A novel strategy for predicting the efficacy of temozolomide treatment for metastatic pheochromocytomas/paragangliomas.

BACKGROUND: There are few studies on the efficacy of temozolomide (TMZ) in the treatment of Metastatic pheochromocytoma / paraganglioma (MPP) patients. And it remains unclear which MPP patients may benefit from TMZ treatment. METHODS: This was a prospective study. MPP patients were enrolled. Patients were treated with TMZ until disease progression or intolerable toxicities. The primary endpoints were disease control rate (DCR) and objective response rate (ORR). Secondary endpoints included biochemical response rate progression-free survival (PFS) and safety. We compared the difference between effective and ineffective groups, to explore which patients are more suitable for TMZ treatment. RESULTS: 62 patients with MPP were enrolled and tumor response were evaluated in 54 patients. The DCR was 83% (35/42), and the ORR was 24% (10/41) among the progressive patients. PFS was 25.2 ± 3.1 months. The most common adverse event was nausea (41/55). We found that 92.9% (13/14) of patients with MGMT methylation greater than 7% respond to treatment. For the patients with MGMT methylation less than 7%, Ki-67 index could be used to guide the use of TMZ in these patients. Among the patients with Ki-67 index less than 5%, 66% (8/12) patients showed respond to treatment, and only 33% (4/12) patients with Ki-67 index more than 5% showed respond to TMZ. CONCLUSIONS: This study indicated that TMZ is a potential choice for the treatment of MPP with the high ability on disease control and well tolerability. We recommended to MGMT methylation analysis test and Ki-67 index to guide TMZ application.

[Clinical features of patients with metastatic pheochromocytoma/paraganglioma].

Objective: To analyze the clinical features of patients with metastatic pheochromocytoma/paraganglioma (PPGL). Methods: A follow-up study. The clinical data of 250 patients with metastatic PPGL treated at Peking Union Medical College Hospital from January 2018 to August 2023 were retrospectively analyzed, including 124 males and 126 females. The clinical features and treatment status of patients with metastatic PPGL were summarized and analyzed. Kaplan-Meier survival curve was used to evaluate patients' prognosis. Results: The age of onset, age of diagnosis, and age of tumor metastasis in patients with metastatic PPGL were (33.1±14.2) years, (35.4±15.2) years, and (40.7±15.3) years, respectively. Metastasis occurred in 26.4%(66/250) of patients at the time of initial diagnosis. Among patients without metastases at the time of initial diagnosis, the time from primary tumor resection to metastasis[M(Q1, Q3)] was 5.0 (3.0, 9.0) years, among which 20.1%(37/184) of patients had metastases more than 10 years after surgery. Most patients showed increased 24-hour urinary norepinephrine and plasma normetanephrine, accounting for 78.2%(176/225) and 78.7%(85/108), respectively. 42.3%(69/163) of patients had increased neuron specific enolase (NSE)levels. Germline mutations were screened in 201 patients, of which 55.2%(111/201) had germline pathogenic mutations. In patients with gene mutations, 76.5%(85/111) had SDHB mutations. 52.0%(130/250) of metastatic PPGL patients had primary sites outside the adrenal gland, with the Ki-67 index of 5% (3%, 8%). There were 85.6%(214/250) patients had multisystem metastasis, with bone metastasis being the most common site of metastasis, accounting for 60.8%(152/250). In terms of treatment, 32.8%(75/229) of patients underwent two treatment regimens and 8.7%(20/229) of patients underwent three treatment regimens. Most patients had a good prognosis, with a 5-year and 10-year survival rate of 88.0% and 84.0%, respectively. However, some patients had rapid disease progression, and as of August 2023, 30 patients died, and the time from diagnosis to death in deceased patients was 2.0 (1.0, 4.0) years. Conclusions: Patients with metastatic PPGL have a high rate of germline mutations, especially those with SDHB mutations. The metastatic PPGL is usually multisystem metastasis with the characteristics of mostly paraganglioma, large lesion diameter and high Ki-67 index.

Novel alternative tools for metastatic pheochromocytomas/paragangliomas prediction.

