[Prenatal genetic diagnosis of the fetuses with isolated corpus callosum abnormality].

Objective: To explore the application value of chromosome karyotype analysis, chromosomal microarray analysis (CMA) and whole exome sequencing (WES) in prenatal diagnosis of isolated corpus callosum abnormality (CCA) fetus. Methods: Fetuses diagnosed with isolated CCA by ultrasound and MRI and receiving invasive prenatal diagnosis in Guangzhou Women and Children's Medical Center and Qingyuan People's Hospital from January 2010 to April 2021 were selected. Karyotype analysis and/or CMA [or copy number variation sequencing (CNV-seq)] were performed on all fetal samples, and WES was performed on fetal samples and their parents whose karyotype analysis and/or CMA (or CNV-seq) results were not abnormal. Results: Among 65 fetuses with isolated CCA, 38 cases underwent karyotype analysis, and 3 cases were detected with abnormal karyotypes, with a detection rate of 8% (3/38). A total of 49 fetuses with isolated CCA underwent CMA (or CNV-seq) detection, and 6 cases of pathogenic CNV were detected, the detection rate was 12% (6/49). Among them, the karyotype analysis results were abnormal, and the detection rate of further CMA detection was 1/1. The karyotype results were normal, and the detection rate of further CMA (or CNV-seq) detection was 14% (3/21). The detection rate of CMA as the first-line detection technique was 7% (2/27). A total of 25 fetuses with isolated CCA with negative results of karyotyping and/or CMA were tested by WES, and 9 cases (36%, 9/25) were detected with pathogenic genes. The gradient genetic diagnosis of chromosomal karyotyping, CMA and WES resulted in a definite genetic diagnosis of 26% (17/65) of isolated CCA fetuses. Conclusions: Prenatal genetic diagnosis of isolated CCA fetuses is of great clinical significance. The detection rate of CMA is higher than that of traditional karyotyping. CMA detection could be used as a first-line detection technique for fetuses with isolated CCA. WES could increase the pathogenicity detection rate of fetuses with isolated CCA when karyotype analysis and/or CMA test results are negative.

[Prenatal diagnosis and clinical outcomes of 297 fetuses with conotruncal defects].

Objective: To analyze the prenatal diagnosis results and pregnancy outcomes of conotruncal defects (CTD) fetuses, and to explore the correlation between the CTD and chromosome diseases. Methods: A total of 297 cases of invasive prenatal diagnosis and chromosome analysis were collected at the Prenatal Diagnosis Center of Guangzhou Women and Children's Medical Center due to CTD from January 1st, 2011 to December 31th, 2019. According to ultrasonic diagnosis, CTD fetuses were divided into 6 subtypes: tetralogy of Fallot (109 cases), pulmonary atresia (30 cases), transposition of the great arteries (77 cases), double outlet right ventricle (53 cases), truncus arteriosus (14 cases) and interrupted aortic arch (14 cases). According to whether they were combined with intracardiac or extracardiac abnormalities, they were divided into simple group (134 cases), combined with other intracardiac abnormalities group (86 cases), combined with extracardiac abnormalities group (20 cases), combined with intracardiac and extracardiac abnormalities group (37 cases) and only combined with ultrasound soft marker group (20 cases), the last 4 groups were referred as non-simple types. The chromosome test results and pregnancy outcomes of each type and group were analyzed retrospectively. Results: Among the 297 CTD fetuses, the chromosome abnormality rate was 17.5% (52/297). There were 21 cases of abnormal chromosome number, 28 cases of pathogenetic copy number variantions and 3 cases of mosaics. All the 19 cases of micropathogenic fragments smaller than 5 Mb were detected by chromosomal microarray analysis (CMA). Among all the subtypes of CTD, the chromosomal abnormality rate of truncus arteriosus was the highest, at 7/14; while the rate of transposition of the great arteries was the lowest, at 5.2% (4/77). There were significant differences in the rate of chromosomal abnormalities between simple and non-simple types [10.4% (14/134) vs 23.3% (38/163); χ²=8.428, P=0.004]. In each group, the chromosomal abnormality rate was the highest in the combined with intracardiac and extracardiac abnormalities group, at 37.8% (14/37), and the lowest in the simple group, at 10.4% (14/134). There was no significant difference in the rate of chromosomal abnormalities in all subtypes of simple group (all P>0.05). Among 112 cases of live birth, 1 case was 22q11.2 microdeletion syndrome, 5 cases of postnatal clinical diagnosis and prenatal ultrasound diagnosis were not completely consistent, 5 cases died after birth. Conclusions: The incidence of chromosomal abnormalities is high in fetuses with CTD. CTD fetuses with concurrent extrapardiac malformations are more likely to incorporate chromosomal abnormalities. CMA technology could be used as a first-line genetic detection method for CTD. After excluding chromosomal abnormalities, most of the children with CTD have good prognosis.

