Two factors affecting the success rate of the second non-invasive prenatal screening after initial no-call result: experience from a single tertiary center in China / 中华医学杂志(英文版)

BACKGROUND@#One inevitable shortcoming of non-invasive prenatal screening (NIPS)/cell-free DNA (cfDNA) sequencing is the uninterpretable ("no-call") result, which is mainly caused by an insufficient fetal fraction. This study was performed to investigate the factors associated with a successful second NIPS in these cases and determine the optimal management for women with initial no-call results.@*METHODS@#We retrospectively analyzed the data of women who underwent NIPS with initial no-call results due to an insufficient fetal fraction from 2017 to 2019 in our center. We compared these women's maternal and pregnancy information with the data of women who had attained a successful second NIPS result and women who had received no-call results for a second time.@*RESULTS@#Among the 33,684 women who underwent NIPS, 137 with a no-call result underwent a retest. Comparison between the 87 (63.50%) women with a successful retest and the other 50 (36.50%) women showed a significant difference in both the initial fetal fraction and maternal body mass index (BMI), whereas the other factors showed no significant differences. In addition, with an initial fetal fraction of < 2.00%, the retest success rate was very limited.@*CONCLUSIONS@#We identified two major factors associated with a successful NIPS retest: the initial fetal fraction and the maternal BMI. These findings suggest the need for specialized management for this subset of women and would be instructional for the counseling for these women.

Rapid screening for Klinefelter syndrome with a simple high-resolution melting assay: a multicenter study / 亚洲男科学杂志(英文版)

Klinefelter syndrome (KS) is the set of symptoms that result from the presence of an extra X chromosome in males. Postnatal population-based KS screening will enable timely diagnosis of this common chromosomal disease, providing the opportunity for early intervention and therapy at the time point when they are most effective and may prevent later symptoms or complications. Therefore, through this study, we introduced a simple high-resolution melting (HRM) assay for KS screening and evaluated its clinical sensitivity and specificity in three medical centers using 1373 clinical blood samples. The HRM assay utilized a single primer pair to simultaneously amplify specific regions in zinc finger protein, X-linked (ZFX) and zinc finger protein, Y-linked (ZFY). In cases of KS, the ratios of ZFX/ZFY are altered compared to those in normal males. As a result, the specific melting profiles differ and can be differentiated during data analysis. This HRM assay displayed high analytical specificity over a wide range of template DNA amounts (5 ng-50 ng) and reproducibility, high resolution for detecting KS mosaicism, and high clinical sensitivity (100%) and specificity (98.1%). Moreover, the HRM assay was rapid (2 h per run), inexpensive (0.2 USD per sample), easy to perform and automatic, and compatible with both whole blood samples and dried blood spots. Therefore, this HRM assay is an ideal postnatal population-based KS screening tool that can be used for different age groups.

Molecular diagnosis of OTC gene mutation in a Chinese family with ornithine transcarbamylase deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect potential mutations of OTC gene in a male infant affected with ornithine transcarbamylase deficiency.</p><p><b>METHODS</b>Genomic DNA were isolated from peripheral blood samples of family members and 100 healthy individuals. Potential mutations of the 10 exons of OTC gene were screened with PCR and Sanger sequencing.</p><p><b>RESULTS</b>A homozygous missense mutation c.917G>C in exon 9, which results in p.R306T, was identified in the infant. Sequencing of the mother and two female members of the family indicated a heterozygous status for the same mutation. The same mutation was not found in other members of the family and 100 healthy controls.</p><p><b>CONCLUSION</b>A missense mutation c.917G>C in the OTC gene is responsible for the pathogenesis of the disease. Identification of the mutation can facilitate prenatal diagnosis and genetic counseling for the family.</p>

Mutational spectrum of phenylalanine hydroxylase gene and identification of novel mutations in patients with hyperphenylalaninemia in Jiangsu province / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the characteristics of phenylalanine hydroxylase (PAH) gene mutations in patients with hyperphenylalaninemia from Jiangsu province by DNA sequencing, and to analyze the spectrum of PAH gene mutations.</p><p><b>METHODS</b>A total of 70 patients and their parents were included in this study. All of the 13 exons and flanking introns of the PAH gene were analyzed with DNA sequencing.</p><p><b>RESULTS</b>Forty five types of mutations were identified, which included 4 novel mutations (L37P, H107R, Q267L, S391T). A total of 125 mutations were identified in 140 alleles (89.3%). All mutations were detected in exons 2-3, 5-7, 9-12 and introns 2, 4, 7 and 8. Most mutations were found in exons 6, 7 and 12. EX6-96A > G, R243Q and R241C were the most common mutations.</p><p><b>CONCLUSION</b>The mutational spectrum of Jiangsu province seems to be different from other regions. The spectrum can offer reliable information for genetic diagnosis of patients with hyperphenylalaninemia.</p>

