Multi-gene Mutations Research Articles

Multi-gene mutations in ultrasound-guided fine-needle aspiration specimens of thyroid micronodules and diagnostic value in thyroid microcarcinomas using next-generation sequencing

Objective: To explore the detection of BRAF, RAS, TERT promoter, and TP53 gene mutations in solitary thyroid micronodule (TMN) specimens obtained by ultrasound-guided fine-needle aspiration (US-FNA) using next-generation sequencing (NGS) technology and assess its diagnostic value in thyroid microcarcinomas (TMC). Methods: On-site recruitment of 428 patients with single suspicious TMC who underwent thyroid ultrasound examination, US-FNA, and NGS from September 2018 to July 2021 at Beijing Tongren Hospital affiliated to Capital Medical University. A total of 147 patients were finally included. NGS was used to detect mutations in the BRAF, RAS, TERT promoter, and TP53 genes in the US-FNA specimens. Comparisons were made between patients with and without gene mutations in terms of age, gender, and the maximum diameter of nodules. The diagnostic efficiency of BRAF mutation for TMC was calculated using the receiver operating characteristic (ROC) curve, with postoperative pathology as the gold standard. Results: The age [M (Q1, Q3)] of the 147 patients was 43.0 (32.0, 51.0) years, and 37 were male (25.2%). Among the 147 US-FNA specimens, 97 (66.0%) were detected with BRAF gene mutations, all of which were p.V600E point mutation; 6 (4.1%) were detected with RAS gene mutation, and no TERT promoter or TP53 gene mutations were detected. Postoperative pathology confirmed that 136 cases were TMC, all of which were papillary thyroid microcarcinomas (PTMC); 11 cases (7.5%)were benign. Among 136 TMC samples, BRAF gene mutations were detected in 97 cases (71.3%). There were no statistically significant differences in age, gender, and maximum nodule diameter between patients with and without BRAF gene mutations (all P>0.05). The sensitivity and specificity of BRAF gene mutation in diagnosing TMC were 71.3% and 100.0%, respectively, with an area under the ROC curve (AUC) (95%CI) of 0.857 (0.789-0.925). For nodules classified as Bethesda Ⅲ-Ⅴ, the sensitivity and specificity were 63.0% and 100.0%, respectively, with an AUC (95%CI) of 0.815 (0.680-0.950). Conclusions: NGS technology can successfully detect multiple gene mutations in US-FNA specimens from TMN patients, especially BRAF gene mutation, and BRAF gene mutation has certain value in diagnosing TMC.

Abstract 2315: AI-enabled precision oncology era: Advanced and interactive interpretation of next-gneneration sequencing (NGS) reports

Abstract Background: Next-generation sequencing (NGS) has been widely adopted in clinical practice, but there are still unmet needs among physicians regarding the interpretation and application of NGS reports. These needs include selecting multi-targeted and combination therapies, keeping up with evolving treatment options and clinical trial results, and incorporating personalized medicine based on real clinical cases. Methods: To address these challenges, we propose using large language model (LLM) to analyze and interpret clinical questions, perform semantic searches in a high-quality database, and provide customized prompts and logical outputs. Our knowledge base includes diagnostic and treatment details for 600+ tumor subtypes, 620,000+ entries, 2,000+ drug details, and 440,000+ clinical trials. The database is updated automatically using a fact engine and medical literature sources. Personalized gene interpretation is incorporated using pretraining, cosine similarity, and context learning prompts. Results: Our tool, named "SmartMTB", enables personalized interpretation of genomic testing reports for precision oncology. Users input the latest clinical features using a selection-based or question-and-answer approach. The tool identifies the treatment stage and genomic variations, provides information on mutation frequencies, sensitivity and resistance of targeted and immunotherapies, and priority selection strategies for multi-gene mutations. It matches similar clinical cases and provides real-time updates on treatment options and clinical trial literature. It also facilitates patient matching for clinical trials and considers economic factors. Conclusion: We present a groundbreaking artificial intelligence (AI) tool in cancer care that revolutionizes precision oncology. This AI system, trained on extensive datasets, swiftly and accurately analyzes and interprets complex genomic and clinical data. It enables clinicians to make informed decisions about personalized treatment strategies, significantly improving patient outcomes in cancer therapy. Citation Format: Hui Chen, Zanmei Xu, Lijuan Chen, Mingmin Wang, Peng Zhang, Fei Pang, Kai Wang. AI-enabled precision oncology era: Advanced and interactive interpretation of next-gneneration sequencing (NGS) reports [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2315.

