Sanger Sequencing Research Articles (Page 1)

Association of interleukins 17A and 22 levels and their common genetic polymorphisms in Guillain Barré syndrome.

Inherited cerebellar ataxias (ICA) are a group of rare, genetically and clinically heterogeneous neurodegenerative disorders, often caused by a broad spectrum of genetic variants, including single nucleotide variants, structural variations, and especially short tandem repeat (STR) expansions. Over time, advances in genomic technologies have profoundly reshaped the diagnostic approach and scientific understanding of ICA. This review traces the evolution of genomic tools, from first-generation methods, such as Sanger sequencing and southern blot, through the revolution brought by short-read second-generation sequencing, including gene panels, exome sequencing (ES), and genome sequencing (GS). These technologies significantly increased diagnostic yield and enabled the discovery of numerous ICA genes, although short-read sequencing remained limited in its ability to detect STR expansions and structural variants accurately. Recently, third-generation long-read sequencing (LRS) technologies, such as Single Molecule Real Time (SMRT) and nanopore sequencing, have emerged, offering reliable detection of all classes of variants, including complex and previously undetectable STR expansions. Optical genome mapping is also highlighted as a powerful, non-sequencing alternative for detecting large SVs and STRs. The manuscript underscores the reciprocal relationship between technological advances and medical knowledge in ICA. With LRS and integrated multi-omics approaches, the field stands at the cusp of a new era in diagnostics, enabling comprehensive variant detection and deeper insights into ICA pathophysiology. However, challenges related to cost, data interpretation, and population biases in reference databases remain.

This study aimed to establish and validate a novel detection method that combines padlock probe technology with fluorescence quantitative PCR (qPCR) for the rapid and specific detection of the SARS-CoV-2 Omicron S371L mutation in the spike protein.Padlock probes and amplification primers were designed and synthesized based on the Omicron S371L mutation site. The probe was designed to anneal with high specificity to the mutation, allowing ligase-mediated circularization only in the presence of the target sequence. The circularized probe then served as a template for qPCR amplification. Assay optimization included probe concentration, ligation temperature, ligase concentration, and ligation time. Analytical sensitivity, specificity, and recovery performance were systematically evaluated. Finally, the method was validated using 30 clinical samples, with results compared to Sanger sequencing.The optimal assay conditions were identified as a padlock probe concentration of 10 nM, ligase at 0.2 U/µL, ligation carried out at 65°C for 30min, and 10 µL of the ligated product used for subsequent qPCR amplification. Under these parameters, the assay achieved a limit of detection of 5.78 fM and demonstrated strong linearity (R² = 0.9669). Specificity testing showed clear differentiation between the mutant template and single-, double-, or triple-base mismatches. In performance evaluations, recovery rates ranged from 89.7% to 108.0% in both PBS and urine samples. For clinical validation, the method showed complete agreement with Sanger sequencing results, yielding positive and negative predictive values of 100%.In summary, the padlock probe-based qPCR assay developed in this study offers a fast and reliable approach for detecting the Omicron S371L mutation in SARS-CoV-2. Because the method bypasses the reverse transcription step that is typically required for RNA targets, it simplifies the workflow without compromising accuracy. These features suggest that the assay could be well suited for broader clinical application, particularly in large-scale screening of emerging SARS-CoV-2 variants.

Von Hippel-Lindau syndrome (VHL) is an autosomal dominant disorder characterized by the development of tumors and cysts in multiple organs. Pathogenic variants in VHL are known to be associated with the development of this syndrome. Therefore, the present study aimed to investigate VHL rearrangements in a family with VHL syndrome. In the proband, who presented with retinal hemangioma, whole exome sequencing (WES) revealed significant genetic variation. However, WES did not detect germline deletion in VHL in the proband's son. Furthermore, multiplex ligation-dependent probe amplification (MLPA)-next-generation sequencing (NGS) analysis showed that the genomes of the proband and her son encompassed a deletion in VHL, extending from exon 2 to exon 3. Via increasing the probe density to gradually approach the breakpoint and following Sanger sequencing analysis, it was verified that an intermediate fragment of 6,662bp was lost, with a reconnection occurring between chromosome 3:10145434 and 10,152,097 (GRCh38/hg38). Overall, the present study demonstrated that the application of MLPA-NGS technology combined with Sanger sequencing could be employed to precisely locate deleted DNA ‌fragments.