OBJECTIVE: The existing prediction models for metastasis in pheochromocytomas/paragangliomas (PPGLs) showed high heterogeneity in different centers. Therefore, this study aimed to establish new prediction models integrating multiple variables based on different algorithms. DESIGN AND METHODS: Data of patients with PPGLs undergoing surgical resection at the Peking Union Medical College Hospital from 2007 to 2022 were collected retrospectively. Patients were randomly divided into the training and testing sets in a ratio of 7:3. Subsequently, decision trees, random forest, and logistic models were constructed for metastasis prediction with the training set and Cox models for metastasis-free survival (MFS) prediction with the total population. Additionally, Ki-67 index and tumor size were transformed into categorical variables for adjusting models. The testing set was used to assess the discrimination and calibration of models and the optimal models were visualized as nomograms. Clinical characteristics and MFS were compared between patients with and without risk factors. RESULTS: A total of 198 patients with 59 cases of metastasis were included and classified into the training set (n = 138) and testing set (n = 60). Among all models, the logistic regression model showed the best discrimination for metastasis prediction with an AUC of 0.891 (95% CI, 0.793-0.990), integrating SDHB germline mutations [OR: 96.72 (95% CI, 16.61-940.79)], S-100 (-) [OR: 11.22 (95% CI, 3.04-58.51)], ATRX (-) [OR: 8.42 (95% CI, 2.73-29.24)] and Ki-67 ≥ 3% [OR: 7.98 (95% CI, 2.27-32.24)] evaluated through immunohistochemistry (IHC), and tumor size ≥ 5 cm [OR: 4.59 (95% CI, 1.34-19.13)]. The multivariate Cox model including the above risk factors also showed a high C-index of 0.860 (95% CI, 0.810-0.911) in predicting MFS after surgery. Furthermore, patients with the above risk factors showed a significantly poorer MFS (P ≤ 0.001). CONCLUSIONS: Models established in this study provided alternative and reliable tools for clinicians to predict PPGLs patients' metastasis and MFS. More importantly, this study revealed for the first time that IHC of ATRX could act as an independent predictor of metastasis in PPGLs.

Deteriorated bone microarchitecture caused by sympathetic overstimulation in pheochromocytoma and paraganglioma.

PURPOSE: Despite the potentially destructive effect of sympathetic activity on bone metabolism, its impact on bone microarchitecture, a key determinant of bone quality, has not been thoroughly investigated. This study aims to evaluate the impact of sympathetic activity on bone microarchitecture and bone strength in patients with pheochromocytoma and paraganglioma (PPGL). METHODS: A cross-sectional study was conducted in 38 PPGL patients (15 males and 23 females). Bone turnover markers serum procollagen type 1 N-terminal propeptide (P1NP) and ß-carboxy-terminal crosslinked telopeptide of type 1 collagen (ß-CTX) were measured. 24-h urinary adrenaline (24hUE) and 24-h urinary norepinephrine levels (24hUNE) were measured to indicate sympathetic activity. High-resolution peripheral quantitative computed tomography (HR-pQCT) was conducted to evaluate bone microarchitecture in PPGL patients and 76 age-, sex-matched healthy controls (30 males and 46 females). Areal bone mineral density (aBMD) was measured by dual-energy X-ray absorptiometry (DXA) simultaneously. RESULTS: PPGL patients had a higher level of ß-CTX. HR-pQCT assessment revealed that PPGL patients had notably thinner and more sparse trabecular bone (decreased trabecular number and thickness with increased trabecular separation), significantly decreased volume BMD (vBMD), and bone strength at both the radius and tibia compared with healthy controls. The deterioration of Tt.vBMD, Tb.Sp, and Tb.1/N.SD was more pronounced in postmenopausal patients compared with the premenopausal subjects. Moreover, subjects in the highest 24hUNE quartile (Q4) showed markedly lower Tb.N and higher Tb.Sp and Tb.1/N.SD at the tibia than those in the lowest quartile (Q1). Age-related bone loss was also exacerbated in PPGL patients to a certain extent. CONCLUSIONS: PPGL patients had significantly deteriorated bone microarchitecture and strength, especially in the trabecular bone, with an increased bone resorption rate. Our findings provide clinical evidence that sympathetic overstimulation may serve as a secondary cause of osteoporosis, especially in subjects with increased sympathetic activity.