Retracted: Analysis of Multigene Mutations in Lung Adenocarcinoma in Zunyi.

[This retracts the article DOI: 10.1155/2021/9939065.].

Oleic acid-induced metastasis of KRAS/p53-mutant colorectal cancer relies on concurrent KRAS activation and IL-8 expression bypassing EGFR activation.

Background: Multigene mutations in colorectal cancer (CRC), including KRAS, BRAF, and p53, afford high metastatic ability and resistance to EGFR-targeting therapy. Understanding the molecular mechanisms regulating anti-EGFR-resistant CRC metastasis can improve CRC therapy. This study aimed to investigate the effects of IL-8 and the activation of KRAS on reactive oxygen species (ROS) production and metastasis of hyperlipidemia-associated CRC harboring mutations of KRAS and p53. Methods: The cytokine array analysis determined the up-expression of secreted factors, including IL-8. The clinical relevance of the relationship between IL-8 and angiopoietin-like 4 (ANGPTL4) was examined in CRC patients from National Cheng Kung University Hospital and TCGA dataset. Expressions of IL-8, ANGPTL4, NADPH oxidase 4 (NOX4), and epithelial-mesenchymal transition (EMT) markers in free fatty acids (FFAs)-treated KRAS/p53 mutant CRC cells were determined. The hyperlipidemia-triggered metastatic ability of CRC cells under treatments of antioxidants, statin, and cetuximab or knockdown of IL-8, KRAS, and EGFR was evaluated in vitro and in vivo. In addition, the effects of antioxidants and depletion of IL-8 and KRAS on the correlation between ROS production and hyperlipidemia-promoted CRC metastasis were also clarified. Results: In this study, we found that free fatty acids promoted KRAS/p53-mutant but not single-mutant or non-mutant CRC cell metastasis. IL-8, the most abundant secreted factor in KRAS/p53-mutant cells, was correlated with the upregulation of NOX4 expression and ROS production under oleic acid (OA)-treated conditions. In addition, the metastasis of KRAS/p53-mutant CRC relies on the ANGPTL4/IL-8/NOX4 axis and the activation of KRAS. The antioxidants and inactivation of KRAS also inhibited OA-induced EMT and metastasis. Although KRAS mediated EGF- and OA-promoted CRC cell invasion, the inhibition of EGFR did not affect OA-induced ANGPTL4/IL-8/NOX4 axis and CRC metastasis. The high-fat diet mice fed with vitamin E and statin or in IL-8-depleted cells significantly inhibited tumor extravasation and metastatic lung growth of CRC. Conclusion: The antioxidants, statins, and targeting IL-8 may provide better outcomes for treating metastatic CRC that harbors multigene mutations and anti-EGFR resistance.

Bt Cry1Ac resistance in Trichoplusia ni is conferred by multi-gene mutations

The three-domain Cry toxin Cry1Ac from Bacillus thuringiensis （Bt） is an important insecticidal toxin in Bt sprays and has been used in transgenic Bt-crops to confer insect resistance. The cabbage looper, Trichoplusia ni, has developed resistance to Bt sprays in commercial greenhouses, and the resistance to Cry1Ac has been previously identified to be associated with altered expression of the APN1 and APN6 genes and be genetically linked to a locus on chromosome 15. In this study, the Cry1Ac resistance locus in T. ni was further finely mapped, and the specific Cry1Ac resistance-conferring mutation in the resistance locus was identified to be a 4 bp frameshift insertion in the ABCC2 gene by whole genome resequencing, midgut transcriptome analysis, candidate gene cDNA sequencing and mutation site genomic DNA sequencing. By CRISPR/Cas9 mutagenesis, a series of ABCC2 and ABCC3 mutant T. ni strains were generated, and the role of ABCC2 in the toxicity of Cry1Ac in T. ni was confirmed. The results from this study also showed that knockout of ABCC2 in T. ni conferred resistance to Cry1Ac at a level lower than that in the greenhouse-derived resistant T. ni strain and that the Cry1Ac resistance-associated alteration of APN1 and APN6 expression was independent of ABCC2 gene mutations, indicating that the altered expression of APN1 and APN6 was controlled by another gene mutation in Cry1Ac resistant T. ni. Furthermore, T. ni larval bioassays showed that the level of Cry1Ac resistance in F1 families from reciprocal crosses of the Cry1Ac resistant strain with an ABCC2 knockout CRISPR strain was significantly higher than that in ABCC2 knockout strain, indicating the presence of additional Cry1Ac resistance-conferring mutation(s) in the Cry1Ac resistant strain. Therefore, the resistance to Cry1Ac in T. ni is conferred by a mutation in ABCC2 and an additional mutation (or mutations) which leads to altered expression of APN1 and APN6. The additional Cry1Ac resistance mutation or mutations remain to be identified.

Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti.

Rhizobia are widespread gram-negative soil bacteria and indispensable symbiotic partners of leguminous plants that facilitate the most highly efficient biological nitrogen fixation in nature. Although genetic studies in Sinorhizobium meliloti have advanced our understanding of symbiotic nitrogen fixation (SNF), the current methods used for genetic manipulations in Sinorhizobium meliloti are time-consuming and labor-intensive. In this study, we report the development of a few precise gene modification tools that utilize the CRISPR/Cas9 system and various deaminases. By fusing the Cas9 nickase to an adenine deaminase, we developed an adenine base editor (ABE) system that facilitated adenine-to-guanine transitions at one-nucleotide resolution without forming double-strand breaks (DSB). We also engineered a cytidine base editor (CBE) and a guanine base editor (GBE) that catalyze cytidine-to-thymine substitutions and cytidine-to-guanine transversions, respectively, by replacing adenine deaminase with cytidine deaminase and other auxiliary enzymes. All of these base editors are amenable to the assembly of multiple synthetic guide RNA (sgRNA) cassettes using Golden Gate Assembly to simultaneously achieve multigene mutations or disruptions. These CRISPR-mediated base editing tools will accelerate the functional genomics study and genome manipulation of rhizobia.

Analysis of Multigene Mutations in Lung Adenocarcinoma in Zunyi.

Objective Driver gene mutation in lung adenocarcinoma patients in Zunyi and its relationship with clinical features were probed in this investigation. Methods In total, with 244 patients with lung adenocarcinoma as study subjects, including 141 males and 103 females, amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) was utilized for detecting multigene mutations. Subsequently, the relationship between gene mutation and clinical characteristics was analyzed. Results The total mutation rate of driver genes was 65.17%, including 48.36% EGFR, 6.15% KRAS, 5.74% ALK, 2.05% HER-2, 1.23% ROS1, 0.82% RET, 0.41% NRAS, and 0.41% BRAF. Among EGFR mutations, 47.46% were EGFR-19-deletion, 42.37% EGFR-21-L858R mutation, 4.24% EGFR-20-T790M mutation, 2.54% EGFR-21-L861Q mutation, 2.54% EGFR-20-insertion, and 0.85% EGFR-18-G719X mutation. Both female patients and nonsmoking patients with lung adenocarcinoma had a higher rate of EGFR mutation. Additionally, 15 patients with multiple mutations in EGFR, including 13 patients with 2 mutations in EGFR and 2 patients with 3 mutations in EGFR, were found. Conclusion Among driver gene mutations in patients with lung adenocarcinoma in Zunyi, EGFR mutation has the highest incidence, followed by ALK fusion and KRAS mutation. Although both mutations and multisite mutations in the other driver genes account for a low proportion, they still have great clinical significance. Multigene mutation detection contributes to the rapid screening of patients with lung adenocarcinoma who respond to targeted therapy.

Multi-gene assay and clinical characteristics research in papillary thyroid carcinoma.