Background This study aims to characterize the potential genetic etiologies in children with developmental delay through whole-exome sequencing (WES) providing assistance for clinical diagnosis, genetic counseling, and reproductive guidance. Methods WES was performed on the proband, followed by Sanger sequencing validation of the identified variant in the parents. Results The proband exhibits global developmental delay, including impaired motor and language development, reduced spontaneous speech, poor coordination, and attention deficits. WES revealed a heterozygous nonsense variant in SETBP1 (c.1630C>T, p.Arg544Ter), which was confirmed as de novo by Sanger sequencing. This variant was classified as pathogenic according to American College of Medical Genetics and Genomics (ACMG) guidelines. The patient was subsequently diagnosed with intellectual disability, autosomal dominant 29 (MRD29). Conclusion The de novo SETBP1 p.Arg544Ter variant was identified as the underlying genetic cause in this case. Our findings underscore the importance of early genetic testing in children with developmental delay to enable precise diagnosis, informed genetic counseling, and evidence-based reproductive planning.

Extreme elevation of serum immunoglobulin E (IgE) concentration is a key marker for detecting immune disorders, including humoral and cellular defects in primary immunodeficiency (PID). IgE antibodies are present in low concentrations in the body and are produced in large amounts when exposed to infections or toxins. However, IgE is also the cause of allergic symptoms and life-threatening anaphylaxis reactions. Early diagnosis of PID associated with elevated IgE may lead to effective or life-saving therapeutic interventions. Therefore, genomic testing-based diagnosis is becoming a widely used diagnostic tool to determine the cause of disease. Three Vietnamese patients with increased IgE expression were collected for genetic analysis at The Allergy, Immunology, and Rheumatology Department, Vietnam National Hospital Pediatrics. Primary immunodeficiency associated with elevated IgE. We performed whole-exome sequencing (WES) to detect novel associated variants and confirmed these by Sanger sequencing. The effects of the variants were predicted using in silico prediction tools. Three novel pathogenic variants including c.2204A > T, p.Asp735Val in the PTPRC gene, c.586T > A, p.Phe196Ile in the UNC119 gene, and c.481C > T, p.Arg161Cys in the IL21R gene were found to be associated with increased IgE. We report novel variants associated with genetic defects that increase IgE found in PID patients. These results emphasize the need for accurate diagnosis and appropriate intervention to improve outcomes and quality of care for individuals with high IgE levels and related immune disorders.

Heart failure (HF) is slightly more common in primary aldosteronism (PA) than in essential hypertension, but early-onset HF remains rare. In such cases, underlying genetic cardiomyopathies should be considered. Autonomously secreted aldosterone and activation of the renin-angiotensin-aldosterone system can lead to extremely high aldosterone levels, worsening cardiac function and creating major therapeutic challenges. A 39-year-old male presented with progressive chest tightness and shortness of breath for 4 months. He had a 7-year history of hypertension and persistent hypokalemia. Electrocardiogram revealed a markedly reduced left ventricular ejection fraction of 18.3%. The patient was diagnosed with PA based on elevated plasma aldosterone concentration, an increased aldosterone-to-renin ratio, and a positive captopril challenge test. Computed tomography and adrenal vein sampling indicated unilateral PA. After initial HF management, the patient underwent laparoscopic adrenalectomy for PA treatment. According to the primary aldosteronism surgical outcome consensus criteria for postoperative evaluation of PA, complete biochemical remission (normalization of aldosterone-to-renin ratio and potassium) and partial clinical remission (stable blood pressure with reduced antihypertensive medication) were achieved 1 month postoperatively and have been maintained since. At the 8-month follow-up, his left ventricular ejection fraction had improved to 45.4% and BNP levels normalized. Whole-exon sequencing revealed a missense mutation of the dystrophin (DMD) gene. Certain DMD mutations are linked to X-linked dilated cardiomyopathy with absent or subclinical skeletal muscle involvement. Sanger sequencing confirmed the hemizygous mutation in the proband. The final diagnosis was poorly controlled PA with early-onset HF, potentially influenced by a coexisting DMD gene missense mutation that may modify both the onset and severity of PA-related HF. Early recognition and surgical treatment of PA with early-onset HF can substantially improve cardiac function, even in the presence of genetic susceptibility to cardiomyopathy. This case underscores the need to consider underlying cardiac genetic disorders in PA patients with atypical or early-onset HF and raises the hypothesis that the identified DMD variant may serve as a potential genetic modifier of HF severity in the context of PA.

