Objective To discover the mutations of rare thalassemia genes by sequencing of α and β-globin genes,to understand the frequency of rare mutations and to enrich thalassemia gene mutation spectrum in Chinese population.Methods For the cases of phenotype and genotype inconsistent,the 1st generation of sequencing was performed for α or β-globin gene coding region sequence analysis.Results A total of 102 patients with rare thalassemia gene mutations were found by sequencing,including 79 cases of β-thalassemia with 35 kinds of mutant types,and 23 cases of α-thalassemia with 11 kinds of mutant types.Conclusion The thalassemia gene sequencing could reveal rare mutations in genes,identify the genotype of patients,provide important support for prenatal diagnosis of rare thalassemia families,and reduce the missing rate and birth rate of children with thalassemia.

ObjectivesTo explore the roles of mean corpuscular volume(MCV),mean corpuscular hemoglobin(MCH) and hemoglobin A2 (HbA2) in the laboratory screening of thalassemia,and to find optimal screening modality for different conditions.Methods From September 2008 to May 2011,1384 subjects underwent thalassemia screening at Department of Obstertrics andGynecology of Nanfang Hospital.Of them,1036 cases were diagnosed with thalassemia (408 α-thalassemia,608 β-thalassemia,and 20 αβ compound thalassemia,thalassemia group) and 348 without thalassemia,non-thalassemia group.All subjects were screened respectively for MCV,MCH and HbA2.Analyses were performed in all subjects to assess the sensitivity,specificity,positive predictive value,negative predictive value and diagnostic accuracy respectively associated with MCV,MCH and HbA2 alone,combination of MCV and MCH,and combination of MCV,MCH and HbA2.Results( 1 ) In the thalassemia group,the sensitivity of MCV alone was 92.9％ (379/408) for α thalassemia,99.3％ (604/608) for β thalassemia and 100.0％(20/20) for αβ compound thalassemia.In the non-thalassemia group,the specificity of MCV alone was 75.0％ (261/348).(2) In the thalassemia group,the sensitivity of MCH alone was 92.9％ (379/408) in α thalassemia,99.0％ (602/608) in β thalassemia and 100.0％ (20/20) in αβ compound thalassemia.In the non-thalassemia group,the specificity of MCH alone was 72.7 ％ (253/348).(3) The sensitivity of Hb A2 alone was 67.4％ (275/408) for α thalassemia,97.5％ (593/608) for 3 thalassemia,and 100％ (20/20) for α3 compound thalassemia while it's specificity was 72.4％ (252/348) in the non-thalassemia group.(4)With positive indexes of MCV,MCH and MCV + MCH,when HbA2 ＞ 3.5％ it had a high value in [β-thalassemia screening,but when HbA2 ＜ 2.5％ it had little value in α-thalassemia screening.(5) As a single marker,MCV and MCH had better sensitivity,specificity,positive predictive value,negative predictive value and diagnosis accuracy than HbA2.MCV + MCH was the best for overall screening,but for [β thalassemia screening,MCV + MCH + HbA2 was the best.ConclusionsMCV and MCH are suitable for epidemic screening in a large population,physical examination and premarital check-up.Hb electrophoresis andthalassemiagenediagnosisarerecommendedforsubjectswithpositiveMCVandMCH indexes.Diagnoses of α and β-thalassemia gene are recommended for pregnant women with positive MCV and MCH indexes.

Objective: To study the pharmacokinetics and brain/plasma concentration ratio of nortriptyline at multiple doses in mice which were pre-treated with physiological saline, piperine and verapamil. Methods: A total of 216 male Kun Ming mice[(25±3) g] were equally divided into 4 groups randomly. Each group was intragastrically administered physiological saline (B), piperine (170 μg/kg), piperine (5 mg/kg) and verapamil (5 mg/kg) for 8 days. On the 8th day, 1 h atfer giving the above drugs, each mice was intraperitoneally injected nortriptyline (13 mg/kg). The mice were sacriifced by picking off eyeballs at the time intervals of 5, 15, 30 min, and 1, 2, 4, 6, 8 and 12 h, andthe cerebra were collected and weighted. Nortriptyline in mouse plasma and brain was determined by HPLC-MS/MS. The pharmacokinetic properties of the plasma, brain and brain/plasma were calculated. Results: hTe AUC0-12 h of brain/plasma concentration ratio in the 170 μg/kg piperine group was significantly lower than that in the other groups (P<0.05), while the AUC0-12 h of brain/plasma concentration ratios in the 5 mg/kg piperine group and the verapamil group were not signiifcantly different from those of untreated mice. Conclusion: Piperine (170 μg/kg) may induce P-glycoprotein expression in the blood-brain barrier, while piperines at 5 mg/kg has no influence on P-glycoprotein expression in the blood-brain barrier.