To investigate the significance of multi-gene assay in papillary thyroid carcinoma (PTC) patients in clinical practice. From April to December 2019, medical records of 68 patients with PTC after the initial surgery were retrospectively collected and analyzed in terms of the relations between gene mutations and clinicopathological characteristics. RET/PTC rearrangement was not detected in BRAF V600E mutation patients (P<0.001). Besides, compared with wild-type patients, BRAF V600E mutation was associated with significantly older age (P=0.001) and a higher rate of extrathyroid invasion (P=0.023). Significantly higher BRAF V600E mutation rates were found in clinical lymph node-negative (P=0.041) and non-metastatic lateral lymph nodes (P=0.027) patients as RET/PTC rearrangement was associated with younger age (P=0.001) and the increasing metastatic number of lymph nodes (P=0.020). Compared to other gene mutations, the multivariate analysis showed that larger tumor size [odds ratio (OR), 8.831; 95% CI: 1.971-35.578; P=0.004], the BRAF V600E mutation alone(OR, 10.567; 95% CI: 1.748-63.873; P=0.010) or in combination with one additional gene mutation (OR, 8.654; 95% CI: 1.453-68.603; P=0.041), and Hashimoto's thyroiditis (OR, 0.112; 95% CI: 0.025-0.499; P=0.004) were all independent predictors for the prevalence of ETE. BRAF V600E mutation was associated with older age and the aggressiveness of PTC but was independent of lymph node metastasis (LNM). RET/PTC rearrangement suggested more LNM in young patients with PTC. BRAF V600E mutation combined with other gene mutations, namely, multi-gene mutations, could indicate a higher aggressiveness in PTC.

Consecutive and automatic detection of multi-gene mutations from colorectal cancer samples by coupling droplet array-based capillary electrophoresis and PCR-RFLP.

The efficacy of targeted therapy is associated with multi-gene mutation status. Carrying out effective multi-genotyping analysis in combination has been a challenge in clinical settings. We therefore developed a droplet-based capillary electrophoresis (CE) system coupled with PCR-restriction fragment length polymorphism (PCR-RFLP) technology to detect multi-gene mutations from a small volume of samples. A 16 × 16 200-nL droplet array for sample encapsulation was constructed on a glass chip. The electrophoresis system consisted of a tapered vertical capillary filled with polyvinylpyrrolidone, a laser-induced fluorescence detector, and a high voltage power supply. Notably, a droplet-based electrokinetic sample introduction method and a "∩" shape capillary were developed to facilitate consecutive droplet sampling using a home-made automatic control module. The DL2000 DNA marker was consecutively separated, achieving high migration time and plate number reproducibility. The system was further applied to detect PCR-RFLP products. For colorectal cancer (CRC) cell lines, KRAS, BRAF, and PIK3CA were genotyped with a sensitivity of 0.25%. For CRC patient specimens, 30 samples were consecutively and automatically multi-genotyped without inter-sample contamination, with a lowest mutation frequency of 0.37%. For the first time, we developed a droplet-based CE system for consecutive DNA analysis with low sample consumption. This automated CE system could be further developed to integrate the full process of gene mutation detection, serving as a more effective platform for individualised therapy.

Significance of detecting antiphospholipid antibodies and genetic mutations of hemostasis for in vitro fertilization

A case of pregravid preparation is described for an in vitro fertilization (IVF) attempt in a woman after four previous IVF failures. Considering her personal and family aggravated medical history, including the family anamnesis, the patient underwent a hemostasis examination including a test for genetic mutations. A high titer of antiphospholipid antibodies of various types and multigene mutations associated with thrombophilia were found. For the treatment of acquired and inherited thrombophilia, vitamin-antioxidant and intravenous immunoglobulin therapy was initiated as well as a course of hirudotherapy. After starting of pregnancy throughout the gestation period, anticoagulant therapy by low molecular weight heparins, antiplatelet, and antioxidant therapy was carried out; in parallel, D-dimer, thrombin-antithrombin and platelet aggregation markers of thrombophilia were monitored. Pregnancy successfully ended by childbirth at 39 weeks.

Efficient ten-gene analysis of NSCLC tissue samples by next-generation sequencing

In the era of personalized medicine, lung cancer is a typical disease which can be treated strategically based on the patient’s histological and molecular diagnosis. Immunohistochemistry (IHC), fluorescence in-situ hybridization (FISH), Sanger sequencing and real-time PCR are techniques commonly used in clinical laboratories. Many patients are required to use several of the above technologies to get a complete diagnosis, which is expensive and timeconsuming. Next generation of sequencing (NGS) has the advantage to simultaneously analyze multigene mutations. The average cost for each patient is affordable if each run contains a certain number of samples. In this study, we tested a 10-gene, 32-mutation detection NGS method, which was used to test 195 samples from non-small cell lung cancer (NSCLC). Sanger sequencing and Amplification-refractory Mutation System (AMRS) PCR were employed to verify Epidermal Growth Factor Receptor (EGFR) and Anaplastic Lymphoma Kinase (ALK) results. This NGS method was partially proved to have a higher sensitivity to detect mutations with low abundance than Sanger sequencing and even ARMS PCR. Using genomic DNA to detect gene fusions may have some disadvantages to miss low abundance or large fragment fusions. As compared to using a few different technologies to analyze multigene mutations, small NGS analysis panel is a clinically applicable, efficient and affordable choice for NSCLC patients.