Development and application of a method for molecular diagnosis of 21-hydroxylase deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a method for elucidating genetic basis of 21-hydroxylase deficiency.</p><p><b>METHODS</b>Sanger sequencing of entire 21-hydroxylase coding gene CYP21A2 was carried out to detect point mutations, and multiplex ligation-dependent probe amplification (MLPA) and locus-specific PCR/enzyme restriction method were used to detect large deletions and conversion mutations.</p><p><b>RESULTS</b>Nine children were analyzed. Point mutations of the CYP21A2 gene have been identified as: IVS2 13A/C>G (9 alleles), p.Arg356Trp (1 allele), Cluster E6 (1 allele), p.Gln318X (1 allele), and Prom conv (1 allele). While the former 4 mutations are pathogenic, the role of Prom conv mutation in the pathogenesis was uncertain. Three cases had entire CYP21A2 gene deletions (3 alleles), three had CYP21A1P/CYP21A2 chimeric mutations (3 alleles). The genotypes of all patients were determined. And all of the mutations were inherited from parents.</p><p><b>CONCLUSION</b>A rational method for detecting point mutations and large deletions/conversions of CYP21A2 gene has been established.</p>

Array-based comparative genomic hybridization detection of copy number variations in a fetus with hypoplastic left-heart syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect the copy number variations (CNVs) of a fetus with hypoplastic left-heart syndrome, and to assess the value of array-based comparative genomic hybridization (array-CGH) for molecular cytogenetic diagnosis.</p><p><b>METHODS</b>The whole genome of a fetus with normal karyotype by G-banding was scanned and analyzed by array-CGH, and the CNVs was confirmed by multiplex ligation-dependent probe amplification (MLPA).</p><p><b>RESULTS</b>Two submicroscopic CNVs [del(11)(q24.1-ter)(121951443-134449216, -12.50 Mb),dup(15)(q26.3)(96889082-100215359, -3.33 Mb)] were identified and mapped by array-CGH. MLPA test confirmed both CNVs.</p><p><b>CONCLUSION</b>Del (11) (q24.1-ter) may contribute to hypoplastic left-heart syndrome of the fetus. For its high-resolution and high-accuracy, array-CGH has provided a powerful tool for detection of genomic imbalance.</p>

Prenatal diagnosis of two fetuses with de novo small supernumerary markers by single nucleotide polymorphism array based comparative genomic hybridization / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the origins of small de novo mosaic supernumerary marker chromosomes (mars) in two fetuses, and to assess the feasibility of single nucleotide polymorphism array-based comparative genomic hybridization (SNP-array CGH) for prenatal molecular cytogenetic diagnosis.</p><p><b>METHODS</b>Two fetuses with de novo were identified. SNP-array marker chromosomes was applied to define the location and range of marker chromosomes. The karyotype of fetus I was determined to be 47,XX,+ mar[23]/46,XX[16], and that of fetus II was 47,XX,+ mar. Multiplex ligation-dependent probe amplification (MLPA) was applied to verify the genomic imbalance found in fetus II. The karyotypes of parents were normal in both families.</p><p><b>RESULTS</b>SNP-array CGH has indicated a 8.3 Mb duplication at 9p21.1-p21.3 in fetus I, and a 10.8 Mb duplication at 15q11.2-q13.3 in fetus II. MLPA has also confirmed a 15q terminal duplication in fetus II.</p><p><b>CONCLUSION</b>Above cases have illustrated that SNP-array CGH is a rapid, powerful and sensitive technique which may be used for identify the origins of marker chromosomes in prenatal diagnosis.</p>

Detection of 9p partial trisomy using array-based comparative genomic hybridization / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect chromosomal aberrations in a child with developmental delay and speech and language disorders in order to explore the underlying genetic causes of congenital malformation, and to investigate the feasibility of array-based comparative genomic hybridization (array-CGH) for molecular genetic diagnosis.</p><p><b>METHODS</b>G-banding and array-CGH were applied to characterize the genetic abnormality in the three family members.</p><p><b>RESULTS</b>G-banding analysis revealed the affected child and the healthy mother are both carriers of inv(9)(p13q13), while the child has carried a chromosome fragment derived from chromosome 13. Array-CGH analysis indicated the derivative chromosome fragment has originated from 9p with breakpoints at around 9p13.1-p24.3.</p><p><b>CONCLUSION</b>Trisomy 9p13.1-p24.3 may be the cause of congenital malformation in the child. For its high resolution and high accuracy, array-CGH is a powerful tool for genetic analysis.</p>