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.

[Preliminary study on the ability of 68Ga-Pentixafor PET/CT to differentiate between adrenal aldosterone-producing adenoma and nonfunctional adenoma].

Objective: To evaluate the ability of 68Ga-Pentixafor (nuclide ligand imaging agents for chemokine receptor 4) PET/CT to differentiate between aldosterone-producing adenoma (APA) and adrenal nonfunctional adenoma (NFA), and to assess how well this imaging method correlates with clinical features and postoperative outcomes. Methods: This was a cross-sectional study involving 73 APA and 12 NFA patients who received 68Ga-Pentixafor PET/CT imaging at Peking Union Medical College Hospital from August 2018 to October 2021. The receiver operating characteristic (ROC) curve was used to evaluate the differential value of visual analysis and the maximum standard uptake value (SUVmax) of the focus on APA and NFA. The related factors of SUVmax, and its predictive effect on postoperative outcomes were analyzed using Pearson or Spearman analysis and χ2 text. Results: 68Ga-Pentixafor PET/CT imaging was positive in 64 APA patients (sensitivity=87.7%) and negative in all 12 NFA patients (specificity=100%). The area under the ROC curve with SUVmax differentiating APA and NFA was 0.932 (P<0.001). When the SUVmax cut-off point was 6.23, the sensitivity was 80.8% and the specificity was 100%. The SUVmax correlated positively with lesion size (r=0.598) and aldosterone/renin activity ratio (r=0.313) and correlated negatively with potassium level (r=-0.286), renin activity (r=-0.240) and age of diagnosis (r=-0.273) (all P<0.05). Of the patients who underwent adrenalectomy and received more than 6 months of post-surgical follow-up, the clinical complete remission rate was higher for 68Ga-Pentixafor PET/CT imaging-positive patients than imaging-negative patients (24/39 vs. 0/4, P=0.031). Conclusions: 68Ga-Pentixafor PET/CT is effective at differentiating between APA and NFA. The SUVmax of 68Ga-Pentixafor PET/CT correlates with age at onset, lesion size, and the severity of clinical manifestations, and is able to predict postoperative outcomes.

Favorable outcome in advanced pheochromocytoma and paraganglioma after hypofractionated intensity modulated radiotherapy.

PURPOSE: The purpose of this study was to review outcomes of patients with advanced/metastatic pheochromocytoma/paraganglioma (PPGL) treated at our institution with Intensity-modulated radiotherapy (IMRT), describe the treatment outcomes, and determine predictors. METHODS: A retrospective study on patients with advanced/metastatic PPGL who received IMRT at Peking Union Medical College Hospital between 2014 and 2019. A total of 14 patients with 17 lesions were included in this study. Ultra-hypofractionated radiation therapy was used for 7 lesions in 5 patients, while hypofractionated radiation therapy was used for 8 lesions in 7 patients. 2 patients got conventional fractionated radiotherapy. Patients who received external beam radiation therapy were given a median total radiation dose of 74.4/130 Gy (BED10/3) in a median of 13 fractions. RESULTS: OS at 2 years was 78% for all patients. For lesions evaluated by RECIST response, at least stable disease of the target lesion was achieved in 94% and distant progression in 28.5%, with an average time to progression of 5.2 months. Patients with locally advanced primary tumors or recurred in situ (n = 8) achieved 100% local control, and none of them got recurrence or distant metastasis after radiotherapy at last follow-up (median 29 months). Of patients with catecholamine-related syndromes (n = 12), 91% of symptomatic lesions improved following radiation therapy and a more than 50% decline in catecholamines. CONCLUSIONS: We have found hypofractionated IMRT effective as an additional therapy for patients with advanced primary tumors or recurrence in situ and not amenable to complete surgical resection.

A Tibetan ice core covering the past 1,300 years radiometrically dated with 39Ar.

