Application of real-time PCR-based multicolor melting curve with automatic analysis system in pregestational and prenatal thalassemia diagnoses.

BACKGROUND: To evaluate the value of the real-time PCR-based multicolor melting curve analysis (MMCA) with an automatic analysis system used in a mass thalassemia screening and prenatal diagnosis program. METHODS: A total of 18,912 peripheral blood samples from 9456 couples and 1150 prenatal samples were detected by MMCA assay. All prenatal samples were also tested by a conventional method. Samples with unknown melting peaks, unusual peak height ratios between a wild allele and a mutant allele, or a discordant phenotype-genotype match were further studied by using multiplex ligation-dependent probe amplification (MLPA) or Sanger sequencing. All MMCA results were automatically analyzed and manually checked. The consistency between MMCA assay and conventional methods among prenatal samples was investigated. RESULTS: Except for initiation codon (T > G) (HBB:c.2T > G), all genotypes of thalassemia inside the scope of conventional methods were detected by MMCA assay. Additionally, 27 carriers with 10 rare HBB variants, 13 with α fusion gene, 1 with a rare deletion in α globin gene, and 1 with rare HBA variant were detected by using MMCA assay. CONCLUSION: MMCA can be an alternative approach used in routine thalassemia carrier screening and prenatal diagnosis for its high throughput, sufficient stability, low cost, and easy operation.

Hb Lepore-Hong Kong: First Report of a Novel δ/ß-Globin Gene Fusion in a Chinese Family.

We describe a new δ/ß fusion gene causing ß-thalassemia (ß-thal) trait and its formation mechanism. The proband was a 39-year-old woman who presented with persistent microcytic microcytosis without iron deficiency. Molecular diagnoses revealed a 뫧 configuration within a 54 bp region between the Cap site (+22) and codon 8, causing a deletion (NG_000007.3: g.63154_70565del). This results in a variant that has been named Hb Lepore-Hong Kong and shows a decreased ß-globin mRNA in carriers compared to that of normal subjects. It is assumed that combination of this variant with ß-thal may cause severe ß-thal syndrome.

Hb Westmead (HBA2: c.369C>G): Hematological Characteristics in Heterozygotes with and without α0-Thalassemia.

Hb Westmead (α122(H5)His>Gln) (HBA2: c.369C>G) is a common α-globin variant causing α-thalassemia (α-thal) in Mainland China. In this study, we report the hematological characteristics in Hb Westmead carriers in a Chinese population. There were 546 individuals carrying Hb Westmead based on their molecular diagnosis: 514 Hb Westmead heterozygotes and 32 compound heterozygotes for Hb Westmead and α0-thal. Compared to common deletional α+-thal, Hb Westmead was associated with higher mean corpuscular hemoglobin (Hb) (MCH) values. Compound heterozygotes for Hb Westmead and α0-thal showed significantly higher Hb, mean corpuscular volume (MCV) and MCH values than subjects with deletional Hb H disease. When compared to α0-thal carriers, compound heterozygotes for Hb Westmead and α0-thal showed similar Hb values, but significantly lower MCV and MCH values. Our results indicate that Hb Westmead is a silent nondeletional α+-thal, with a deficiency of α-globin chain milder than deletional α+-thal, and compound heterozygotes for Hb Westmead/α0-thal have a phenotype similar to simple α0-thal.

Hematological Characteristics of Hb Constant Spring (HBA2: c.427T>C) Carriers in Mainland China.

Hb Constant Spring (Hb CS) (HBA2: c.427T>C) is a common α-globin variant causing α-thalassemia (α-thal) phenotypes in mainland China. In this study, we evaluated the efficiency of erythrocyte parameters and capillary electrophoresis (CE) in the determination of Hb CS in blood samples from Hb CS carriers. Based on molecular diagnosis, there were 462 patients carrying Hb CS: 411 Hb CS heterozygotes, seven carried Hb H-Hb CS disease, 18 compound heterozygotes for Hb CS/α+-thal, and 26 double heterozygotes for Hb CS and ß-thalassemia (ß-thal). Forty-three cases had no Hb CS peak visible on CE, including all 26 cases of double heterozygotes for Hb CS and ß-thal, and 17 cases of heterozygotes carrying only Hb CS. Hb CS heterozygotes, those without a Hb CS peak, presented with lower hemoglobin (Hb), mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) values than those with a Hb CS peak. The MCV <80.0 fL yielded a detection rate of 87.8% for screening individuals carrying Hb CS. Therefore, we emphasize that if one partner of a couple has tested positive for α0-thal, the other should be subjected to detailed screening for this nondeletional allele using molecular analysis, regardless of his/her red cell indices and electrophoretic chromatogram.