Cytogenetic analysis of a complex chromosomal imbalance 14q+ in a fetus featuring multiple congenital defects / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze chromosomal imbalance in a fetus presenting with congenital heart disease and mild lateral ventriculomegaly, and to investigate the correlation between genotype and phenotype. The etiology of the fetal congenital diseases was determined, and the feasibility of array-based comparative genomic hybridization (array-CGH) application in molecular cytogenetic diagnosis was evaluated.</p><p><b>METHODS</b>Following conventional G-banding analysis, array-based comparative genomic hybridization (array-CGH) was applied to delineate the precise location and size of genomic imbalance.</p><p><b>RESULTS</b>A de novo 46, XY, -14, +der14(q31)? karyotype was identified in the fetus by G-banding analysis. Array-CGH has verified the chromosomal imbalance to be 46, XY, -14, +der(12; 14) (p13; q32.33)del(14) (q32.33→ qter).</p><p><b>CONCLUSION</b>del(14)(q32.33→ qter) is probably the predominant cause of the fetal congenital disease. For its high resolution and accuracy, array-CGH has provided a powerful tool for prenatal diagnosis and genetic counseling.</p>

Second trimester screening for trisomy 21 using ADAM12-S as a maternal serum marker / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the value of a disintegrin and metalloproteinase 12 secreting form (ADAM12-S) as a maternal serum marker in second trimester screening for trisomy 21 (Down syndrome, DS), and to develop an appropriate prenatal DS screening protocol.</p><p><b>METHODS</b>Serum samples were collected from 53 pregnant women carrying a trisomy 21 fetus and 621 pregnant women with matched gestational age and weight carrying a healthy fetus. ADAM12-S concentrations were determined with a time-resolved fluorescence immunoassay (TRFIA). Curve fitting by weighted regression and other statistical methods were conducted, and the model was optimized for prenatal trisomy 21 screening program in second trimester. ADAM12-S alone or in combination with other two- or three-combination test was selected as a serum marker for prenatal second-trimester screening of trisomy 21 by calculation of detection rate (DR) and false positive rate (FPR).</p><p><b>RESULTS</b>By comparison, the median multiple of the median (MoM) value of ADAM12-S in DS pregnancy group was higher than that of the control group (P< 0.01). When FPR = 5%, the DR of ADAM12-S was 28.3%, and the positive and negative likelihood ratios were 5.66 and 0.75, respectively. The DR of three-combination test of ADAM12-S, alpha-fetoprotein (AFP) and free beta subunit of human chorionic gonadotropin (β-HCG) has increased to 52.80% from 39.62% of the conventional two-combination test (AFP and free β-HCG). For women with a risk between 1/300 and 1/1000 by two-combination test for DS, the DR has increased from 39.62% to 47.12%, but FPR only increased by 0.8% after adding ADAM12-S as a maternal serum marker.</p><p><b>CONCLUSION</b>Considering the increased DR of pregnancies with a risk between 1/300 and 1/1000 in second trimester, ADAM12-S may provide a feasible maternal serum maker when combined with AFP and free β-HCG. The cost-effectiveness ratio is reasonable.</p>

Application of multiplex ligation-dependent probe amplification for rapid detection of aneuploidies in prenatal diagnosis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the applicability of multiplex ligation-dependent probe amplification (MLPA) for rapid detection of aneuploidies in prenatal diagnosis.</p><p><b>METHODS</b>A total of 561 prenatal samples were analyzed in parallel by MLPA and traditional karyotyping. Another 20 clinical samples with known common chromosome abnormalities were also determined by MLPA to evaluate the accuracy and reliability of MLPA. The results obtained from MLPA were compared with that from traditional karyotyping.</p><p><b>RESULTS</b>The results were available within 48 h. A total of 38 aneuploidies were identified by MLPA, including 20 cases of trisomy 21, 10 cases of trisomy 18, 1 case of trisomy 13, 4 cases of Turner syndrome, 1 case of Klinefelter syndrome, 1 case of 47, XYY trisomy and 1 case of 48,XYY, +18. MLPA was able to detect all the expected aneuploidies with 100% accuracy. The results obtained from MLPA agreed with traditional karyotyping. Among 561 prenatal samples, the results of 550 samples were concordant with those of karyotyping, and the coincidence rate of MLPA was 98.04%.</p><p><b>CONCLUSION</b>MLPA is a rapid, simple and reliable method for detection of the most common chromosome aneuploidies in prenatal diagnosis. MLPA is a valuable tool in prenatal clinical practice.</p>