CrossPlan: systematic planning of genetic crosses to validate mathematical models.

Mathematical models of cellular processes can systematically predict the phenotypes of novel combinations of multi-gene mutations. Searching for informative predictions and prioritizing them for experimental validation is challenging since the number of possible combinations grows exponentially in the number of mutations. Moreover, keeping track of the crosses needed to make new mutants and planning sequences of experiments is unmanageable when the experimenter is deluged by hundreds of potentially informative predictions to test. We present CrossPlan, a novel methodology for systematically planning genetic crosses to make a set of target mutants from a set of source mutants. We base our approach on a generic experimental workflow used in performing genetic crosses in budding yeast. We prove that the CrossPlan problem is NP-complete. We develop an integer-linear-program (ILP) to maximize the number of target mutants that we can make under certain experimental constraints. We apply our method to a comprehensive mathematical model of the protein regulatory network controlling cell division in budding yeast. We also extend our solution to incorporate other experimental conditions such as a delay factor that decides the availability of a mutant and genetic markers to confirm gene deletions. The experimental flow that underlies our work is quite generic and our ILP-based algorithm is easy to modify. Hence, our framework should be relevant in plant and animal systems as well. CrossPlan code is freely available under GNU General Public Licence v3.0 at https://github.com/Murali-group/crossplan. Supplementary data are available at Bioinformatics online.

MISSA 2.0: an updated synthetic biology toolbox for assembly of orthogonal CRISPR/Cas systems

Efficient generation of plants carrying mutations in multiple genes remains a challenge. Using two or more orthogonal CRISPR/Cas systems can generate plants with multi-gene mutations, but assembly of these systems requires a robust, high-capacity toolkit. Here, we describe MISSA 2.0 (multiple-round in vivo site-specific assembly 2.0), an extensively updated toolkit for assembly of two or more CRISPR/Cas systems. We developed a novel suicide donor vector system based on plasmid RK2, which has much higher cloning capacity than the original, plasmid R6K-based system. We validated the utility of MISSA 2.0 by assembling multiple DNA fragments into the E. coli chromosome, and by creating transgenic Arabidopsis thaliana that constitutively or inducibly overexpress multiple genes. We then demonstrated that the higher cloning capacity of the RK2-derived MISSA 2.0 donor vectors facilitated the assembly of two orthogonal CRISPR/Cas systems including SpCas9 and SaCas9, and thus facilitated the creation of transgenic lines harboring these systems. We anticipate that MISSA 2.0 will enable substantial advancements in multiplex genome editing based on two or more orthogonal CRISPR/Cas9 systems, as well as in plant synthetic biology.

Recurrent mutations of MAPK pathway genes in multiple myeloma but not in amyloid light-chain amyloidosis.

Clinically applicable platforms revealing actionable genomic alterations may improve the treatment efficacy of myeloma patients. In this pilot study, we used a high depth targeted sequencing panel containing 83 anti-cancer drug target genes to sequence genomic DNAs extracted from bone marrow aspirates of 23 patients with myeloma and 12 patients with amyloid light-chain (AL) amyloidosis. Mutation analysis revealed NRAS as the most commonly mutated gene (30%, 7/23) in myeloma patients followed by KRAS (26%, 6/23) and BRAF (22%, 5/23). However, no significant mutations were found in the 12 patients with AL amyloidosis. Notably, 6 of the 23 myeloma patients showed multi-site and/or multi-gene mutations in NRAS, KRAS, or BRAF, indicating compound aberrations in the Mitogen activated protein kinase (MAPK) pathway. Gene panel sequencing also revealed cytogenetic abnormalities associated with prognosis in myeloma patients. In conclusion, our pilot study suggests that targeted gene sequencing may have an important prognostic value for myeloma patients for the identification of actionable genomic alterations and cytogenetic aberrations.