[Analysis of families with fetal congenital abnormalities but negative prenatal diagnosis by whole exome sequencing].

Objective: To evaluate the value of whole exome sequencing (WES) in prenatal clinical application. Methods: A total of 1 152 cases of congenital abnormal [including structural malformation, nuchal translucency (NT) thickening and intrauterine growth restriction] with traditional prenatal diagnosis [including G-band karyotype analysis and chromosome microarray analysis (CMA)] negative were analyzed. The congenital abnormal fetuses were divided into retrospective group and prospective group according to the time of WES detection, that is whether the pregnancy termination or not. According to the specific location of fetal malformation and their family history, the cohort was divided into subgroups. The clinical prognosis of all fetuses were followed up, and the effect of WES test results on pregnancy decision-making and clinical intervention were analyzed. According to the follow-up results, the data of fetuses with new phenotypes in the third trimester or after birth were re-analyzed. Results: Among 1 152 families who received WES, 5 families were excluded because of nonbiological parents. Among the remaining 1 147 families, 152 fetuses obtained positive diagnosis (13.3%,152/1 147), including 74 fetuses in the retrospective group (16.1%,74/460) and 78 fetuses in the prospective group (11.4%,78/687). In fetuses with negative CMA and G-band karyotype analysis results but new phenotypes in the third trimester or after birth, the positive rate by WES data re-analysis was 4.9% (8/163). A total of 34 (21.3%, 34/160) fetuses were directly affected by the corresponding positive molecular diagnosis. Among 68 cases of live births with diagnostic variation grade 4, 29 cases (42.7%, 29/68) received appropriate medical intervention through rapid review of WES results. Conclusions: WES could increase the detection rate of abnormal fetuses with negative G-banding karyotype analysis and CMA by 13.3%. Prenatal WES could guide pregnancy decision-making and early clinical intervention. It might be an effective strategy to pay attention to the special follow-up of the third trimester and postnatal fetus and to re-analyze the WES data.

[Whole exome sequencing and analysis of a Chinese family with familial pulmonary sarcoidosis].

Objective: To analyze the clinical features and the results of the whole exome sequencing (WES) of a Chinese family containing both pulmonary sarcoidosis patients and healthy members, and to find potent genes and variants that may be involved in the pathogenesis of sarcoidosis. Methods: Three patients with pulmonary sarcoidosis and 1 healthy member was included from a Chinese Han family in the north of China diagnosed in November 2016, which characterized as 2 consecutive generations including 2 males and 1 female, aged from 23 to 69 years old. The proband is Ⅱ-6. Pulmonary sarcoidosis was diagnosed by clinical features, imaging and pathological findings, and clinical data such as family history were collected. Whole blood samples were taken and WES (Illumina NovaSeq S2) was performed. The pathogenicity analysis and gene annotation analysis were performed by ExAC, SIFT, Polyphenv2, Metascape databases. Results: It was found that 27 genes were highly pathogenic in the database filtering result. After gene annotation analysis, we found that ZC3H12A gene can negatively regulate the differentiation of Th17 cells, which may be involved in the onset of pulmonary sarcoidosis. Sanger sequencing confirmed the c.1361 A>G variant in 3 sarcoidosis patients but normal in healthy member. Conclusions: In patients with familial pulmonary sarcoidosis, the genetic background could regulate immune response which is one of the pathogenic mechanisms of sarcoidosis. The whole exome test and gene ontology analysis showed that Ⅱ-2, Ⅱ-6 and Ⅲ-1 pulmonary sarcoidosis patients in this family were all shared the same variant on ZC3H12A gene, which played a pivotal role in differentiation of Th17 cells and is a potent pathogenesis gene in this Chinese pulmonary sarcoidosis family.

[Application of chromosomal microarray analysis for fetuses with talipes equinovarus].

