Classic congenital adrenal hyperplasia with unilateral functional adrenal cortical adenoma: case report.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders related to adrenal steroid biosynthesis, and mainly caused by mutations in the CYP21A2 gene encoding 21-hydroxylase. Adrenal tumors are common in CAH, but functional adrenal tumors are rare. Here, we report a 17-year-old female with virilized external genitalia and primary amenorrhea, accompanied by a right adrenal tumor. Her 17-OHP level was normal, cortisol and androgen levels were significantly elevated, and the tumor pathology showed adrenal cortical adenoma. Gene testing for CYP21A2 showed c.518T > A in exon 4 and c.29313C > G in intron 2. The possibility of untreated classic CAH with 21-OH deficiency causing functional adrenal cortical adenoma should be considered. When clinical diagnosis highly considers CAH and cannot rule out the influence of functional adrenal tumors' secretion function on 17-OHP, gene mutation analysis should be performed.

Congenital Adrenal Hyperplasia with Combined 21-hydroxylase deficiency and 17α-hydroxylase/17,20-lyase deficiency: An undervirilized male.

21-hydroxylase deficiency stands as the most prevalent form of congenital adrenal hyperplasia, primarily resulting from mutations in the CYP21A2 gene. On the other hand, mutations within the CYP17A1 gene lead to 17α-hydroxylase/17,20-lyase enzyme deficiencies. The scarcity of 17-OH deficiency is noteworthy, accounting for less than 1% of all congenital adrenal hyperplasia cases. The male patient, born from a first-degree cousin marriage, exhibited several symptoms, including left undescended testis, micropenis, penile chord, left sensorineural hearing loss, and gynecomastia. He reported micropenis as a concern at the age of 13.5 years. His hormone profile revealed high levels of serum 17-hydroxyprogesterone, progesterone, and pregnenolone. In this case with a 46 XY karyotype, suspicions arose regarding Cytochrome P450 oxidoreductase deficiency due to ambiguous genitalia and an atypical hormone profile. Analysis unveiled two distinct homozygous and pathogenic variants in the CYP21A2 and CYP17A1 genes. Notably, mineralocorticoid precursors escalated, while cortisol and sex steroid precursors decreased during the high (250 mcg) dose ACTH stimulation test. The mutation c.1169C > G (p.Thr390Arg) in CYP17A1, which is the second documented case in literature, stands out due to its unique set of accompanying features. Mutations occurring in CYP21A2 and CYP17A1 result in complete or partial enzyme deficiencies, and the detection of homozygous mutations in two different enzyme systems within the steroidogenic pathway is noteworthy.

Improved Genetic Characterization of Congenital Adrenal Hyperplasia by Long-Read Sequencing Compared with Multiplex Ligation-Dependent Probe Amplification Plus Sanger Sequencing.

Genetic analysis of congenital adrenal hyperplasia (CAH) has been challenging because of high homology between CYP21A2 and its pseudogene CYP21A1P. This study aimed to evaluate the clinical utility of long-read sequencing (LRS) in diagnosis of CAH attributable to 21-hydroxylase deficiency by comparing with multiplex ligation-dependent probe amplification plus Sanger sequencing. In this retrospective study, 69 samples, including 49 probands from 47 families with high-risk of CAH, were enrolled and blindly subjected to detection of CAH by LRS. The genotype results were compared with control methods, and discordant samples were validated by additional Sanger sequencing. LRS successfully identified biallelic variants of CYP21A2 in the 39 probands diagnosed as having CAH. The remaining 10 probands were not patients with CAH. Additionally, LRS directly identified two pathogenic single-nucleotide variations (SNVs; c.293-13C/A>G and c.955C>T) in the presence of interference caused by nearby insertions/deletions (indels). The cis-trans configuration of two or more SNVs and indels identified in 18 samples was directly determined by LRS without family analysis. Eight CYP21A1P/A2 or TNXA/B deletion chimeras, composed of five subtypes, were identified; and the junction sites were precisely determined. Moreover, LRS determined the exact genotype in two probands who had three heterozygous SNVs/indels and duplication, which could not be clarified by control methods. These findings highlight that LRS could assist in more accurate genotype imputation and more precise CAH diagnosis.

Biochemical monitoring of 21-hydroxylase deficiency: a clinical utility of overnight fasting urine pregnanetriol.