Comparison of second-trimester maternal serum free-beta-human chorionic gonadotropin and alpha-fetoprotein between normal singleton and twin pregnancies: a population-based study / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The second-trimester maternal serum screening in twin pregnancy is still controversial, as the serum marker levels in twins are not as clear as those in singletons. This study aimed to evaluate the relationship between the levels of the second-trimester maternal serum free beta-human chorionic gonadotropin (free beta-HCG) and alpha-fetoprotein (AFP) in normal twin and singleton pregnancies and to estimate feasible analysis methods for utilizing these markers in second trimester screening for twin pregnancy.</p><p><b>METHODS</b>On the basis of a prospective population-based study of second-trimester maternal serum screening, the concentrations of maternal serum AFP and free beta-HCG of 195 normal twin pregnancy and 26,512 singleton controls at gestational weeks 15 to 20 were measured by time-resolved fluoroimmunoassay in one laboratory. The levels of markers were compared between the twins and singletons using weight-correction and gestational age-specific model.</p><p><b>RESULTS</b>According to the research protocol, 95 communities were randomly sampled, which covered the whole Jiangsu province, the east of China. A total of 26 803 pregnant women (98%), from the target population accepted prenatal screening for maternal serum AFP, beta-HCG detection, and all babies were followed up for at least six months. There were 197 (0.73%) twin pregnancies, of which one case had fetal trisomy 18, and one case with fetal anencephaly. The others were normal twin pregnancy. From a total enrollment of 26 803 women participants, 26 512 women with normal singleton pregnancies were selected as the model controls. The other 291 pregnancies, including trisomy 21, neural tube defect (NTD), trisomy 18, and other fetal abnormalities, were excluded. No significant differences were found in the medians of gestational age-specific maternal serum free beta-hCG and AFP in normal twin pregnancy comparing with twice those in model controls with the exception of the medians for free beta-hCG during the 16th gestational week (P = 0.012).</p><p><b>CONCLUSION</b>The weight-correction and gestational age-specific levels of Chinese Han population maternal serum free beta-hCG and AFP in normal twins were twice the levels as those in the singleton controls during the 17-19 gestational weeks.</p>

Prenatal genetic study of fetuses with congenital heart diseases / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the genetic abnormalities of fetuses with congenital heart diseases (CHD), and to provide guidance for the management of pregnancy and genetic counseling.</p><p><b>METHODS</b>Eighty-one fetuses with CHD detected by fetal echocardiography were analyzed by karyotyping after amniocentesis, cordocentesis or chorionic sampling. Then 22q11.2 deletion/duplication was detected by a competitive fluorescent multiplex short tandem repeat assay in 47 CHD fetuses without chromosomal abnormalities. With fluorescence in situ hybridization (FISH) using LSI dual color DNA probe, the deletion/duplication status was confirmed.</p><p><b>RESULTS</b>Thirty-four of 81 CHD fetuses had chromosomal anomalies, and 1 of the 47 CHD fetuses without chromosomal anomalies had duplication at chromosome 22q11. The incidence of aneuploidy associated CHD was 43.2%. The rate of chromosomal anomalies is higher in the cases associated with extra-cardiac anomalies than in that with isolated CHD (64.5% versus 28.0%). In the 35 fetuses with chromosomal abnormalities, 19 (54.3%) were trisomy 18.</p><p><b>CONCLUSION</b>Chromosomal abnormalities occurred in 43.2% of CHD cases and trisomy 18 is the most common aneuploidy. The likelihood of chromosomal anomaly increases when there is extracardiac involvement. Testing for the 22q11.2 microdeletion/duplication is recommended in all CHD fetuses without chromosomal anomalies. It is important for the further management of pregnancy and genetic counseling.</p>

Evaluating the applicability of MoM in second trimester maternal serum to screening for Down's syndrome in Jiangsu province / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the applicability of MultiCalc software to prenatal screening of Down's syndrome in Jiangsu province, China.</p><p><b>METHODS</b>The gestational age-specific median of maternal serum marker was calculated by means of regression method. Regression functions for adjustment of Multiple of the Median (MoM) by weight were established for our own population.</p><p><b>RESULTS</b>Before the adjustment by weight, the average level of alpha fetal protein(AFP) was 16% higher and the free beta-human chorionic gonadotrophin (beta-hCG) was 14% higher than those of the Caucasian in MultiCalc software respectively. But when the AFP and free beta-hCG results were converted to weight-adjusted MoM levels, the values were 0.99 and 1.02 respectively. The median of MoM of AFP and the free beta-hCG were 1.00 through the regression model of gestational age and weight adjustment.</p><p><b>CONCLUSION</b>There was no difference of average weight-adjusted MoM levels between the Jiangsu population and the Caucasian, and the MultiCalc software was applicable to maternal serum screening for Down's syndrome of Jiangsu.</p>