Ice cores from alpine glaciers are unique archives of past global and regional climate conditions. However, recovering climate records from these ice cores is often hindered by the lack of a reliable chronology, especially in the age range of 100 to 500 anni (a) for which radiometric dating has not been available so far. We report on radiometric 39Ar dating of an ice core from the Tibetan Plateau and the construction of a chronology covering the past 1,300 a using the obtained 39Ar ages. This is made possible by advances in the analysis of 39Ar using the laser-based detection method atom trap trace analysis, resulting in a twofold increase in the upper age limit of 39Ar dating. By measuring the anthropogenic 85Kr along with 39Ar we quantify and correct modern air contamination, thus removing a major systematic uncertainty of 39Ar dating. Moreover, the 85Kr data for the top part of the ice core provide information on firn processes, including the age difference between the ice and its enclosed gas. This first application of 39Ar and 85Kr to an ice core facilitates further ice cores from nonpolar glaciers to be used for recovering climate records of the Common Era, a period including pronounced anomalies such as the Little Ice Age and the Medieval Warm Period.

[Recurrence and metastasis of pheochromocytoma/paraganglioma after tumor resection and clinical characteristics analysis].

Objective: The purpose of this study is to investigate the incidence of recurrence or metastasis of pheochromocytoma/paraganglioma (PPGL) patients after primary tumor resection, and to compare the differences of clinical indicators between patients with or without recurrence or metastasis. Methods: This study is a retrospective study. All 157 patients were followed up after tumor resection in Peking Union Medical College Hospital from 2008 to 2016. We obtained the basic information [age of diagnosis, gender, height, weight and body mass index (BMI)], the onset status of PPGL (age of onset, course of disease, family history, tumor location, whether the tumor is bilateral or multiple, and preoperative blood pressure), clinical and pathological features of PPGL tumor (tumor size, whether it could adhere, invade or infiltrate during operation, whether the tumor capsule is smooth and complete on the postoperative pathological diagnosis, whether there is infiltration growth and cystic necrosis on tumor pathology and Ki-67 index), and laboratory examination results [24 hours urinary norepinephrine (NE), epinephrine (E), dopamine (DA) before operation]. According to the outpatient or telephone follow-up, the postoperative incidences of recurrence and metastasis were explored, and the basic information, status of onset, clinical and pathological characteristics of tumors, and laboratory test results of patients were compared. Results: A total of 157 patients, 69 males and 88 females, were with an average age of (42.4±13.4) years old. There were 103 patients with PCC and 54 with PGL. The average follow-up time was (9.5±2.0) years. Of the 103 patients with PCC, 13 (12.6%) had postoperative recurrence and 9 (8.7%) had distant metastasis. Compared with the patients without recurrence and metastasis, the onset age of the 13 patients with recurrence was younger [(27.3±15.7) years vs (39.3±12.2) years, P=0.003], the course of disease was longer [48.0 (23.0, 141.0) months vs 12.0 (4.0, 60.0) months, P=0.010]. The tumor size of 9 patients with distant metastasis was larger [8.0 (6.1, 12.8) cm vs 5.0 (4.0, 7.0) cm, P=0.027]. Of the 54 patients with PGL, 8 (14.8%) had postoperative recurrence and 5 (9.3%) had distant metastasis. Compared with the patients without recurrence and metastasis, the course of disease of the 8 patients with recurrence was longer [90.0 (36.3, 165.0) months vs 24.0 (8.0, 72.0) months, P=0.009], and the proportion of primary tumors with multiple lesions was higher (4/8 vs 4.4%, P=0.003). The preoperative diastolic blood pressure was higher in 5 patients with distant metastasis [(146.0±32.1) mmHg vs (120.6±25.3) mmHg, P=0.043] (1 mmHg=0.133 kPa), and the proportion of primary tumors with multiple lesions was higher (2/4 vs 4.4%, P=0.029). Conclusion: PPGL patients are prone to have recurrence or metastasis. PPGL patients with postoperative recurrence or distant metastasis had younger onset age, longer course of disease, larger tumor size and higher proportion of multiple lesions.

Selective use of Peptide Receptor Radionuclide Therapy following comparative imaging of Ga-68 DOTATATE PET/CT against I-131 MIBG scintigraphy in a small Asian cohort of Adult Neuroblastoma.