PURPOSE OF REVIEW: 21-Hydroxylase deficiency (21-OHD), the most common form of congenital adrenal hyperplasia, is an autosomal recessive disorder caused by pathogenic variants in CYP21A2 . Although this disorder has been known for several decades, many challenges related to its monitoring and treatment remain to be addressed. The present review is written to describe an overview of biochemical monitoring of this entity, with particular focus on overnight fasting urine pregnanetriol. RECENT FINDINGS: We have conducted a decade-long research project to investigate methods of monitoring 21-OHD in children. Our latest studies on this topic have recently been published. One is a review of methods for monitoring 21-OHD. The other was to demonstrate that measuring the first morning PT level may be more practical and useful for biochemical monitoring of 21-OHD. The first morning pregnanetriol (PT), which was previously reported to reflect a long-term auxological data during the prepubertal period, correlated more significantly than the other timing PT in this study, with 17-OHP, before the morning medication. SUMMARY: In conclusion, although the optimal method of monitoring this disease is still uncertain, the use of overnight fasting urine pregnanetriol (P3) as a marker of 21-OHD is scientifically sound and may be clinically practical.

Rapid detection of common variants and deletions of CYP21A2 using MALDI-TOF MS.

Newborn screening (NBS) for congenital adrenal hyperplasia (CAH) based on hormonal testing is successfully implemented in many countries. However, this method cannot detect non-classic CAH and has high false positive rates. We have developed a novel MALDI-TOF MS assay that can identify common variants and deletions of CYP21A2 in the Chinese population. Thirty-seven clinical patients with CAH confirmed by Sanger sequencing and MLPA analysis were detected by MALDI-TOF MS assay. Two CYP21A2 variants were detected in 30 patients and one CYP21A2 variant was detected in 7 patients. The MALDI-TOF MS assay detected 67 mutant alleles in 37 patients with a detection rate of 90.5%. Sanger sequencing revealed that three variants in seven patients were not included in the designed panel. Eleven distinct CYP21A2 variants were identified, including five missense variants, two nonsense variants, two large gene deletions, one splice variant, and one frameshift variant. The most frequent variant was c.293-13C > G (37.84%), followed by c.518T > A (21.62%) and exon 1-7 deletion (17.57%). The high-throughput MALDI-TOF MS assay that can simultaneously detect common variants and deletions of CYP21A2. This assay can be used for population-based genetic screening and rapid detection of suspected patients, and is expected to be a valuable complement to biochemical-based testing for the detection of CAH.

Genetic Disruption of cyp21a2 Leads to Systemic Glucocorticoid Deficiency and Tissues Hyperplasia in the Teleost Fish Medaka (Oryzias latipes).

cytochrome P-450, 21-hydroxylase (cyp21a2), encodes an enzyme required for cortisol biosynthesis, and its mutations are the major genetic cause of congenital adrenal hyperplasia (CAH) in humans. Here, we have generated a null allele for the medaka cyp21a2 with a nine base-pair insertion which led to a truncated protein. We have observed a delay in hatching and a low survival rate in homozygous mutants. The interrenal gland (adrenal counterpart in teleosts) exhibits hyperplasia and the number of pomca-expressing cells in the pituitary increases in the homozygous mutant. A mass spectrometry-based analysis of whole larvae confirmed a lack of cortisol biosynthesis, while its corresponding precursors were significantly increased, indicating a systemic glucocorticoid deficiency in our mutant model. Furthermore, these phenotypes at the larval stage are rescued by cortisol. In addition, females showed complete sterility with accumulated follicles in the ovary while male homozygous mutants were fully fertile in the adult mutants. These results demonstrate that the mutant medaka recapitulates several aspects of cyp21a2-deficiency observed in humans, making it a valuable model for studying steroidogenesis in CAH.

A Humanized and Viable Animal Model for Congenital Adrenal Hyperplasia-CYP21A2-R484Q Mutant Mouse.

Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive disorder impairing cortisol synthesis due to reduced enzymatic activity. This leads to persistent adrenocortical overstimulation and the accumulation of precursors before the blocked enzymatic step. The predominant form of CAH arises from mutations in CYP21A2, causing 21-hydroxylase deficiency (21-OHD). Despite emerging treatment options for CAH, it is not always possible to physiologically replace cortisol levels and counteract hyperandrogenism. Moreover, there is a notable absence of an effective in vivo model for pre-clinical testing. In this work, we developed an animal model for CAH with the clinically relevant point mutation p.R484Q in the previously humanized CYP21A2 mouse strain. Mutant mice showed hyperplastic adrenals and exhibited reduced levels of corticosterone and 11-deoxycorticosterone and an increase in progesterone. Female mutants presented with higher aldosterone concentrations, but blood pressure remained similar between wildtype and mutant mice in both sexes. Male mutant mice have normal fertility with a typical testicular appearance, whereas female mutants are infertile, exhibit an abnormal ovarian structure, and remain in a consistent diestrus phase. Conclusively, we show that the animal model has the potential to contribute to testing new treatment options and to prevent comorbidities that result from hormone-related derangements and treatment-related side effects in CAH patients.

Case report: Development of central precocious puberty in a girl with late-diagnosed simple virilizing congenital adrenal hyperplasia complicated with Williams syndrome.

Congenital adrenal hyperplasia (CAH) and Williams Syndrome (WS; MIM # 194050) are distinct genetic conditions characterized by unique clinical features. 21-Hydroxylase deficiency (21-OHD; MIM #201910), the most common form of CAH, arises from mutations in the CYP21A2 gene, resulting in virilization of the external genitalia in affected females, early puberty in males, and short stature. Williams syndrome, caused by a microdeletion of 7q11.23, presents with distinctive facial features, intellectual disability, unique personality traits, early puberty, and short stature. This case report describe the clinical features of a 4-year-old girl referred due to progressive virilization and developmental delay. Genetic analysis confirmed concurrent CAH and WS, identifying a novel mutation in the CYP21A2 gene (c.1442T>C). Following corticosteroid therapy initiation, the patient developed central precocious puberty. This case report delves into the pubertal change patterns in a patient affected by overlapping genetic conditions, providing valuable insights in to the intricate clinical manifestation and management of these rare complex disorders.

Genetic diagnosis of endocrine disorders in Cyprus through the Cyprus Institute of Neurology and Genetics: an ENDO-ERN Reference Center.

The report covers the current and past activities of the department Molecular Genetics-Function and Therapy (MGFT) at the Cyprus Institute of Neurology and Genetics (CING), an affiliated Reference Center for the European Reference Network on Rare Endocrine Conditions (Endo-ERN).The presented data is the outcome of > 15 years long standing collaboration between MGFT and endocrine specialists from the local government hospitals and the private sector. Up-to-date > 2000 genetic tests have been performed for the diagnosis of inherited rare endocrine disorders. The major clinical entities included Congenital Adrenal Hyperplasia (CAH) due to pathogenic variants in CYP21A2 gene and Multiple Endocrine Neoplasia (MEN) type 2 due to pathogenic variants in the RET proto-oncogene. Other rare and novel pathogenic variants in ANOS1, WDR11, FGFR1, RNF216, and CHD7 genes were also found in patients with Congenital Hypogonadotropic Hypogonadism. Interestingly, a few patients with Disorders of Sexual Differentiation (DSD) shared rare pathogenic variants in the SRD5A2, HSD17B3 and HSD3B2 while patients with Glucose and Insulin Homeostasis carried theirs in GCK and HNF1A genes. Lastly, MGFT over the last few years has established an esteemed diagnostic and research program on premature puberty with emphasis on the implication of MKRN3 gene on the onset of the disease and the identification of other prognosis biomarkers.As an Endo-ERN member MGFT department belongs to this large European network and holds the same humanistic ideals which aim toward the improvements of health care for patients with rare endocrine conditions in respect to improved and faster diagnosis.

Giant Bilateral Adrenal Myelolipomas in a Non-Compliant Patient with Congenital Adrenal Hyperplasia.

BACKGROUND 21-hydroxylase deficiency, an essential enzyme for glucocorticoid and mineralocorticoid synthesis, is the cause of congenital adrenal hyperplasia (CAH) in more than 95% of cases. It is an autosomal recessive disorder encoded by the CYP21A2 gene, categorized into classical forms, which encompass the salt-wasting (SW) and simple virilizing (SV) forms, as well as the nonclassical form (NC). The aim of medical treatment is to replace missing glucocorticoids and, if necessary, mineralocorticoids, while also reducing elevated adrenal androgens. CASE REPORT We present the case of a 42-year-old woman with CAH who discontinued therapy during adolescence and was admitted to hospital with fatigue, nausea, and severe abdominal pain. A CT scan showed an extreme enlargement of the adrenal glands. Laboratory tests revealed elevated levels of 17-hydroxyprogesterone and other adrenal androgens, along with normal plasma metanephrine levels. Decreased morning cortisol levels suggested partial adrenal insufficiency requiring glucocorticoid replacement therapy. Due to the development of several serious complications and clinical deterioration, the multidisciplinary team recommended bilateral removal of masses measuring 300×250×200 mm on the right side and 250×200×200 mm on the left side. Histological and immunochemical examination confirmed the presence of giant myelolipomas with adrenal cortex hyperplasia. CONCLUSIONS Adrenal tumors, particularly myelolipomas, have a higher prevalence in patients with CAH. Our case report provides further evidence of the suspected link between non-compliant CAH therapy and the development of myelolipomas, along with promotion of their pronounced growth.

