasticity, and neural stem cell proliferation and differentiation. Biallelic TNR variants were reported to cause neurodevelopmental disorders with developmental delay, hypotonia, spasticity, and a variety of motor abnormalities. Here, we describe two Japanese siblings sharing novel compound heterozygous TNR missense variants (NM_003285.3:c.[1783 G > A];[3766 C > T] p.[(Asp595Asn)];[(Arg1256Cys)]) identified by exome and Sanger sequencing. The elder brother had dystonia, while the younger sister was asymptomatic except for adult-onset restless legs syndrome. Their development and intellect were normal. A total of 15 patients, including 13 previously reported patients, showed diverse phenotypic variability and severity, even among individuals sharing the same variants, indicating variable expressivity and reduced penetrance possibly influenced by genetic or environmental modifiers. Our findings extend the clinical spectrum of TNR-related disease and highlight the need for further accumulation of clinical cases and functional studies to understand genotype-phenotype correlations and the pathogenesis of diseases.

Thyroid hormones are central to regulating metabolism, growth, and development, yet their complex interactions with socioeconomic, metabolic, and genetic factors remain understudied in diverse populations. We compared thyroid profiles - free triiodothyronine (FT3), free thyroxine (FT4), and thyroid-stimulating hormone (TSH) in Indian adolescents with anthropometric traits, metabolic markers, and socioeconomic status (SES). We observed that adolescents from higher SES backgrounds exhibited greater metabolic dysregulation, altered thyroid profiles, and abnormalities in lipid and adipokine levels. Subclinical (16.1%) and clinical hypothyroidism (1.1%) were found to be prevalent in this population but were not associated with obesity. Instead, they showed links with dyslipidemia and altered adipokine profiles. To investigate the genetic basis of thyroid traits, we conducted an exome-wide association study (ExWAS, N = 4324), and a two-staged genome-wide association study (GWAS, N = 4854). The ExWAS revealed two novel loci for TSH (GYS2 and CEP162) and fifteen novel loci for FT4, including ZNF467, P3H3, CRLF3, SPATA2L, MEFV, THNSL2, COL27A1, COL28A1, IGSF3, ZNF732, MOG, GABBR1, HPF1, LOC440563, and SPEG. The GWAS identified novel associations at near-genome-wide significance for TSH (ACTL7B) and FT4 (LINC00648, YTHDC1, and C2CD4B). We also replicated established associations in FOXE1 and IGFBP5. Our findings suggest that SES, metabolic health, and genetics jointly influence thyroid function in Indian adolescents. The identification of population-specific loci emphasizes the importance of ancestry-informed genetic studies and supports the development of precision interventions to enhance pediatric thyroid health.

The CACNA1H gene, which encodes the T-type calcium channel Cav3.2, is known to confer susceptibility to childhood absence epilepsy (CAE) and has been implicated in various neurological disorders. However, its pathogenic significance, especially in childhood intractable epilepsies, has not been comprehensively explored. We performed whole-exome sequencing on a 4-year-old boy diagnosed with epilepsy with myoclonic-atonic seizures (EMAtS), and identified two missense variants in CACNA1H. One was a novel variant, p.D949H, inherited from the father, while the other was a known variant, p.R788C, inherited from the mother. Because the latter was previously reported to alter Cav3.2 channel function and contribute to the pathogenesis of CAE and idiopathic generalized epilepsy, we evaluated the former's functional impact using two-electrode voltage clamp analysis in Xenopus laevis oocytes. While the current-voltage relationship of the D949H mutant channel was not significantly different from that of the wild-type channel, the time constant of recovery from inactivation was significantly prolonged in the D949H variant (671.7 ± 52.0 ms vs. 455.5 ± 28.2 ms), indicating moderately impaired properties of the mutant. Notably, neither the D949H nor the R788C variant was associated with epilepsy in either parent, suggesting that these variants were not sufficient to cause epilepsy on their own, and that the compound heterozygous state of CACNA1H contributed to the EMAtS phenotype in the proband. Our findings highlight the genetic complexity of EMAtS and underscore the importance of accumulated functional impacts of modifier variants in severe epileptic diseases, even when individual variants are not pathogenic.