NOTCH3, a key regulator of vascular smooth muscle cell function within the Notch signaling pathway, is essential for maintaining small artery integrity, especially in cerebral microvasculature. Pathogenic heterozygous missense variants in NOTCH3 cause CADASIL, an autosomal dominant small-vessel disease marked by extracellular domain aggregation, granular osmiophilic material deposition, and vascular smooth muscle cell dysfunction, leading to early-onset ischemic strokes, migraine with aura, and progressive cognitive decline. We report a 12-year-old Moroccan girl, born to first-degree consanguineous parents, presenting with global developmental delay, spastic tetraparesis, epilepsy, and bilateral periventricular white matter abnormalities. Whole-exome sequencing identified a novel homozygous frameshift variant in NOTCH3 (NM_000435.3:c.2985_2991del; NP_000426.2:p.(Gln996ArgfsTer274)), confirmed by Sanger sequencing, with heterozygous carrier parents. Emerging evidence shows that biallelic loss-of-function variants, unlike the gain-of-function variants underlying CADASIL, define a distinct autosomal recessive leukodystrophy characterized by early-onset pyramidal signs, epilepsy, and white matter abnormalities, thereby expanding the phenotypic and molecular spectrum of NOTCH3-related disorders. This work highlights the functional divergence between dominant and recessive NOTCH3 variants and supports the inclusion of NOTCH3 in gene panels for early-onset leukodystrophies, especially in consanguineous populations.

Hermansky-Pudlak Syndrome (HPS) is a rare autosomal recessive syndromic type of albinism. It is characterized by oculocutaneous hypopigmentation, platelet dysfunction, and variable systemic involvement depending on the specific subtype. To date, eleven distinct HPS types have been identified, with HPS3 being among the milder forms. Clinical and ophthalmic examinations, followed by whole exome sequencing (WES), Sanger sequencing, and segregation analysis, were performed. Here, we report a case of a 26-year-old Lebanese male patient born to consanguineous parents who presented with oculocutaneous albinism without a history of bleeding or other systemic involvement. WES identified a novel homozygous nonsense variant in the HPS3 gene (NM_032383.5): c.998T > A; p.(Leu333Ter) that co-segregated with the phenotype. The platelet function analysis (PFA-100) revealed a prolonged collagen/epinephrine closure time, accompanied by a normal collagen/ADP response. We report one of the very few HPS3 cases in the Middle East and North Africa region (MENA) and the Arab regions, caused by a novel homozygous nonsense variant associated with platelet dysfunction. In contrast to the founder mutations described in Puerto Rican and Ashkenazi Jewish populations, HPS3 cases in this region appear to result from distinct mutational events, indicating the absence of a common ancestral origin.

We revise the molecular variation and taxonomy of an assemblage of lineages of endemic Malagasy cophyline microhylid frogs, previously considered to be allied to Platypelis tetra. Based on DNA sequences obtained from the P. tetra holotype via a museomics approach and further complementary DNA sequences of other specimens obtained by Sanger sequencing, we genetically characterize the species and reveal at least two additional genetic lineages associated to it. These differ by uncorrected pairwise distances of 4.4–5.0% in a fragment of the mitochondrial 16S rRNA gene and require taxonomic revision in future work. A further lineage previously assigned to P. tetra differs by 16S distances of 7.1–7.8% and occupies an isolated phylogenetic position. We here formally name and describe this new species from the Sorata Massif as Platypelis efatra sp. nov. and thereby increase the number of nominal Platypelis species to 18. We provide a description of its skeleton based on a micro-computed tomography scan of a paratype, which is the first detailed osteological description of a Platypelis species, and identify some possibly diagnostic osteological differences compared to P. tetra. We furthermore provide an updated advertisement call description for P. tetra from its type locality and briefly discuss the status of a divergent population from Ambodivoangy that we tentatively assign to P. tetra pending future study.