High carrier frequency of CYP21A2 gene mutations in Southern India - underscoring the need for genetic testing in Congenital Adrenal Hyperplasia.

PURPOSE: Congenital Adrenal Hyperplasia (CAH) is one of the highly prevalent autosomal recessive endocrine disorders. The majority of CAH cases result from mutations in the CYP21A2 gene, leading to 21-hydroxylase deficiency. However, with the pseudogene-associated challenges in CYP21A2 gene analysis, routine genetic diagnostics and carrier screening in CAH are not a part of the first-tier investigations in a clinical setting. Furthermore, there is a lack of data on the carrier frequency for 21-OH deficiency. Therefore, this study is aimed at investigating the carrier frequency of common pseudogene derived CYP21A2 mutations in Southern India. METHODS: Recently, a cost-effective Allele-specific PCR based genotyping for CYP21A2 hotspot mutations has been demonstrated to be a highly specific and sensitive assay at the authors' center. Leveraging this approach, a total of 1034 healthy individuals from South India underwent screening to identify the carrier frequency of nine hotspot mutations in the CYP21A2 gene. RESULTS: In this study, it was observed that 9.76% of the subjects were carriers for one or more of the nine different CYP21A2 mutations. Among the carriers, the most common was the large 30 kb deletion, followed by II72N, E6 CLUS, and I2G mutations. CONCLUSION: We have identified a high prevalence of CYP21A2 mutation carriers in Southern India. These findings emphasize the importance of implementing and expanding cost-effective genetic diagnostics and carrier screening throughout India. Such initiatives would play a crucial role in managing the disease burden, enabling early intervention, and establishing guidelines for CAH newborn genetic screening in the country. This study represents the first carrier screening data on CYP21A2 hotspot mutations from India and is the largest study conducted till date in this context.

Evaluating the efficacy of a long-read sequencing-based approach in the clinical diagnosis of neonatal congenital adrenocortical hyperplasia.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders predominantly characterized by impaired corticosteroid synthesis. Clinical phenotypes include hypoadrenocorticism, electrolyte disturbances, abnormal gonadal development, and short stature, of which severe hyponadrenocorticism and salt wasting can be life-threatening. Genetic analysis can help in the clinical diagnosis of CAH. However, the 21-OHD-causing gene CYP21A2 is arranged in tandem with the highly homologous CYP21A1P pseudogene, making it difficult to determine the exact genotypes using the traditional method of multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing or next-generation sequencing (NGS). We applied a long-read sequencing-based approach termed comprehensive analysis of CAH (CACAH) to 48 newborns with CAH that were diagnosed by clinical features and the traditional MLPA plus Sanger sequencing method for retrospective analysis, to evaluate its efficacy in the clinical diagnosis of neonatal CAH. Compared with the MLPA plus Sanger sequencing method, CACAH showed 100 % consistency in detecting SNV/indel variants located in exons and exon-intron boundary regions of CAH-related genes. It can directly determine the cis-trans relationship without the need to analyze parental genotypes, which reduces the time to diagnosis. Moreover, CACAH was able to distinguish different CYP21A1P/CYP21A2 and TNXA/TNXB chimeras, and detect additional variants (CYP21A2 variants c.-121C > T, c.*13G > A, c.*52C > T, c.*440C > T, c.*443 T > C, and TNXB variants c.12463 + 2 T > C, c.12204 + 5G > A). We also identified the TNXB variant c.11435_11524 + 30del alone instead of as a part of the TNXA/TNXB-CH-1 chimera in two newborns, which might be introduced by gene conversion. All of these characteristics enabled clinicians to better explain the phenotype of subjects and manage them more effectively. CACAH has a great advantage over the traditional MLPA and Sanger sequencing methods, showing substantial potential in the genetic diagnosis and screening of neonatal CAH.