OBJECTIVE: To investigate the application of fetuses with talipes equinovarus(TE)using chromosomal microarray analysis(CMA)technology. METHODS: From May 2012 to June 2015, 54 fetuses were found with TE and with or without other structural anomalies by prenatal ultrasound. Karyotyping was taking for them all, and the fetuses with normal karyotypes took another CMA test. The data were analyzed with CHAS software. Finally all the cases were followed up to know about their pregnancy outcomes. RESULTS: One of the 54 cases was detected with abnormal karyotype which was trisomy 18(2%, 1/54). CMA was undertaken to the remaining fetuses, they were divided into 2 groups, including isolated TE group(n= 38)and complex TE group(n=15). The detection rate of clinical significant copy number variations(CNV)by CMA was 11%(6/53), while isolated and complex TE group were 5%(2/38)and 4/15, respectively(P= 0.047). Of the 53 cases, 51 cases were successfully followed up. Eleven cases were found without TE after birth, and the false positive rate(FPR)of TE was 22%(11/51). CONCLUSIONS: Whole-genome high-resolution CMA increased the detection rate by 11% in fetuses with TE. With the FPR and the detection rate of the clinical significant CNV of 2 groups, whole-genome CMA could be recommended to the fetuses with complex TE group but normal karyotypes. A series of ultrasonic tests should be suggested to the isolate TE group, while with the abnormal ultrasound, fetuses would be suggested to have CMA test for decreasing the rates of invasive prenatal diagnosis and FPR.

Effect of mutations in a simian virus 40 PolyA signal enhancer on green fluorescent protein reporter gene expression.

Our previous studies have shown that tandem Alu repeats inhibit green fluorescent protein (GFP) gene expression when inserted downstream of the GFP gene in the pEGFP-C1 vector. We found that the 22R sequence (5'-GTGAAAAAAATGCTTTATTTGT-3') from the antisense PolyA (240 bp polyadenylation signal) of simian virus 40, eliminated repression of GFP gene expression when inserted between the GFP gene and the Alu repeats. The 22R sequence contains an imperfect palindrome; based on RNA structure software prediction, it forms an unstable stem-loop structure, including a loop, a first stem, a bulge, and a second stem. Analysis of mutations of the loop length of the 22R sequence showed that the three-nucleotide loop (wild-type, 22R) induced much stronger GFP expression than did other loop lengths. Two mutations, 4TMI (A7→T, A17→T) and 5AMI (A6→T, T18→A), which caused the base type changes in the bulge and in the second stem in the 22R sequence, induced stronger GFP gene expression than 22R itself. Mutation of the bulge base (A17→T), leading to complete complementation of the stem, caused weaker GFP gene expression. Sequences without a palindrome (7pieA, 5'-GTGAAAAAAATG CAAAAAAAGT-3', 7pieT, 5'-GTGTTTTTTTTGCTTTTTTTGT-3') did not activate GFP gene expression. We conclude that an imperfect palindrome affects and can increase GFP gene expression.

Detection of infections of the eye with Chlamydia trachomatis by the polymerase chain reaction.

The aim of this study was to test the diagnostic feasibility of the polymerase chain reaction (PCR) for detection of infections with Chlamydia trachomatis in eye swabs from patients with conjunctivitis, and to establish the basic technique of the PCR for epidemiological survey. The results of the PCR were compared with the Mikro Trak immunofluorescence assay (IFA). From 49 specimens of patients with conjunctivitis, 31 were found positive by PCR (63%) and 23 by IFA (47%). On the other hand, in 10 normal eye specimens and 10 non-Chlamydia trachoma conjunctivitis specimens no Chlamydia trachomatis was detected.

Acoustic signals and acoustic behaviour of Chinese river dolphin (Lipotes vexillifer).

Chinese river dolphin's (Lipotes vexillifer) underwater acoustic signals can be divided into two classes, short-duration signal (pulse train or single pulses) and long-duration signal (whistle). In this paper, time waveforms of these signals are presented and the characteristics of the signals are discussed. The experimental results on this dolphin's acoustic behaviour are given. A sinusoidal pulse with duration of about 300 ms is used as the calling signal. In a certain frequency range, as soon as a calling signal is transmitted the dolphin answers with 2 or 3 whistles. The frequency range of sensitive response is from 4 to 7 KHz, in which the answering rate reaches 100%. When the dolphin's acoustic signals recorded on the tape are transmitted into the water it swims directly toward the transducer. When noise and music are transmitted the dolphin does not respond at all.