Classical twin studies can be used to disentangle the extent to which phenotypic variance of a given complex trait is determined by genetic and environmental variance. The designs widely employ 'ACDE' structural equation models where partitioned variances, including that of additive (A) and dominance (D) genetic components, are estimated and where A is taken as reflective of the narrow-sense heritability. Here, it is illustrated in a clear and accessible manner that it is in reality impossible to reliably partition the genetic variance into A and D components using the models which are consequently very open to overestimating the additive genetic variance. This essay should serve as a reminder that classical twin studies can approximate the total (broad-sense) heritability of complex traits, but that leveraging the findings with molecular measurement-based methods is necessary to reliably partition genetic variance components; brief examples based on recent relevant findings is also presented.

Congenital central hypoventilation syndrome (CCHS) is primarily caused by dominant PHOX2B mutations, with recessive LBX1 or MYO1H mutations being rare. Among PHOX2B mutations, polyalanine repeat expansion mutations (PARMs) are common, whereas non-PARMs (NPARMs) are less frequent. PHOX2B mutations are believed to act through loss-of-function mechanisms combined with dominant-negative and/or toxic gain-of-function effects. However, the role of PHOX2B haploinsufficiency remains unclear. We investigated the role of PHOX2B deletion and other genetic modifiers in CCHS. Among 93 patients without PHOX2B mutations, four were found to carry PHOX2B deletions via multiplex ligation-dependent probe amplification. Two had typical CCHS, whereas two siblings presented with mild sleep hypoventilation following CCHS symptoms in infancy. After ruling out pathogenic variants in LBX1 and MYO1H, we explored potential modifiers by analyzing sequence and methylation changes in the wild-type PHOX2B promoter and 3' untranslated region (3'UTR), and the coding regions of PHOX2A and MIR204. One female patient with CCHS carried a 3'UTR haplotype predicted to reduce PHOX2B expression via MIR204 interaction. To date, 15 informative cases with PHOX2B deletions (eight males, seven females) have been reported. Respiratory phenotypes included: CCHS (n = 5), CCHS with obstructive sleep apnea (OSA) (n = 1), OSA alone (n = 2), mild central sleep apnea (n = 1), mild central sleep hypoventilation or apnea following CCHS symptoms in infancy (n = 3), and asymptomatic (n = 3). These indicate that although a heterozygous PHOX2B deficiency alone is insufficient to cause CCHS, it may delay or impair the development of respiratory control.

AKT1 (Protein Kinase B alpha) is a serine/threonine kinase that plays a pivotal role in regulating various cellular processes. To elucidate the role of the AKT1 gene in signaling pathways, this study generated AKT1 knockout (KO) HT-1080 cells using the CRISPR/Cas9 system. Gene-editing efficiency was validated through Sanger DNA sequencing and insertion/deletion (InDel) analysis. Quantitative real-time PCR and Western blot analyses were performed to evaluate the expression levels of AKT1 mRNA and protein, as well as to examine the expression of AKT1 downstream effectors: mTOR, BCL-2, and FOXO1. The AKT1 single-guide RNA sequence was successfully cloned into the CRISPR/Cas9 vector, leading to the establishment of AKT1 KO cells. InDel analysis identified eight editing types, with two dominant populations. The expression levels of AKT1 mRNA and protein were significantly reduced in the KO cells. The expression levels of mTOR, BCL-2, and FOXO1 were significantly altered in the KO cells compared to normal cells. These findings highlight the impact of AKT1 disruption on signaling pathways and provide fundamental insights into the regulatory role of the AKT1 gene.

Single nucleotide polymorphisms in microRNA genes (miRNA-SNPs) can alter miRNA maturation or target mRNA recognition, resulting in gain- or loss-of-function, and are associated with various diseases. This study aimed to identify miRNA-SNPs or somatic mutations in miRNA gens that could serve as biomarkers for the onset or progression of adult T-cell lymphoma-leukemia (ATLL), using next-generation sequencing (NGS) targeting 1809 pre-miRNA genes. Genomic DNA extracted from peripheral blood samples from 31 ATLL patients with low human T-cell leukemia virus type-1 (HTLV-1) proviral loads and 28 healthy subjects was analyzed. Fourteen miRNA-SNPs with significantly different allele frequencies were between the two groups were identified. To determine whether the observed variants were germline or somatic, miRNA-SNPs detected in blood-derived DNA were compared with those from saliva-derived DNA in 6 out of 31 patients. Concordant results between the two sources suggested the variants were germline SNPs. Furthermore, comparison of blood-derived DNA samples from 10 ATLL patients collected during low and high HTLV-1 proviral load periods revealed 10 somatic mutations in pre-miRNA genes, including pre-mir-142, present only in high proviral load samples. These somatic mutations may serve as markers of ATLL progression. In conclusion, out comprehensive NGS analysis identified both germline miRNA-SNPs and somatic mutations that may act as biomarkers for the onset or progression of ATLL. Future studies with larger cohorts will be essential to validate their clinical utility.