Though anti-histone H3.3G34W antibody is highly sensitive to giant cell tumours of bone, a subset of tumours which are negative on immunohistochemistry harbours variant H3F3A mutations. In this study, the diagnostic utility of anti-histone H3.3G34V and R immunohistochemistry was assessed along with H3F3A gene mutation analysis by Sanger sequencing. This study also attempted to establish an algorithmic approach to analysis of H3.3G34W immunohistochemically negative tumours. Immunohistochemistry was performed using anti-histone H3.3G34R and anti-histone H3.3G34V antibodies in 35 anti-histone H3.3G34W IHC negative tumours. Simultaneously, all the tumours were subjected to Sanger sequencing to detect mutations in H3F3A gene. Of the 35 tumours tested, 14 showed positive staining for H3.3G34V and one for H3.3G34R immunohistochemistry. Gene mutation analysis by sequencing detected 3 additional cases of H3.3G34W mutation not detected by immunohistochemistry. Of the 14 tumours with H3.3G34V immunohistochemical expression, 11 showed corresponding H3.3G35V mutation on sequencing and a single tumour with H3.3G34R expression showed corresponding H3.3G35R mutation. In addition, sequencing detected 5 cases of H3.3G35L mutation which were negative on immunohistochemistry. Combining immunohistochemistry and sequencing results for the overall 309 GCTBs diagnosed during the study period, the most frequent H3F3A gene mutation was H3.3G34W/H3.3G35W (277/309, 89.6%). Other variants included H3.3G34V/H3.3G35V (14/309,4.5%), H3.3G34L/H3.3G35L (5/309,1.6%), and H3.3G34R/H3.3G35R (1/309,0.3%), while 3.9% (12/309) had the wild-type gene. Immunohistochemistry with H3.3 G34W, H3.3 G34R and G34V can be used as a surrogate for detection of corresponding H3F3A gene mutations. These antibodies should be used as first-line tests for confirming the diagnosis with sequencing being restricted only to tumors negative on immunohistochemistry.

Background Oliver-McFarlane syndrome (OMCS) is an extremely rare congenital disorder that presents with hypogonadotropic hypogonadism, long eyelashes and eyebrows, pigmentary retinopathy, peripheral nerve axon neuropathy and other associated features. It is currently known that OMCS is linked to variants in the patatin-like phospholipase domain containing 6 ( PNPLA6 ) gene, but the specific pathogenic mechanism is still unclear. Methods We performed Whole exome sequencing (WES) on the proband and his parents, followed by validation of the findings through Sanger sequencing and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis. Results Sanger sequencing identified two compound heterozygous variants in the PNPLA6 (NM_006702.5) gene in the proband: c.3184G>A (p.Val1062Met) and c.2704-18C>G. According to the ACMG guidelines, the c.3184G>A variant is classified as likely pathogenic, while the c.2704-18C>G variant is discovered for the first time. Segregation analysis further revealed that the c.3184G>A variant was inherited from the father, whereas the c.2704-18C>G variant was derived from the mother—consistent with an autosomal recessive inheritance pattern. RT-PCR detected that the c.2704-18C>G variant caused a 29bp deletion upstream of exon 26, resulting in a splice site mutation (p.His902Alafs108). Conclusion We report a case from China of PNPLA6 gene variants leading to Oliver-McFarlane syndrome, with the patient exhibiting typical characteristics of OMCS. Our findings further substantiate the pathogenicity of PNPLA6 gene variation in OMCS, broadening the established genotypic spectrum of the PNPLA6 gene. These findings enhance the understanding of its pathogenesis and offer perspectives for clinical diagnosis and management.

Background D-bifunctional protein deficiency (D-BPD) is a rare fatal autosomal recessive peroxisomal disorder caused by biallelic pathogenic mutations in the hydroxysteroid 17-beta dehydrogenase 4 ( HSD17B4 ) gene; it is characterized by hypotonia, seizures, and facial dysmorphisms during the neonatal period. Case presentation In this report, we describe a female neonate from China who was diagnosed with D-BPD. The patient presented with neonatal asphyxia, hypotonia, weak reflexes, and feeding difficulty after birth. Seizures occurred on the fifth day of life and were initially treated with phenobarbital. However, the seizures reoccurred and became more difficult to control because of their increased frequency, duration, and anticonvulsive drug resistance. Whole-genome sequencing (WGS) revealed novel compound heterozygous mutations c.1145G>A(p.Gly382Asp)/c.1193C>G(p.Ser398*) in exon 13 of the HSD17B4 gene, which was confirmed by parental Sanger sequencing. Neither variant has been reported previously. Very-long-chain fatty acid (VLCFA) testing revealed markedly elevated levels of hexacosanoic acid (C26:0), tetracosanoic acid/docosanoic acid (C24:0/C22:0), and C26:0/C22:0. The patient was managed with formula nasogastric feeding and antiepileptic therapy. At 7 months of age, she demonstrated severe psychomotor retardation, inability to grasp and manipulate objects, no language development, hearing loss, and poor visual response. Conclusion We described the incidence of D-BPD in a Chinese neonate caused by novel biallelic pathogenic variants in HSD17B4 , which expands its mutational spectrum.