[Correlation of age, body mass index and obesity-related biochemical indexes with semen quality in males intending for a second child].

OBJECTIVE: To explore the influence of age, body mass index (BMI) and obesity-related biochemical indexes on semen quality in adult males intending to have a second child in Guangzhou. METHODS: We conducted a questionnaire investigation among 632 adult males seeking medical advice on their intention for a second child at Guangzhou Women and Children's Medical Center from August 2017 to July 2018. We obtained their lipid metabolism indicators and semen parameters, and analyzed the correlation of semen quality with age, BMI, obesity-related biochemical indexes, living environment and occupation. RESULTS: Age, BMI, season of sperm extraction, living environment and occupation all influenced the semen quality of the males. Age was correlated negatively with the percentage of progressively motile sperm (PMS) (r = －0.109, P < 0.05), BMI positively with the semen volume (r = 0.103, P < 0.05) but negatively with the percentage of morphologically normal sperm (MNS) (r = －0.138, P < 0.05), high-density lipoprotein (HDL) negatively with PMS (r = －0.168, P < 0.01) and the percentage of immotile sperm (IM) (r = －0.135, P < 0.05), low-density lipoprotein (LDL) negatively with the semen volume (r = －0.124, P < 0.01), PMS (r = －0.127, P < 0.05), sperm concentration (r = －0.121, P < 0.05) and total sperm count (r = －0.210, P < 0.01) but positively with IM (r = 0.140, P < 0.01). Multivariate regression analysis showed BMI and LDL to be independent factors influencing the semen volume, uric acid to be an independent factor influencing semen liquefaction time, age, HDL and LDL to be independent factors influencing PMS, age, and HDL to be independent factors influencing IM, LDL to be independent factors influencing total sperm count, while BMI and TG to be independent factors influencing MNS. CONCLUSIONS: Age, BMI, season of sperm extraction, living environment and occupation may affect the semen quality of the males in Guangzhou.

KFL1 Gene Variants in α-Thalassemia Individuals with Increased Fetal Hemoglobin in a Chinese Population.

Krüppel-like factor 1 (KLF1) is a pleiotropic erythroid transcription factor that is a regulator of definitive erythropoiesis. The aim of this study was to detect KLF1 gene variants in α-thalassemia (α-thal) carriers with an increased Hb F level in a Chinese population, and determine the changes of hematological parameters as a result of interactions between KLF1 gene mutations and α-thal. Subjects with α-thal and Hb F levels of ≥1.0% were selected for further investigation. Direct sequencing was used to detect KLF1 gene mutations. Hematological parameters of subjects with α-thal and concomitant KLF1 gene mutations and those with α-thal alone were compared. The KLF1 gene variants were detected in 46 of 275 (16.7%) individuals with α-thal and Hb F levels of ≥1.0%. The detection rate of KLF1 gene mutations rose correspondingly when the Hb F level increased. For α0-thal carriers, significantly lower mean corpuscular volume (MCV) and mean corpuscular hemoglobin (Hb) (MCH) values were observed in KLF1 gene mutation-positive carriers than that in KLF1 gene mutation-free carriers; conversely, significantly higher Hb A2 and Hb F levels were observed in the former condition rather than in the latter condition. The results of this study indicate that KLF1 gene variants are common in Chinese subjects with α-thal and increased Hb F levels, and KLF1 gene mutations decreased the red blood cell (RBC) indices in α-thal carriers as that in normal adults.

Detection of rare thalassemia mutations using long-read single-molecule real-time sequencing.

Gap- polymerase chain reaction (PCR), reverse dot-blot assay (RDB), real-time PCR based multicolor melting curve analysis (MMCA assay), multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing are conventional methods to diagnose thalassemia but all of them have limitations. In this study, we applied single-molecule real-time (SMRT) sequencing following multiplex long-range PCR to uncover rare mutations in nine patients and their family members. The patients with different results between Gap-PCR and MMCA assay or with phenotype not matching genotype were included. Using SMRT sequencing, we first identified the carriers with αααanti3.7/HKαα, -α762bpα/αα (chr16:172,648-173,409), ααfusion/αQSα (in a trans configuration), two cases with novel gene rearrangements and another case with a novel 341 bp insertion in α-globin gene cluster, respectively. One carrier with --SEA/αααanti4.2, and two carriers with the coexistence of globin variant and an α-globin gene duplication were also found. Most importantly, we could determine two defects in α-globin gene cluster being a cis or trans configuration in a single test. Our results showed that SMRT has great advantages in detection of α-globin gene triplications, rare deletions and determination of a cis or trans configuration. SMRT is a comprehensive and one-step method for thalassemia screening and diagnosis, especially for detection of rare thalassemia mutations.