Early-onset hippocampal sclerosis is a major cause of focal epilepsy, yet many genetic contributors remain unknown. We investigate a 12-year-old girl with recurrent focal seizures, progressive memory impairment, and MRI evidence of left hippocampal atrophy with ipsilateral parietal gliosis. Singleton exome sequencing revealed a novel homozygous NOL10 variant (NM_024894.4: c.682 A > C; p.Asn228His), absent from population and clinical databases; both parents were heterozygous carriers. The variant alters a highly conserved residue within the WD-repeat domain. Proband fibroblasts maintained normal NOL10 transcript levels but exhibited nucleoplasmic mislocalization and loss of interaction with AATF and NGDN, key partners in small ribosomal subunit biogenesis. Structural modeling and ΔΔG calculations predicted that N228H is strongly destabilizing. Functionally, proband cells showed specific impairment of 40S maturation with reduced 40S, 80S and polysome content, accompanied by G/G arrest and increased cell death. Network and expression analyses place NOL10 at the center of nucleolar rRNA processing and ribosome assembly, with substantial expression across hippocampal subfields, supporting selective vulnerability of hippocampal neurons. Collectively, our data implicate biallelic NOL10:c.682 A > C as a novel, likely pathogenic cause of neurodevelopmental disorder characterized by hippocampal sclerosis and gliosis, and highlight disrupted ribosome biogenesis as a plausible disease mechanism.

The m.3243 A > G mitochondrial DNA variant is a major pathogenic variant associated with various clinical phenotypes, including hearing impairment and diabetes. This study retrospectively analyzed clinical data from 37 patients with the m.3243 A > G variant to clarify the relationship between clinical characteristics and hearing loss. Most patients developed post-lingual, late-onset sensorineural hearing loss (SNHL), with flat-type audiometric configurations being the most common. Blood heteroplasmy levels were negatively correlated with age at genetic testing (R² = 0.6303), and age-adjusted heteroplasmy levels were inversely associated with age at onset of hearing loss (p = 0.029). A significant difference in clinical characteristics was observed between patients with hearing loss and/or diabetes alone and those with multiple organ involvement, with the latter group showing a lower BMI (p = 0.031) and more severe hearing loss (p < 0.001). Two cases initially presenting with maternally inherited deafness and diabetes progressed to MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes), demonstrating the importance of early systemic evaluation. Cochlear implantation was considered for advanced hearing loss, although systemic complications were a challenge. Our findings suggest that hearing loss may be an early risk factor of systemic mitochondrial disease, particularly in lean individuals. Comprehensive assessment, including BMI and genetic testing, may aid in the early diagnosis and management of patients with m.3243 A > G variant.

Congenital heart disease and ectodermal dysplasia syndrome (CHDED syndrome) (MIM: 617364) is an autosomal dominant disorder cause by PRKD1 gene pathogenic variants, characterised mainly by congenital heart defects (CHD) and ectodermal dysplasia, along with other variable clinical features (including skeletal defects). Whole exome sequencing was performed on a 7-year-old male proband with CHD, born of non-consanguineous Asian Indian origin couple with affected father and unaffected mother. We identified a novel heterozygous splice variant in PRKD1 (c.1906-1 G > T) in the proband, inherited from the affected father. Transcript analysis confirmed that the PRKD1 splice variant caused the complete skipping of exon 14. Interestingly, the proband exhibited a novel extended phenotype, which include CHD - TOF, and sagittal craniosynostosis, thus broadening the phenotypic spectrum of CHDED syndrome. Affected father showed CHD (septal defects) and no craniosynostosis. Further functional studies are required to elucidate the association of PRKD1 sequence variants with craniosynostosis observed in the proband.