Developmental and epileptic encephalopathy 9 (DEE9) is an X-linked genetic disorder characterized by the onset of seizures during infancy. Mutations in protocadherin 19 (PCDH19) are the main cause of DEE9. Our study aims to demonstrate the diagnostic process and long-term follow-up of a female pediatric case presenting with recurrent seizures. In the present study, a female child presented with recurrent epileptic seizures and findings of abnormal synchronous discharges on electroencephalograms. Whole exome sequencing (WES) was performed on the proband and her parents to identify potential genetic variants. A heterozygous variant (NM_001105243: c.695A>G) in PCDH19 was identified and validated using Sanger sequencing. Based on clinical features and genetic analyses, the patient was diagnosed with PCDH19-female limited epilepsy. Furthermore, a 4-year follow-up was conducted to assess the impact of the pathogenic variant on phenotype and treatment outcomes. The patient exhibited normal intelligence, which differed with the clinical features reported in other studies involving the same variant. WES confirmed the diagnosis of DEE9, and subsequent follow-up highlighted the effectiveness of the treatment. Therefore, genetic testing can improve the diagnosis of DEE9, particularly in cases with atypical symptoms, and provide valuable insights for genetic counseling and clinical treatment strategies.

Background Autosomal recessive polycystic kidney disease (ARPKD) is a rare, inherited disorder primarily affecting the kidneys and liver. Disease-causing variants in PKHD1 lead to a disruption of the encoded protein fibrocystin/polyductin. This study aims to identify disease causing variants in PKHD1 in families from the Dhofar region of Oman. Methods We conducted a case series of six families with antenatal diagnoses of ARPKD and postnatal deaths. Genetic testing was performed on neonates using Sanger sequencing and next-generation sequencing (NGS) to detect variants in PKHD1. In silico analysis of mutational consequences was performed. Results 5 distinct homozygous variants in the PKHD1 gene were identified, including three pathogenic frameshift variants (c.6111_6112delTT, c.7011dupT and c.9550dupT), a nonsense variant (c.340C>T) and a homozygous deletion spanning exons 58-60 of the PKHD1. These alleles have not been reported in previous studies. In silico modelling identified pathogenic alleles, predicted to lead to either truncated protein products or nonsense-mediated decay. Discussion Our findings identify disease causing PKHD1 variants in this genetically distinct Dhofar population, potentially due to factors such as geographical isolation, consanguinity, and founder effects. The identification of previously undescribed variants underscores the need for regional genetic studies in understanding ARPKD and its genotype-phenotype correlations. Conclusion This study reveals distinct PKHD1 disease-causing variants in the Dhofar region of Oman, contributing to the broader genetic understanding of ARPKD. These findings highlight the value of region-specific genetic research in identifying new disease causing variants.

BackgroundCircRNAs have a potential regulatory effect on Renal cell carcinoma (RCC). However, there is still a significant lack of research on their action pathways and mechanisms in the treatment of RCC. The aim of this study is to identify the effects of Circ_001024 on the proliferation, migration, and invasion of RCC cells, and to elucidate the molecular mechanisms by which Circ_001024 promotes the malignant biological behaviors of RCC cell lines.MethodsThe expression of Circ_001024 in RCC was detected using quantitative real-time polymerase chain reaction (qRT-PCR) To confirm Circ_001024’s structure and properties, tests using Sanger sequencing, RNase R, and actinomycin D were performed. The localization in RCC cells was identified using the Fluorescence in situ hybridization (FISH) assay. Bioinformatics analysis, Spearman correlation analysis, dual-luciferase reporter assay, and RNA pull-down assay were used to verify the interactions among Circ_001024, its targeted miR-145-3p, and the targeted protein GLUT5,and these interactions were further validated by rescue experiments. In addition, Western blot (WB) was used to detect the expression changes of the signaling pathway target protein GLUT5, and the clinicopathological characteristics of GLUT5 in RCC were analyzed.ResultsThe gene Circ_001024 showed high expression levels in RCC, predominantly found in the cytoplasm of RCC cells. Forced expression of Circ_001024 accelerated RCC progression by enhancing cell proliferation, migration, and invasion, whereas Circ_001024 knockdown elicited the reverse effects. miR-145-3p could interact with both Circ_001024 and the targeted protein GLUT5. Overexpression of miR-145-3p could partially reverse the changes in the proliferation, migration, and invasion abilities caused by overexpression of Circ_001024. Moreover, the expression of GLUT5 was associated with the World Health Organization/International Society of Urological Pathology (WHO/ISUP) grade, but not with age, gender, tumor diameter, or TNM staging.ConclusionCirc_001024 is highly expressed in RCC. Within RCC, Circ_001024 can promote the proliferation, migration, and invasion of RCC cells through the competitive inhibition of miR-145-3p, which controls GLUT5 expression.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s12885-025-14878-3.