Adult neuroblastoma (AN) is rare with an extremely poor prognosis. No standard therapy exists for this entity and treatment options are limited in recurrent or refractory disease. 131I-MIBG has been used in combination with myeloablative therapy before autologous bone marrow transplantation or in a salvage therapy setting. However, myelotoxicity is a dose-limiting factor in heavily pre-treated patients and response is not always sustained. Somatostatin receptor scintigraphy and theranostics with radiolabelled somatostatin receptor analogues are becoming more commonplace with the recognition of these receptors in over 90% of neuroblastoma cells. We describe three AN patients assessed for somatostatin receptor status and the novel use of 177Lu-based peptide recep-tor radionuclide therapy (PRRT) in two of them and a literature review.

[Prevalence and related factors of dyslipidemia in patients with 21-hydroxylase deficiency].

Objective: To analyze the prevalence and the related factors of dyslipidemia in 21-hydroxylase deficiency (21-OHD) patients. Methods: A total of 205 patients with 21-OHD were recruited in Peking Union Medical College Hospital from January 2016 to January 2018. The basic information, glucocorticoid replacement therapy, and laboratory examination results of patients were obtained from medical records. The genotypes of CYP21A2 were identified by Sanger sequencing and multiplex ligation dependent probe amplification. The prevalence of dyslipidemia among 21-OHD patients, basic information and related hormone levels of 21-OHD patients with different status of blood lipid were described. Logistic regression model was used to analyze the related factors of dyslipidemia in 21-OHD patients. Results: The age of subjects was 17.0 (8.3, 25.0) years old, including 51 males (24.9%). According to CYP21A2 genotypes, there were 16 cases in Null group, 26 cases in Group A, 105 cases in group B, 27 cases in group C, and 31 cases in group D. The incidence of dyslipidemia was 29.3% (60/205), among which 37.3% (19/51) in male and 26.6% (41/154) in female patients, respectively. The M (Q1, Q3) of total cortisol level (nmol/L) and body mass index (kg/m2) of male 21-OHD patients with dyslipidemia were 0.17 (0.06, 0.35) and 25.76 (17.01, 30.45), respectively, which were higher than those with ortholiposis [0.04 (0.02, 0.21) and 18.83 (16.53, 23.88)] (all P<0.05). The M (Q1, Q3) of progesterone level (nmol/L), body mass index (kg/m2) and age (years) of female 21-OHD patients with dyslipidemia were 74.40 (50.97, 98.52), 23.09 (21.78, 27.78) and 23.00 (16.50, 28.00), respectively, which were higher than those with ortholiposis [52.81 (33.41, 68.85), 21.55 (18.63, 25.71) and 18.00 (9.50, 25.00)] (all P<0.05). The risk of dyslipidemia increased by 5.0% [OR (95%CI): 1.05 (1.01, 1.09)] for every 1 nmol/L increase of progesterone. Conclusion: The incidence of dyslipidemia is high in 21-OHD patients, and progesterone level is positively correlated with dyslipidemia.

[Clinical characteristics of testicular adrenal rest tumor and factors associated with its occurrence in 21-hydroxylase deficiency patients].

Objective: To analyze the occurrence and clinical characteristics of testicular adrenal rest tumor (TART) in 21-hydroxylase deficiency (21-OHD) patients, and further explore the possible factors related to the occurrence of TART. Methods: Twenty-seven male 21-OHD patients who visited Peking Union Medical College Hospital from January to December 2018 were enrolled and their clinical and biochemical data were collected. The CYP21A2 mutations were identified by Sanger sequencing and multiple ligation probe amplification (MLPA). Patients were divided into different subgroups according to the residual activity of 21-hydroxylase: Null (residual enzymatic activity 0, 3 cases), group A (0-<1%, 9 cases), group B (1%-5%, 7 cases), group C (20%-50%, 2 cases) and group D (6 cases). The ultrasound of testis was used to detect whether there was TART and its morphological characteristics. Results: Among 27 patients with 21-OHD [average age (17.4±9.3) years], 55.6% (15/27) had TART lesions, most of them were adolescents, and the youngest was only 4 years old. The lesions were mostly bilateral and hypoechoic. The levels of 17α-hydroxyprogesterone (17-OHP) and progesterone in patients with TART were higher than those in patients without TART [17-OHP: 199.6 (62.1, 232.7) nmol/L vs 7.4 (3.2, 105.0) nmol/L, P=0.003; progesterone: 97.1 (42.0, 126.8) nmol/L vs 5.4 (0.7, 20.0) nmol/L, P=0.004]. There was a correlation between the occurrence of TART and genotype of CYP21A2. Patients with Null and A genotypes were more likely to have TART than those with B and C genotypes (8/12 vs 4/9, P=0.021). Conclusions: TART is common in 21-OHD male patients, which is related to 17-OHP and CYP21A2 genotype. It is of great significance for the early screening of TART in 21-OHD patients.