NADf Chip, a Two-Color Microarray for Simultaneous Screening of Multigene Mutations Associated with Hearing Impairment in North African Mediterranean Countries

Hearing impairment (HI) is the most frequent sensory defect. Genetic causes are involved in two thirds of prelingual cases. Moreover, the autosomal recessive HI frequency is increased in countries where there is a high rate of consanguinity, such as in North African Mediterranean countries. This population shares several features, including history and social behavior, that promote the spread of founder mutations. HI is characterized by tremendous heterogeneity in both the genetic and clinical aspects. The identification of the causal mutation is important for early diagnosis, clinical follow-up, and genetic counseling. Addressing the extreme genetic heterogeneity of HI using classic molecular methods would be expensive and time-consuming. We designed a cost-effective North African Deafness chip for rapid and simultaneous analysis of 58 mutations using multiplex PCR coupled with dual-color arrayed primer extension. These mutations are found in North African HI patients and are distributed over 31 exons and five introns in 21 distinct genes. Assay specificity was initially optimized using 103 archived DNA samples of known genotypes. Blind validation of HI-unrelated patients revealed mutant alleles in 13 samples, and these mutations were confirmed by Sanger sequencing. The North African Deafness chip allows for simultaneous genotyping of eight different samples, at a minimal cost and in a single day, and is therefore amenable to large-scale molecular screening of HI in North Africa.

Multiple gene mutations in patients with type 2 autoimmune pancreatitis and its clinical features.

BackgroundIt is now clear that there are two histological types (type 1 and type 2) of autoimmune pancreatitis (AI P). The histological substance of type 1 AI P is known as lymphoplasmacytic sclerosing pancreatitis (LPSP) or traditional AIP, and type 2 AIP is characterized by distinct histology called idiopathic duct centric pancreatitis (IDCP). Serum IgG4 increase is considered as a marker for type 1 AI P. Far less is known about type 2 and it lacks predicting markers, so it easily leads to missed diagnosis and misdiagnosis.The aim of this studyThe aim of this study was to describe multi-gene mutations in patients with type 2 AI P and its clinical features.Material and methodsThree unrelated patients with type 2 AI P, 10 cases with type 1 AIP, 15 cases with other chronic pancreatitis and 120 healthy individuals were studied. The mutations and polymorphisms of 6 genes involved in chronic pancreatitis or pancreatic cancer — PRSS1, SPINK1, CFTR, MEN1, PKHD1, and mitochondrial DNA – were sequenced. Information of clinical data was collected by personal interview using a structured questionnaire.ResultsNovel mutations were found in the genes encoding for MEN1 (p.546 Ala > The) and PKHD1 (c. 233586 A > G and c. 316713 C > T) from patients with type 2 AIP. What is more, the serum TCR (T cell receptor) level is relatively higher in patients with type 2 AIP than in patients with type 1 AIP and other chronic pancreatitis or normal controls. Weight loss was the major manifestation and no patients had extrapancreatic involvement in type 2 AIP.ConclusionsType 2 AIP may occur with multi-gene mutations. For screening purposes, it is more reasonable to evaluate TCR levels in serum.

A Rapid Method for Simultaneous Screening of Multi-Gene Mutations Associated with Hearing Loss in the Korean Population

Hearing loss (HL) is a congenital disease with a high prevalence, and patients with hearing loss need early diagnosis for treatment and prevention. The GJB2, MT-RNR1, and SLC26A4 genes have been reported as common causative genes of hearing loss in the Korean population and some mutations of these genes are the most common mutations associated with hearing loss. Accordingly, we developed a method for the simultaneous detection of seven mutations (c.235delC of GJB2, c.439A>G, c.919-2A>G, c.1149+3A>G, c.1229C>T, c.2168A>G of SLC26A4, and m.1555A>G of the MT-RNR1 gene) using multiplex SNaPshot minisequencing to enable rapid diagnosis of hereditary hearing loss. This method was confirmed in patients with hearing loss and used for genetic diagnosis of controls with normal hearing and neonates. We found that 4.06% of individuals with normal hearing and 4.32% of neonates were heterozygous carriers. In addition, we detected that an individual is heterozygous for two different mutations of GJB2 and SLC26A4 gene, respectively and one normal hearing showing the heteroplasmy of m.1555A>G. These genotypes corresponded to those determined by direct sequencing. Overall, we successfully developed a robust and cost-effective diagnosis method that detects common causative mutations of hearing loss in the Korean population. This method will be possible to detect up to 40% causative mutations associated with prelingual HL in the Korean population and serve as a useful genetic technique for diagnosis of hearing loss for patients, carriers, neonates, and fetuses.