This study is to screen 23 blank O/W type microemulsion (ME) samples, that is 15 samples from our laboratory, and 8 samples from literature; compare the conductivity-water content curve (CWCC) method and visual method in determining the critical water content during O/W type MEs' formation, to analyze the deficiency and the feasibility of visual method and to exploxe scientific meanings of CWCC method in judging the critical water content of O/W type MEs during formation. The results show that there is a significant difference between the theoretical feasible CWCC method and visual method in determining the critical water content (P<0.001), and the results judged by conductivity is higher than that by eye-based water content. Therefore, this article firmly confirmed the shortcomings of visual method and suggested that the eye-base "critical water content" may falls into continuous ME stage during O/W MEs' formation. Further more, the CWCC method has theoretical feasibility and scientific meanings in determining the critical water content of O/W type MEs during formation.

Objective To summarize the geographical distribution,phenotype and genotype data of 206 thalassemia families underwent prenatal diagnosis to provide information for clinical genetic counseling and avoid the birth of severe thalassemia children.MethodsTotally,206 thalassemia families were collected from Southern Medical University Nanfang Hospital from January 2008 to December 2009.Genomic DNA was extracted from peripheral blood,villus,amniotic fluid or cord blood from the couples or the fetuses.Gap-polymerase chain reaction (gap-PCR) and reverse dot blot (RDB) technology were used to detect the common α and β-thalassemia mutations.DNA sequencing was used to detect the rare mutations.Follow-up visit were done half a year after the fetuses were born. Results The 206 thalassemia families came from 12 provinces and areas across China,including Heilongjiang province.Mutations detected in α-thalassemia families included --SEA/,-α3.7/,-α4.2/,αCS α/ and αQS α/,which were all included in the testing kit. While there were 4 kinds of β-thalassemia mutations,Gγ+ (A γδβ)0,-28(A→C),CD54-58(-TATGGGCAACCCT) and CD37(G→A),could not be identified with routine testing kit. The 57 α-thalassemia families consisted of 11(19.3％) severe thalassemia,induding 8 Bart's hydrops syndrome and 3 Hb H disease,26(45.6％) heterozygote and 20(35.1％) normal infants,and the 149 β-thalassemia majors families consisted of 28 (18.8％) severe thalassemia,82(55.0％) heterozygote and 39 (26.2％) normal infants.Among the β-thalassemia heterozygotes,there was one 13-trisomy.Follow-up visit found that babies with Bart ' s hydrops syndrome (n =8),Hb H disease (n =3),β-thalassemia majors (n =28) and β thalassemia heterozygote combined with 13-trisomy(n=1) were aborted.Conclusions Thalassemia was found in some north area other than south of China,which should be paid more attention by clinicians.Gap-PCR and PCR-RDB technology are effective measures for thalassemia prenatal diagnosis in identifying major thalassemia fetuses before their birth,thus reduce the birth rate of thalassemia baby.But missed diagnosis might exist during the screening,so it is necessary to perform DNA sequencing on those patients with positive symptoms and negative common genetic diagnostic results.At the same time,prenatal diagnosis of chromosomal disorders should not be neglected for high-risk families.

OBJECTIVETo detect copy number changes of α-globin gene, and analyze molecular mechanism of the impacts of fetal hemoglobin (HbF) levels for α-globin gene copy numbers loss or increase.

METHODSA total of 15 cases with combined increased levels of fetal hemoglobin with β-thalassemia were collected. Firstly, three common α-thalassemia deletions were validated by Gap-PCR. Secondly, the largest deletions of the β-globin gene cluster were detected by multiplex ligation-dependent probe amplification (MLPA).

RESULTSAmong the 15 cases, there was 1 case with duplication of the α-globin gene cluster, 3 cases of SEA heterozygote deletion of the α-globin gene, 1 cases of α 3.7 deletion heterozygote of the α-globin gene, 1 case of alpha 4.2 deletion homozygote of the α-globin gene, 1 case of deletion homozygote in the like α-globin gene. A compound heterozygous for SEA and α 3.7 of the α-globin gene was also detected. However, 7 cases showed no copy numbers loss and increase of the the α-globin gene cluster.

CONCLUSIONAdditional α-globin gene can produce excessive α-chain, which can aggravate imbalance for α and β-chain, and cause clinical symptoms in patients with β-thalassemia. Yet, copy number loss or mutation in α-globin gene will cause a milder clinical phenotype.