Tubulin proteins form microtubules, which are critical for neuronal cell migration. In humans, there are at least 27 tubulin genes. Pathogenic variants in these genes cause tubulinopathies, which are characterized by diverse neurodevelopmental symptoms and brain malformations. This study analyzed the genetic variants and clinical characteristics of 12 patients (3 males, 9 females) with confirmed β-tubulinopathies. A retrospective chart review indicated that diagnoses were made via exome, genome, or targeted next-generation panel sequencing. Most patients (11/12) showed significant developmental delay and hypotonia. Other neurological symptoms included ocular motility disorders (7/12), ataxia (6/12), seizures (3/12), and microcephaly (2/12). The median age at symptom onset was 10 months (range 0 - 24). Corpus callosum abnormalities were the most common brain malformation, present in 10 patients, including one case of complete agenesis. Basal ganglia abnormalities and cerebellar hypoplasia were each observed in 9 patients. Cortical abnormalities, white matter changes, and brainstem hypoplasia were each present in 8 patients. Severe lissencephaly was not observed. Ten pathogenic or likely pathogenic missense variants were identified in five β-tubulin genes (TUBB2A, TUBB2B, TUBB3, TUBB4A, and TUBB5). Most variants were de novo, but one was a maternally inherited variant, c.211 G > A in TUBB3, which was associated with milder features. A novel variant in TUBB2A, c.1234 G > A p.(Glu412Lys), was also identified. These genetic variations were associated with a broad phenotypic spectrum of β-tubulinopathies, including complex brain malformations and neurodevelopmental disorders. Despite its rarity, tubulinopathy may be considered in the differential diagnosis for patients presenting with developmental delay and brain malformations.

Next-generation sequencing (NGS) offers a fast, cost-effective, and scalable solution for pharmacogenomic allele assignment but faces challenges in accurately identifying variants and haplotypes in regions with high sequence similarity. This study aimed to evaluate the performance of three NGS-compatible genotyping tools - PyPGx, Stargazer, and Aldy - for CYP2D6 annotation and investigated the CYP2D6 genetic distribution in 1008 whole-genome sequences from the 1000 Vietnamese Genome Project (VN1K) data. A benchmark dataset was constructed using 8556 diverse CYP2D6 alleles and 122 samples with complex structural variations. Tools were then assessed for haplotype, diplotype, and phenotype concordance across sequencing coverages of 8x, 30x, and 60x. Then, all three tools were subsequently applied to genotype CYP2D6 in the VN1K dataset. Overall, Aldy outperformed others, achieving haplotype and phenotype accuracies of 89.56% and 96.59%, respectively, even at low coverage (8x). In comparison, Stargazer and PyPGx achieved diplotype concordance rates of 51.33% and 47.77% under the same coverage level. According to the output of Aldy - the best performance tool in analysing the CYP2D6 genetic distribution in VN1K data, the results revealed a predominance of reduced-function alleles and a high prevalence of intermediate metabolizer phenotypes, underscoring the need for population-specific pharmacogenomic strategies and highlighting Aldy's potential in advancing precision medicine.

Gardos channelopathies are rare hereditary hemolytic anaemias caused by mutations in the KCNN4 gene, which encodes the calcium-activated potassium channel (KCa3.1) in red blood cells. In this study, we report three unrelated Indian patients with unexplained chronic hemolytic anaemia. Whole exome sequencing revealed distinct KCNN4 mutations: a homozygous c.5G > A mutation (p.Gly2Asp) in Case I, a compound heterozygous condition with the Hb Nottingham mutation (HBB: c.296T > G) and a splice-site mutation in KCNN4 (c.931-1G > C) in Case II, and homozygous c.541A > T mutation (p.Ser181Cys) in Case III. All three patients presented with chronic anaemia, indirect hyperbilirubinemia, reticulocytosis, and recurrent blood transfusions. Red cell enzyme studies (G6PD, PK, GPI) showed normal activities, and flow cytometry-based EMA binding was normal. Haemoglobin electrophoresis by HPLC was normal, except in Case II, and tested positive for unstable haemoglobin using a heat instability test. Flow cytometry revealed significantly elevated intracellular calcium levels and reactive oxygen species (ROS) in all cases, indicating oxidative stress under osmotic stress. In Case III, a Percoll density gradient assay demonstrated dehydrated erythrocytes, supporting the diagnosis. This study expands the mutation spectrum of Genetic diagnosis using NGS, which is essential for appropriate clinical management and genetic counselling in unexplained cases of hemolytic anaemia. Elevated intracellular calcium levels play a key role in hemolysis, suggesting that calcium-modulating therapies could aldehyleviate symptoms.