Background/Objectives: TP53 mutations in advanced epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer could worsen prognosis. Therefore, we aimed to investigate the clinical significance of TP53 mutations and p53 expression in these patients. Methods: Patients with advanced/metastatic EGFR-mutated lung adenocarcinoma treated with first-line tyrosine kinase inhibitors were retrospectively enrolled. Sanger sequencing was performed to detect TP53 mutations and immunohistochemical staining was used to verify p53 protein expression levels. Kaplan-Meier and Cox proportional hazards analyses were used to estimate survival and hazard ratio (HR) with 95% confidence interval (CI). Results: The study involved 83 patients with adequate tumor samples for TP53/p53 analysis. Patients with tumor p53 immunostaining ≥50% showed significantly better overall survival (OS) (HR: 0.49 [95% CI: 0.30–0.81], p < 0.001), but TP53 mutations were not associated with inferior progression-free survival (PFS) or OS (missense vs. wild-type [PFS, HR: 0.68 (95% CI: 0.40–1.15), p = 0.151; OS, HR: 0.88 (95% CI: 0.56–1.42), p = 0.599]). Areas under the receiver operating characteristic curves of TP53 mutations with different cut-off values for p53 positivity were 0.51–0.56. The Kaplan-Meier survival analysis revealed significant survival benefits in patients with EGFR L858R substitution and tumor p53 immunostaining ≥50% (median PFS: 8.0 vs. 5.3; median OS: 20.4 vs. 15.3 months; log-rank p = 0.025 and 0.049, respectively). Conclusions: Tumor p53 immunostaining (≥50%) was associated with better OS, especially in patients with TP53 mutations or L858R. Prospective clinical trials are required to explore the prognostic significance of p53 expression in the genomic era of TP53 mutations.

The Signal Transducer and Activator of Transcription 5A (STAT5A) gene plays a crucial role in the development of the mammary gland and milk production by mediating prolactin (PRL) signaling. These functions make STAT5A a promising candidate gene for further research, especially in milk-producing animals. The aim of this study was to determine the relationship between polymorphisms in the 1720 bp segment spanning exons 6-9 of the STAT5A gene and the milk yield of 192 Anatolian buffalo raised in the Afyonkarahisar region. The total of 13 polymorphisms were identified through Sanger sequencing, including five in the 7th intron, seven in the 8th intron, and only one of them in the 8th exon. The relationship between adjusted milk yields and SNPs was analyzed using ANOVA (one-way analysis of variance). The association between milk and SNP 12237 (g+12237 C>T) was located in exon 8 of the STAT5A gene. Therefore, milk yield was found to be significant (P

Genetic analysis of a family with skeletal muscle ion channelopathy and hereditary spastic paraplegia type 7 caused by SCN4A and SPG7 double mutations.

Background Thalassemia is one of the most common monogenic disorders worldwide and classified as α-thalassemia and β-thalassemia. Conventional methods for diagnosis of thalassemia, constrained by their focus on commonly known genotypes, can easily lead to missing or misdiagnosis of rare thalassemia genotypes. Case report We report the case of a 32-year-old pregnant woman with abnormal hematological parameters. Conventional gap polymerase chain reaction (Gap-PCR) and PCR-reverse membrane hybridization (PCR-RDB) were performed to analysis the 23 common thalassemia variants, but did not identify any pathologic variants. Next, we used PacBio third-generation sequencing platform based on single molecule real-time technology (SMRT) for this woman and her newborn and identified a novel 9.159-kb large deletion (chr16:165599-174758, GRCh38) of α-globin gene loci, which disrupted HBA2 gene. And the breakpoints of the deletion were validated by gap-PCR and sanger sequencing. Conclusion Our study identified a novel large deletion, which expanded the α-thalassemia gene variant spectrum and confirmed the efficiency of SMRT technology in detecting rare thalassemia variants, provided genetic counseling and prenatal intervention in clinical rare patients.