[The analysis of clinical characteristics of 34 novel coronavirus pneumonia cases in Ningxia Hui autonomous region].

Objective: To analyze the clinical characteristics of 34 COVID-19 cases and to provide the basis for the prevention and control of the epidemic disease. Methods: Thirty-four COVID-19 patients diagnosed with RT-PCR in the isolation ward of the Fourth People's Hospital of Ningxia Hui autonomous region (infectious diseases hospital) from the January 22 to February 4, 2020 were selected as the research subjects. The clinical data were collected. Excel was used to describe the relationship between clinical classification and age distribution, contact history and date of onset. SPSS 25.0 statistical software was used for analysis. The general information, clinical symptoms, blood test, virus nucleic acid test results, epidemiology, CT imaging, treatment and prognosis were analyzed. Results: There were 5 mild cases (5/34), 24 ordinary cases (24/34), 5 severe cases (5/34). The elderly was more common in severe patients. The main clinical symptoms were fever (27/34)and dry cough(26/34). The peripheral blood showed normal or decreased leukocyte count (33/34), decreased lymphocyte count (12/34). The increase of C-reactive protein (CRP) and D-dimer was related to the severity of the disease. Some patients had mild liver and kidney damage. Six patients were diagnosed through 3 or more times of nucleic acid tests. Sixteen cases had Wuhan related history,13 cases were close contacts, 5 cases had no confirmed route. The clustered infections were found in 6 families. In typical cases, CT showed single or multiple patchy ground glass shadow with thickening of interlobular septum. In severe cases, diffuse lesions of both lungs were found, with ground glass shadow, consolidation shadow and strip shadow coexisting. Thirty-four patients were treated with interferon-α and Lopinavir/Ritonavir with good prognosis. Conclusions: The clinical characteristics of COVID-19 were similar to that of general viral pneumonia but with strong infectivity. Close contact and family aggregation caused disease outbreaks. COVID-19 could not be excluded if two nucleic acid tests were negative and high-resolution CT was helpful for differential diagnosis. Early detection, early isolation, early diagnosis and early treatment are important for good prognosis. The effectiveness of antiviral drugs needs to be further verified.

[Detection of CYP21A2 gene mutations and the differences in the levels of hormones in patients with 21-hydroxylase deficiency].

Objective: To analyze the detection of CYP21A2 gene mutations in 21-hydroxylase deficiency (21-OHD) patients, so as to determine the accuracy of clinical diagnosis. Methods: Totally, 514 patients with 21-OHD who visited Peking Union Medical College Hospital from January 2015 to January 2018 were enrolled and their clinical and biochemical data were collected. DNAs were extracted from peripheral blood leukocytes and CYP21A2 mutations were detected by Sanger sequencing and multiple ligation probe amplification (MLPA) technique. We divided 514 patients into three groups: two mutations of CYP21A2 alleles (group A), one mutation of CYP21A2 (group B), and no mutation of CYP21A2 (group C). Results: Mutation was detected in each allele of CYP21A2 gene in 401 (78.0%) patients, ninety (17.5%) had only one mutant allele and 23 (4.5%) had no mutation. There was no significant difference between the patients with different clinical phenotypes and the number of CYP21A2 gene mutations detected. In male, the cortisol of the patients with simple virilizing 21-OHD in group A [0.04 (0.02, 0.20) nmol/L] was lower than that of group B [0.24 (0.17, 0.28) nmol/L] and the difference was statistically significant (P=0.014). In female, 17-hydroxyprogesterone (17-OHP) of patients with salt wasting 21-OHD in group A [153.7 (90.1, 204.5) nmol/L] was higher than that of group B [38.2 (31.0, 183.3) nmol/L] and C [42.6 (27.8, 48.1) nmol/L] and the differences were statistically significant (both P<0.05). The progesterone of patients with simple virilizing 21-OHD in group C [23.0 (8.6, 33.2) nmol/L] was lower than that of gourp A [57.8 (34.4, 110.2) nmol/L] and B [63.6 (31.4, 110.8) nmol/L] and the difference were statistically significant (both P<0.05). The 17-OHP of patients with non-classical 21-OHD in group C [24.5 (20.4, 54.2) nmol/L] was lower than that of group A [158.7 (59.1, 187.6) nmol/L] and B [147.8 (131.9, 179.3) nmol/L]. The difference were statistically significant (both P<0.05). Conclusions: Mutations of two alleles have not been found in all patients with clinically diagnosed 21-OHD. Other congenital adrenal hyperplasia (CAH) types which can cause similar changes in 17-OHP and other hormones may be misdiagnosed as 21-OHD. Therefore, 21-OHD cannot be diagnosed with help of 17-OHP level only, and gene detection plays a vital role in the differential diagnosis of different CAH types.