Appraise operative outcome for acute central cervical spinal cord injuries without fracture and dislocation / 中华外科杂志

<p><b>OBJECTIVE</b>To evaluate the surgical outcome for acute central cervical spinal cord injuries without fracture and dislocation.</p><p><b>METHODS</b>A retrospective study was performed on 52 patients with acute central cervical cord injuries without fracture and dislocation from 2000 to 2005. All of patients underwent cervical anterior or posterior decompression, fusion and inter fixation. Spinal function was evaluated by ASIA (American Spinal Injury Association) guidelines. Pre- and post-operation ASIA scores were analyzed using liner correlation and regression. The neurological function was recorded during followed-up. The average follow-up was 29 months (range, 12 to 42).</p><p><b>RESULTS</b>After operation, the ASIA scores increased significantly (P<0.01). Finally, ASIA motor, pin pricking and light touching scores of the 41 patients were 91 +/- 7, 107 +/- 6 and 107 +/- 6 respectively.</p><p><b>CONCLUSION</b>Decompression and inter fixation for injured segment can make a stable and broad space for spinal cord, promoting early neurological recovery and long-term improvement.</p>

Detection and related analysis to chromosome 22q11 microdeletion in patients with congenital heart diseases / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To ascertain 5 short tandem repeat (STR) markers as qualified tools for detecting chromosome 22q11 deletion and to understand the prevalence and clinical importance of the deletions in patients with congenital heart diseases (CHD) from Chinese Han population.</p><p><b>METHODS</b>The authors selected 5 new tetranucleotide repeat markers, 22D_4_1, 22D_4_2, 22D_4_3, 22D_4_4 and D22S873 located in the proximal region of chromosome 22q11 deletion. One hundred and sixty-three unselected CHD patients and their unaffected parents were analyzed by genotyping of these new tetranucleotide STR markers to detect 22q11 deletion. With fluorescence in situ hybridization (FISH, LSI dual color DNA probe), the deletion status was confirmed in all patients with deletions and some patients without deletions.</p><p><b>RESULTS</b>The heterozygosity of these STR markers in normal population was more than 0.7, except for 22D_4_1 and 22D_4_2 that were 0.65 and 0.52 respectively. Twelve cases of 163 CHD patients (7.36%) had the deletions at chromosome 22q11. The deletions were confirmed in 9 of 12 patients by FISH, except for 2 cases who had unique nested deletion and 1 case who had nested distal deletion. One hundred and ten patients were associated with ventricular septal defect (VSD); and 9 (8.18%) of these cases had microdeletion. Twenty-one patients were associated with tetralogy of Fallot (TOF); and 3 (14.3%) of these cases had microdeletion.</p><p><b>CONCLUSION</b>This study demonstrated that genotyping of 5 STR markers was a useful mean of detecting 22q11 microdeletion in clinical diagnosis owing to its rapid experimental procedure, cost effectiveness and high resolution. 22q11 deletion was common in CHD patients, particularly in VSD and TOF patients, from Chinese Han population.</p>

Effect of trial transfer on embryo transfer techniques / 中华男科学杂志

<p><b>OBJECTIVE</b>To evaluate the effect of trial transfer on embryo transfer.</p><p><b>METHODS</b>Embryo transfer guided by ultrasound was performed in 114 cycles. And trial transfer was accomplished in 101 of them prior to embryo transfer. The clinical pregnancy rate and embryo implantation rate were compared between the group with trial transfer and the group without, and also among the sub-groups formed according to the difference between the trial transfer position and the actual embryo depth.</p><p><b>RESULTS</b>There was no statistically significant difference in the clinical pregnancy rate and embryo implantation rate between the group with trial transfer and that without trial transfer(59.41% vs 61.54%, 35.81% vs 31.43%, P > 0.05), nor was there between the two subgroups of the group with trial transfer (61.54% vs 58.67%, 40.79% vs 34.09%, P > 0.05).</p><p><b>CONCLUSION</b>As far as modern embryo transfer techniques are concerned, trial transfer can not provide accurate guidance for the depth of embryo transfer.</p>