Quantitative Characterization of Ectopic Adrenal Gene Expression in Fetal Testes in 21-Hydroxylase Deficient Mice.

Testicular adrenal rest tumors (TART) are a frequent and fertility impairing long-term complication in males with classic congenital adrenal hyperplasia. Due to lack of clear experimental data on their origin, they are hypothesized to be derived from ectopic adrenocortical cells within testicular tissue mainly growing upon stimulation by chronically elevated levels of adrenocorticotropin (ACTH). Alternatively, a more totipotent embryological origin has been discussed as the potential source of these tumors. The aim of this study was to quantify alterations of ectopic expression of adrenocortical genes (CYP11B1, CYP11B2, CYP21, MC2R) and the Leydig cell specific marker (INSL3) in testicular tissue of fetal 21-hydroxylase deficient (21OHD) mice. Timed-pregnancy studies were performed using H-2aw18 (aw18)-mice. Testes and adrenals of E15.5 and E18.5 mouse fetuses were used for real-time PCR and immunohistochemistry. Gene expression levels were analyzed for genotype-dependent alterations and compared with immunohistochemistry. While enzymes of steroidogenesis showed a significant increased expression in adrenals of 21OHD mice at both E15.5 and E18.5 compared to wild-type (WT) mice, expression levels were unaltered in testes of 21OHD mice. When compared to WT adrenals a significant increase of INSL3 expression in adrenals of 21OHD mice at E15.5 and E18.5 was detected. Cells with adrenocortical properties in mice fetal testis differ from in situ adrenocortical cells in gene expression and growth at E15.5 and E18.5. These findings suggest that the different local regulation and different local niche in adrenals and testes influence growth of aberrant adrenal cells.

Familial ApoB-specific familial hypobetalipoproteinemia in a patient with non-classical congenital adrenal hyperplasia. / Hipobetalipoproteinemia familiar ApoB específica en una paciente con hiperplasia suprarrenal congénita no clásica.

Familial hypobetalipoproteinaemia is a disorder of lipid metabolism characterized by low levels of total cholesterol, low-density lipoprotein cholesterol and apolipoprotein B. ApoB-related familial hypolipoproteinemia is an autosomal condition with a codominance inheritance pattern. Non-classical congenital adrenal hyperplasia is an autosomal recessive disorder due to mutations in the CYP21A2, a gene encoding for the enzyme 21-hydroxylase, which results in an androgen excess production from adrenal source. We here present the case of a 25-year-old woman with NCAH showing decreased levels of total-cholesterol, low-density lipoprotein cholesterol and triglycerides. Her parent had digestive symptoms and severe hepatic steatosis with elevated liver enzymes, as well as decreased levels of total and low-density lipoprotein cholesterol. A genetic-molecular study of the proband identified a mutation in the APOB gene, which allowed a diagnosis of heterozygous ApoB-related hypolipoproteinaemia to be made.

Genotype-Specific Cortisol Reserve in a Cohort of Subjects With Nonclassic Congenital Adrenal Hyperplasia (NCCAH).

CONTEXT: Recent guidelines suggest that patients with nonclassic congenital adrenal hyperplasia (NCCAH) stop glucocorticoid therapy after achieving adult height. However, these guidelines do not differentiate between NCCAH genotype groups. OBJECTIVE: Compare ACTH-stimulated cortisol and 17-hydroxyprogesterone (17OHP) levels, and the rate of partial cortisol insufficiency in subjects with NCCAH carrying one mild and one severe (mild/severe) mutation vs subjects with biallelic mild (mild/mild) mutations. METHODS: Retrospective evaluation of the medical records of 122 patients who presented with postnatal virilization and were diagnosed with NCCAH. Patients underwent standard intravenous 0.25 mg/m2 ACTH stimulation testing. Those with stimulated 17OHP level ≥40 nmol/L were screened for the 9 most frequent CYP21A2 gene mutations followed by multiplex ligation-dependent probe amplification. A stimulated cortisol level below 500 nmol/L was defined as partial cortisol deficiency. RESULTS: Patients were subdivided into 3 genotype groups: 77 carried the mild/mild genotype, mainly homozygous for p.V281L mutation; 29 were compound heterozygous for mild/severe mutation, mainly p.V281L/p.I2Splice, and 16 were heterozygous for p.V281L, and were excluded from statistical evaluation. Stimulated cortisol levels were significantly lower in the mild/severe than in the mild/mild group (mean ± SD, 480 ± 90 vs 570 ± 125 nmol/L, P < .001). The mild/severe group exhibited a significantly higher rate of partial cortisol insufficiency (21/28, 75% vs 28/71, 39%, P = .004). Peak 17OHP was significantly higher in the mild/severe group (198 ± 92 vs 118 ± 50 nmol/L, P < .001). CONCLUSION: The high rate of partial adrenal insufficiency in the mild/severe group underscores the need to carefully consider the value of glucocorticoid therapy cessation and the importance of stress coverage in this group.