[Analysis of copy number variation of CYP21A2 gene and the type of CYP21A1P/CYP21A2 fused gene in patients with 21-hydroxylase deficiency].

Objective: To analyze the copy number variation of CYP21A2 gene in 21-hydroxylase deficiency (21-OHD) patients, and identify the three copy repetition, single copy deletion of CYP21A2 gene and the type and proportion of CYP21A1P/CYP21A2 fused gene in 21-OHD patients. Methods: A total of 424 patients (140 males and 284 females) with 21-OHD who visited Peking Union Medical College Hospital from January 2015 to January 2018 were enrolled and the average age was (17.1±12.4) years. All clinical and biochemical data were collected. DNAs were extracted from peripheral blood leukocytes, and CYP21A2 gene mutation and copy number variation were detected by Sanger sequencing and multiple ligation probe amplification (MLPA). Results: Of 424 21-OHD patients, 287 (67.7%) had two copies of CYP21A2 gene, 137 (32.3%) had copy number variation, of which 1 patients (0.2%) had 3 copies of CYP21A2 gene and 136 (32.1%) were carriers of large deletion/rearrangement mutation of CYP21A2 gene. Three pathogenic mutations including a truncated Q319X protein mutation were detected in the patient with 3 copies of CYP21A2 gene. Of 136 patients with large deletion/rearrangement mutation of CYP21A2 gene, 82 (60.3%) carried fused CYP21A1P/CYP21A2 gene, and the remaining 54 harbored the one allele deletion of CYP21A2. The most common types of fused CYP21A1P/CYP21A2 gene were CH-5, CH-1 and CH-2, with the frequency being 31.7% (26 cases), 26.8% (22 cases) and 19.5% (16 cases), respectively, and followed by CH-4 and CH-7, with the incidence being 8.5% (7 cases) and 4.9% (4 cases), respectively. In addition, two cases of CH-3, CH-6 and CH-8 and one case of CH-9 were detected. Conclusions: This is the first study to detect the occurrence of CYP21A2 gene copy number variation and fused CYP21A1P/CYP21A2 gene in a large cohort of 21-OHD patients. The number of CYP21A2 gene copies in 21-OHD patients includes 2 copies, 1 copy deletion and 3 copies duplication. One copy deletion of CYP21A2 includes one allele deletion of CYP21A2 gene and fused CYP21A1P/CYP21A2 gene. In patients with 3 copies of CYP21A2 gene, pathogenic mutations should be verified in all 3 copies of CYP21A2 gene to make the precise diagnosis. Therefore, the accurate molecular diagnosis of 21-OHD patients should take both genotype and copy number variation of CYP21A2 into account.

Pancreatic ß-cell function is inhibited by miR-3666 in type 2 diabetes mellitus by targeting adiponectin.