Prevalence of late-onset hypogonadism among middle-aged and elderly males in China: results from a national survey.

This study aimed to propose an operational definition of late-onset hypogonadism (LOH) that incorporates both clinical symptoms and serum testosterone measurements to evaluate the prevalence of LOH in aging males in China. A population-based sample of 6296 men aged 40 years-79 years old was enrolled from six representative provinces in China. Serum total testosterone (TT), sex hormone-binding globulin (SHBG), and luteinizing hormone (LH) were measured and free testosterone (cFT) was calculated. The Aging Males' Symptoms (AMS) scale was used to evaluate the LOH symptoms. Finally, 5078 men were included in this analysis. The TT levels did not decrease with age (P = 0.59), and had no relationship with AMS symptoms (P = 0.87 for AMS total score, P = 0.74 for ≥ 3 sexual symptoms). The cFT levels decreased significantly with age (P < 0.01) and showed a negative association with the presence of ≥ 3 sexual symptoms (P = 0.03). The overall estimated prevalence of LOH was 7.8% (395/5078) if a cFT level <210 pmol l-1 combined with the presence of ≥ 3 sexual symptoms was used as the criterion of LOH. Among them, 26.1% (103/395) and 73.9% (292/395) had primary and secondary hypogonadism, respectively. After adjustment for confounding factors, primary and secondary hypogonadism was positively related to age and comorbidities. Body mass index was an independent risk factor for secondary hypogonadism. The results suggest that the AMS total score is not an appropriate indicator for decreased testosterone, and that the cFT level is more reliable than TT for LOH diagnosis. Secondary hypogonadism is the most common form of LOH.

[Screening Abnormal Hemoglobin Diseases for Couples of Childbearing Age in Guangzhou City by HPLC].

OBJECTIVE: To investigate the incidence and common types of abnormal hemoglobin diseases of the couples at childbearing age by using high performance liquid chromatography (HPLC) in Guangzhou city. METHODS: The couple of childbearing age in the Birth defect intervention project and Free pre pregnancy health examination were screened from October 2008 to February 2016 in Guangzhou city. The HPLC was used to detect abnormal hemoglobin; The Gap-PCR and reverse dot blot(RDB) were used to detect thalassemia gene deletions. RESULTS: The detection rate of hemoglobinopathies was 1.14% in the couples of childbearing age in Guangzhou city. 8 kinds of abnormal hemoglobin were detected: Hb E in 102 cases(17 cases with α thalassemia) , Hb Q-Thailand in 20 cases(18 cases with α thalassemia, 2 cases with α and ß thalassemia), Hb D-Iran in 4 cases(2 cases with α thalassemia, 1 case with α and ß thalassemia), Hb G-Honolulu in 3 cases(1 case with α thalassemia), Hb J-Bangkok in 2 cases(2 cases with α thalassemia), Hb Osu-Christiansborg in 1 case(with α thalassemia), Hb Hasharon in 1 case(with α thalassemia), Hb Koln in 1 case(with α thalassemia). CONCLUSION: The incidence of abnormal hemoglobin diseases in Guangzhou area has been found to be more high, 8 kinds of abnormal hemoglobin are found by HPLC, the HPLC is an effective way to screen the couples of childbearing age for ß thalassemia and hemoglobinopathies.

[Experimental study on treatment of endometriosis with xiaochaihu decoction].