Adult ; Asian Continental Ancestry Group ; genetics ; China ; DNA Copy Number Variations ; Female ; Fetal Hemoglobin ; metabolism ; Humans ; Infant ; Male ; Mutation ; Pedigree ; alpha-Globins ; genetics ; beta-Thalassemia ; genetics ; metabolism

Objective To investigate pregnancy outcomes and neurodevelopment in fetuses with ventriculomegaly. Methods This was a cohort study of 173 gravidas with singleton pregnancy who were diagnosed with fetal ventriculomegaly by ultrasound in Prenatal Diagnostic Center of Nanfang Hospital Affiliated to Southern Medical University from March 2010 to July 2016. Thirty normal gravidas who received antenatal care in the same hospital and at the same period were selected as control. Clinical data were collected. Gravidas who had chosen to continue their pregnancy were followed up to monitor the variations of fetal ventricular. Fetal mild and moderate ventriculomegaly were respectively defined as a ventricular atrial width of 10-12 mm and >12 mm but <15 mm. Isolated ventriculomegaly (IVM) indicated those without any other ultrasound abnormalities, otherwise the case would be defined as non-isolated ventriculomegaly (NIVM). Among the 173 gravidas, 54 cases were mild IVM, 53 mild NIVM, 26 moderate IVM and 40 moderate NIVM. Fetuses with chromosome abnormalities were excluded from the study. Neonatal behavioral neurological assessment (NBNA) was used to analyze the neonatal neurodevelopment at the age of 7 days, and Bayley scales of infant development was used to evaluate the development of nervous system at the age of 6 months through analyzing their mental development index (MDI) and psychomotor development index (PDI). Statistical methods included t test, χ2 test (or Fisher's exact test), nonparametric test, Mann-Whitney test and multiple Kruskal-Wallis H test. Results (1) Among the 107 fetuses with mild ventriculomegaly, 72.9％ (78), 23.4％ (25) and 3.7％ (4) of them regressed, stabilized and progressed,respectively; however, among the 66 moderate cases, the figures were 45.4％ (30), 37.9％ (25) and 16.7％ (11) respectively (χ2=15.769, P<0.001). For those in the IVM and NIVM subgroups within the moderate ventriculomegaly group, significant difference was shown [17(65.4％), 8(30.8％) and 1(3.8％) vs 13(32.5％), 17(42.5％) and 10(25.0％), χ2=8.552, P=0.014], but not within the mild groups (χ2=2.412, P=0.299). (2) There were 164 gravidas who continued their pregnancy and delivered. Significant differences in NBNA score were observed between the ventriculomegaly group and the control (37.70±1.80 vs 38.53±1.38, t= - 2.424, P<0.05). Numbers of neonates with NBNA score < 36 and ≥ 36 points were 5(4.7％) and 101(95.3％) in the mild group, and 8(13.8％) and 50(86.2％) in the moderate group (χ2=4.231, P=0.004). There was significant difference in NBNA score between the IVM and NIVM subgroup within neither mild nor moderate group (χ2 were 0.210 and 0.201, P were 1.000 and 0.720). (3) Totally, 137 cases completed the assessment of nervous system development at the age of 6 months. There was significant difference in PDI score between the ventriculomegaly group and the control (90.50±10.85 vs 95.80±9.65, t= - 2.471, P=0.014), but not in MDI score (95.42+11.20 vs 99.50+12.00, t= - 1.786, P=0.076). (4) The comparison of the proportion of excellent, average and poor PDI scores: Significant differences were found between the IVM and NIVM subgroup within the moderate ventriculamegaly group and in the different intrauterine outcome groups [IVM vs NIVM groups: 3(15.0％), 16(80.0％) and 1(5.0％) vs 1(3.1％), 24(75.0％) and 7(21.9％),Z= - 2.097, P=0.036;intrauterine regression, stable and progress group: 9(10.6％), 75(88.2％) and 1(1.2％);3(6.5％), 37(80.4％) and 6(13.1％) vs 0, 2(2/6) and 4(4/6), χ2=19.808, P<0.001], but not between the mild and moderate vetriculamegaly group, or between the subgroups within the mild ones (Z were - 1.869 and - 1.946, P were 0.062 and 0.052). (5) The comparison of the proportion of excellent, average and poor scores of MDI: Significant difference was only found among the different intrauterine outcome groups[13(15.3％), 71(83.5％), 1(1.2％); 2(4.4％), 41(89.1％), 3(6.5％) vs 0, 5(5/6), 1(1/6); χ2=7.980, P=0.018], but not in any other comparisons (all P>0.05). Conclusions Prognosis of fetal ventriculomegaly is affected by co-existed abnormalities and intrauterine progression. Fetus with mild ventriculomegaly can also have risk of abnormal neural development, suggesting that we should pay much attention to such cases and a regular follow-up is required.