Type 2 diabetes mellitus (T2D) is a common endocrine and metabolic disorder, and poses threats to human health worldwide. Recently, microRNAs (miRNAs) have been suggested to play important roles in the pathophysiology of T2D. In this study, we explored the role of miR-3666 in T2D. miR-3666 was significantly down-regulated in the serum of T2D patients when compared to that of healthy volunteers, and miR-3666 expression level was negatively correlated with blood glucose levels of T2D patients. Overexpression of miR-3666 inhibited cell proliferation, reduced insulin secretion, and promoted cell apoptosis of pancreatic ß-cell line (INS-1 cells). On the other hand, knockdown of miR-3666 had the opposite effects in INS-1 cells. The bio-informatics analysis using TargetScan revealed that adiponectin (ADIPOQ) was a downstream target of miR-3666, and the interaction between miR-3666 and ADIPOQ was validated by luciferase reporter assay. In addition, miR-3666 negatively regulated the mRNA and protein expression of ADIPOQ. Overexpression of ADIPOQ promoted insulin secretion after glucose stimulation, promoted cell proliferation, inhibited cell apoptosis, and partially abolished the effects of miR-3666 overexpression on insulin secretion, cell proliferation, and cell apoptosis of INS-1 cells. In conclusion, our results revealed that miR-3666 inhibited pancreatic cell proliferation, reduced insulin sensitivity, and promoted apoptosis by targeting ADIPOQ.

[Molecular and clinical study on homozygous or heterozygous large deletion of CYP21A2 gene in 21-OHD patients].

Objective: To explore the relationship between homozygous or heterozygous large deletion of CYP21A2 gene and clinical manifestation in 21-hydroxylase deficiency (21-OHD) patients. Methods: The records of 100 patients with 21-OHD were collected between June 2016 and December 2017 in Peking Union Medical College Hospital. Large deletion of CYP21A2 gene was detected by multiplex ligation probe amplification (MLPA). The biochemical results and clinical symptoms of patients with homozygous or heterozygous large deletion were analyzed in order to investigate the influence of complete or single allele deletion of CYP21A2 gene on 21-OHD patients. Results: Large deletion of CYP21A2 gene was detected in 33 patients by MLPA, including 13 males and 20 females, with an median age of 10 (6,16) years. Two of them had simultaneous deletions of two alleles. Among 31 patients with heterozygous deletion, 16 were combined with gene mutations that severely affected 21-hydroxylase enzyme activity (I2G and Q318X), 15 with mutations that retained part enzyme activity (I172N and P30L). Two patients with complete deletion of CYP21A2 gene had no significant difference in biochemical and clinical manifestations compared with those with single allele deletion combined with another gene mutation that severely affected enzyme activity. Both kinds of patients above were manifested as severe salt-wasting type. Patients with a single allele deletion and a mutation retaining part enzyme activity were manifested as mild simple-viralizing type. Conclusion: Large deletion of CYP21A2 gene could appear in 21-OHD patients and the phenotype is similar to that of salt-wasting patients with heterozygous large deletion.

[Diagnostic value of multiplex ligation dependent probe amplification combined with Sanger sequencing in 21-hydroxylase deficiency].

Objective: To study the procedure of CYP21A2 gene mutation detection in 21-hydroxylase deficiency (21-OHD) patients. Methods: The detail clinical and biochemical data of 51 patients with 21-OHD [18 males and 33 females, with an average age of (16.4±9.9) years] were collected between December 2016 and December 2017 at Department of Endocrinology, Peking Union Medical College Hospital. Multiplex ligation dependent probe amplification (MLPA) and Sanger sequencing of the CYP21A2 gene were used to clarify the cause of 21-OHD. The genotype-phenotype correlation was also analyzed. Results: The incidences of large deletion, 8 bp deletion, I2G, I172N and F306+T were 19.6% (20/102), 1.0% (1/102), 30.4% (31/102), 25.5% (26/102) and 1.0%(1/102), respectively, and the detection rate of gene mutation in 51 21-OHD patients was 77.5% (79/102) by MLPA test. Except large and 8 bp deletion, all above mutations contained in MLPA and other 8 mutations, including P31L, Q319X, R361L, R357W, V282L, R484Q, G425S and R342W were detected, and the detection rate was 79.4% (81/102) by Sanger sequencing of CYP21A2. MLPA combined with direct sequencing identified mutations in all patients. Genotype correlated well with clinical phenotype in 21-OHD patients. Conclusions: When MLPA or CYP21A2 gene sequencing were used alone to diagnose the cause of 21-OHD, gene mutations in all patients could not be detected. The combination of the two methods can complement each other and fully clarify the underlying causes of 21-OHD.