Targeted long-read sequencing for comprehensive detection of CYP21A2 mutations in patients with 21-hydroxylase deficiency.

BACKGROUND: 21-Hydroxylase deficiency (21-OHD) is caused by pathogenic CYP21A2 variations. CYP21A2 is arranged in tandem with its highly homologous pseudogene CYP21A1P; therefore, it is prone to mismatch and rearrangement, producing different types of complex variations. There were few reports on using only one method to detect different CYP21A2 variants simultaneously. AIMS: Targeted long-read sequencing method was used to detect all types of CYP21A2 variants in a series of patients with 21-OHD. METHODS: A total of 59 patients with 21-OHD were enrolled from Peking Union Medical College Hospital. Long-range locus-specific PCR and long-read sequencing (LRS) were performed to detect the pathogenic variants in CYP21A2. RESULTS: Copy-number variants of CYP21A2 were found in 25.4% of patients, including 5.1% with 3 copies of CYP21A2, 16.9% with 1 copy of CYP21A2, and 3.4% with 0 copy of CYP21A2. The remaining 74.6% of patients had 2 copies of CYP21A2. Pathogenic variants were identified in all 121 alleles of 59 patients. Specifically, single-nucleotide variants and small insertions/deletions (< 50 bp) were detected in 79 alleles, of which conversed from CYP21A1P were detected in 63 alleles, and rare variants were found in the other 16 alleles. Large gene conversions (> 50 bp) from pseudogene were detected in 10 alleles, and different chimeric genes (CYP21A1P/CYP21A2 or TNXA/TNXB) formed by large deletions were detected in 32 alleles. Of all variants, p.I173N was the most common variant (19.0%). CONCLUSIONS: Our study demonstrated that targeted long-read sequencing is a comprehensive method for detecting CYP21A2 variations, which is helpful for genetic diagnosis in 21-OHD patients.

A MinION-based Long-Read Sequencing Application With One-Step PCR for the Genetic Diagnosis of 21-Hydroxylase Deficiency.

CONTEXT: Recently developed long-read sequencing (LRS) technology has been considered an option for CYP21A2 analysis. However, the clinical use of LRS for CYP21A2 analysis is limited. OBJECTIVE: This study's objective is to develop an efficient and low-cost LRS system for CYP21A2 screening. METHODS: A DNA fragment library was prepared in a single polymerase chain reaction (PCR) that covers the entire CYP21A2 gene and all known junctions caused by TNXB gene structural rearrangements, yielding a single 8-kb product of CYP21A2 or CYP21A1P/CYP21A2 chimera. After barcoding, the PCR products were sequenced on a MinION-based platform with Flongle Flow Cell R9.4.1 and R10.4.1. RESULTS: The reference genotypes of 55 patients with 21-hydroxylase deficiency (21OHD) were established using the conventional method with multiplex ligation-dependent probe amplification (MLPA) and nested PCR. LRS using Flongle Flow Cell R9.4.1 yielded consistent results. Additionally, the recently updated LRS "duplex" analysis with Flongle flow cell R10.4.1 was tested to reveal an advantage of accurately sequencing a variant located on the homopolymer region. By introducing a barcode system, the cost was reduced to be comparable to that of conventional analysis. A novel single-nucleotide variation was discovered at the acceptor site of intron 7, c.940-1G > C. We also identified a subtype of the classical chimeric junction CH2, "CH2a," in the region from the latter part of intron 5 to exon 6. CONCLUSION: We successfully established a novel low-cost and highly accurate LRS system for 21OHD genetic analysis. Our study provides insight into the feasibility of LRS for diagnosing 21OHD and other genetic diseases caused by structural rearrangements.