OBJECTIVE: To explore the effect of Xiaochaihu Decoction (XCHD) on ectopic endometrium in rats with endometriosis and its mechanism. METHODS: The rat model of endometriosis was established and rats were divided into 5 groups, the low dosage of XCHD group (5 g x kg(-1) x d(-1) , n = 10) , the medium dosage of XCHD group (10 g x kg(-1) x d(-1), n =10), the high dosage of XCHD group (15 g x kg(-1) x d(-1), n = 10) , the danazol group(0. 1 g x kg(-1) x d(-1), n =9) and the control group(20 ml x kg(-1) x d(-1) distilled water, n = 10). After treated for four successive weeks, the effect of XCHD on ectopic endometrium was evaluated by observing the changes in transmission electron microscopy. Fas and Caspase-3 protein expression in endometrium and endometriotic tissue were observed by immunohistochemistry method. RESULTS: Ectopic endometrial growth was markedly inhibited (P < 0. 05 or P < 0. 01) in all the XCHD groups; Fas and Caspase-3 protein expressions of ectopic endometrial tissue were higher than those in the endometrium. CONCLUSION: XCHD could inhibit the growth of the ectopic endometrium in rats with experimentally induced endometriosis. The therapeutic mechanism may be related to promoting the apoptosis in ectopic endometrial tissue by increasing Fas protein expression.

Association of Alcohol Consumption with Markers of Prostate Health and Reproductive Hormone Profiles: A Multi-Center Study of 4,535 Men in China.

BACKGROUND: The effect of alcohol consumption on prostate health and reproductive hormone profiles has long been investigated and currently, no consensus has been reached. Additionally, large studies focusing on this topic are relatively rare in China. PURPOSE: To investigate the association of alcohol consumption with prostate measurements and reproductive hormone profiles in Chinese population; and to examine the relationship between hormone levels and prostate measurements. METHODS: This cross-sectional study included 4535 men from four representative provinces of China. Demographic details, family history of prostate disease, tobacco and alcohol consumption, as well as International Prostate Symptom Score (I-PSS) were collected through a questionnaire. Total prostate specific antingen (total PSA), free PSA, free PSA/total PSA ratio (f/tPSA), and reproductive hormones were measured in serum. Multi-variable regression models were used to test for association of alcohol consumption with markers of prostate health, used to test for association of alcohol consumption with reproductive hormones, and reproductive hormones with markers of prostate health. RESULTS: Alcohol consumption had no obvious impact on total PSA concentration and I-PSS. Current drinkers had lower level of free PSA (ß = -0.11, p = 0.02) and f/tPSA (ß = -0.03, p = 0.005), former drinkers also had lower level of free PSA (ß = -0.19, p = 0.02) when compared with never drinkers. Lower Luteinizing hormone (LH) (ß = -1.05, p = 0.01), sex hormone-binding globulin (SHBG) (ß = -4.71, p = 0.01) and higher estradiol (ß = 7.81, p = 0.01) was found in current drinkers than never drinkers, whereas higher LH (ß = 1.04, p = 0.04) and free testosterone (FT) (ß = 0.03, p = 0.02) was detected in former drinkers than never drinkers. Furthermore, LH was positively associated with f/tPSA (ß = 0.002, p = 0.006), SHBG was also positively related with free PSA (ß = 0.003, p = 0.003) and f/tPSA (ß = 0.0004, p = 0.01). Both total testosterone (TT) and FT were inversely related with I-PSS (OR = 0.97, 95% CI, 0.95-0.98; OR = 0.23, 95% CI, 0.11-0.45, respectively). CONCLUSIONS: Alcohol consumption could affect serum free PSA concentration and also f/tPSA ratio, and also acts as an endocrine disruptor on the male reproductive hormone profiles. LH and SHBG were positively related with fPSA and f/tPSA, and higher level of TT and FT may be helpful for improving participants' subjective symptoms.

[Effects of Xiaochaihu Tang, a traditional Chinese medicinal preparation, on ectopic endometrium in rats].

OBJECTIVE: To investigate the effects of Xiaochaihu Tang (XCHT), a traditional Chinese medicinal preparation, on ectopic endometrium in rats. METHODS: In rat models of endometriosis, the effect of XCHT of ultrastructure of the ectopic endometrium was observed by transmission electron microscopy. RESULTS: After treatment with XCHT at moderate (500 g/L) or high dose (750 g/L), ectopic endometrial growth was significantly inhibited (P<0.01); small-dose XCHT (250 g/L) was less effective (P<0.05). The volume of endometrium implant in the control group remained unchanged (P>0.05). The glandular cells in the endometrial implant after therapy with moderate- or high-dose XCHT showed characteristic features of apoptosis, presented by decreased cell size, karyopyknosis, cytoplasm and nuclear chromatin condensation, increased density and presence of apoptotic bodies. Some stromal cells displayed degenerative and necrotic changes. CONCLUSION: XCHT may inhibit the growth of ectopic endometrium by inducing cell death in the form of apoptosis and necrosis.