[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 as a diagnostic adjunct to clinical testing in fetuses with structural abnormalities.

OBJECTIVES: To evaluate the diagnostic yield of prenatal whole exome sequencing (WES) for monogenic disorders in fetuses with structural malformations and normal results on cytogenetic testing, and to describe information on pathogenic variants that is provided by WES. METHODS: Karyotyping, chromosomal microarray analysis (CMA) and WES were performed sequentially on stored samples from a cohort of 3949 pregnancies with fetal structural abnormalities detected on ultrasound and/or magnetic resonance imaging, referred between January 2011 and December 2015. Diagnostic rates of the three techniques were investigated overall, for phenotypic subgroups and for proband-only vs fetus-mother-father samples. Information on pathogenic variants was identified by WES. RESULTS: Overall, 18.2% (720/3949) of fetuses had an abnormal karyotype. Pathogenic copy number variants were detected on CMA in 8.2% (138/1680) of fetuses that had a normal karyotype result. WES performed on a subgroup of 196 fetuses with normal CMA and karyotype results revealed the putative genetic variants responsible for the abnormal phenotypes in 47 cases (24%). The molecular diagnosis rates for fetus-mother-father and proband-only samples were 26.5% (13/49) and 23.1% (34/147), respectively. Variants of uncertain significance were detected in 12.8% (25/196) of fetuses, of which 22 were identified in the fetal proband-only group (15%; 22/147) and three in the fetus-mother-father group (6.1%; 3/49). The incidental finding rate was 6.1% (12/196). CONCLUSIONS: WES is a promising method for the identification of genetic variants that cause structural abnormalities in fetuses with normal results on karyotyping and CMA. This enhanced diagnostic yield has the potential to improve the clinical management of pregnancies and to inform better the reproductive decisions of affected families. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

[Relationship between overnight urinary sodium to potassium ratio and the risk of cardiovascular disease].

Objective: To explore the relationship between overnight urinary sodium to potassium ratio and the risk of cardiovascular disease (CVD). Methods: A subsample of 10 percent of the participants (35-59 years old) from the People's Republic of China-United States Collaborative Study of Cardiovascular and Cardiopulmonary Epidemiology (prospective survey) were used. Three consecutive overnight urine samples were collected in the autumn of 1983-1984 and the spring in 1985-1986, respectively. Urinary sodium and potassium were detected and calculated for 8 hours excretion. The occurrences of cardiovascular events were recorded in 2 years interval from 1987-1988 until December 31, 2005. Participants were divided into first ratio group, second ratio group, and third ratio group based on the tertiles of sodium to potassium ratio. Cox proportional hazard regression model was used to determine the relationship between sodium to potassium ratio and risk of CVD. In addition, participants were divided into 2 subgroups by the median of overnight urinary sodium and potassium, and then combined each other for 4 subgroups including low sodium-low potassium group, low sodium-high potassium group, high sodium-low potassium group, and high sodium-high potassium group, to explore the relationship between different sodium-potassium combinations and the risk of CVD. Results: A total of 954 participants were included in the final analysis, of whom 459 (48.1%) were males. There were 318 cases in the first, second and third ratio group, respectively. There were 347 cases in low sodium-low potassium group and high sodium-high potassium group, and 130 cases in low sodium-high potassium group and high sodium-low potassium group. After a median follow-up of 18.6 (18.3, 19.3) years, cardiovascular events occurred in 81 participants, including 64 stroke and 20 coronary heart disease events. Multivariate analysis showed that comparing with the first ratio group, the hazard ratios (HR) in the second and the third ratio groups were 2.04 (95%CI 1.06-3.95, P=0.034) and 2.07 (95%CI 1.07-4.03, P=0.032), respectively. The CVD risk in low sodium-low potassium group was 24% higher than the low sodium-high potassium group (reference), but this result did not reach statistical significant level (P=0.685). The risks in high sodium-high potassium group (HR=3.32, 95%CI 1.26-8.76,P=0.015) and high sodium-low potassium (HR=3.04, 95%CI 1.05-8.83, P=0.041) group were both significantly increased. Conclusions: Overnight urinary sodium to potassium ratio is positively correlated with the risk of cardiovascular events. High urinary sodium plays a more important role for the increased risk of cardiovascular events than low potassium.

[Analysis of long-term compliance to continuous positive airway pressure in patients with obstructive sleep apnea].

OBJECTIVE: To explore the continuous positive airway pressure (CPAP) therapy compliance in patients with obstructive sleep apnea (OSA). METHODS: This prospective study recruited a group of subjects from May 2009 to December 2013 who were diagnosed and had accepted CPAP treatment in Sleep Center of Guangdong General Hospital, and the patients were followed-up regularly for long-term and assessed the CPAP treatment compliance. The patients were diagnosed, had pressure titration and CPAP treatment through out of center sleep test. The subjects were followed-up for 1 st, 3rd, 6th, 12th month, and each year regularly after accepting the CPAP treatment in Sleep Center by face to face follow-up with specialist physicians. Physicians followed-up the patients' subjective symptoms, CPAP adherence, patient education and side effect solutions. The patients were classified into good and poor compliance groups, and statistical analysis was done between the two groups. RESULTS: There were 77 cases enrolled until December 2015, only 73 patients completed the study. The patients were followed-up about 2-6 years, the average was (3.93±1.29) years, the compliance accounted for 54.8% (40/73), and the average compliance was (4.02±1.87) hours/night. The trend of the long-term compliance showed that there was a gradual increase within the first 3 months of CPAP treatment and then the compliance decreased; it then increased gradually after the first two years. The good compliance group showed that the compliance increased gradually in the initial 3 months, and then fell; from the first year to the 3rd year, the compliance was stable; after the 3rd year there was a drop and the compliance tended to increase again after the 4th year. The poor compliance group showed the compliance had a downward trend from the beginning of the first two years, then after a brief rise, the compliance decreased linearly. Multivariate analysis showed that long-term compliance was not associated with age, daytime sleepiness (ESS), oxygen desaturation index (ODI), anxiety, depression (P>0.05), etc. However, it was associated with the time of the titration treatment (P<0.001), the time of the flow monitored (P<0.01) and the number of the pressure titration within one week (P<0.05). CONCLUSIONS: Long-term compliance shows a curve change, the increased compliance is related with the regular follow-up. Long-term compliance can be predicted by the degree of cooperation with the initial diagnosis and treatment.

Systolic Blood Pressure and Mortality in Community-Dwelling Older Adults: The Role of Frailty.

OBJECTIVES: To investigate whether frailty modifies the association of systolic blood pressure (SBP) with cardiovascular mortality and all-cause mortality in community-dwelling older adults. DESIGN: A prospective cohort study. SETTING: A population-based study of nationally representative older Chinese adults in a community setting. PARTICIPANTS: This study included participants aged 65 years or older from the Chinese Longitudinal Healthy Longevity Survey 2002-2014 and followed up to 2018. MEASUREMENTS: Participants were divided into two groups according to a frailty index based on the accumulation of a 44-items deficits model. The association between SBP and mortality was analyzed using multivariable-adjusted Cox proportional hazards models. RESULTS: Among 18,503 participants included, the mean age was 87.2 years and the overall median follow-up time was 42.7 months. We identified 7808 (42.2%) frail participants (mean frailty index=0.33), in which 7533 (96.5%) died during the follow-up. Effect modification by frailty was detected (P for interaction=0.032). Among frail participants, a U-shaped association was found with hazard ratios of 1.16 (95% CI, 1.02-1.32) for SBP < 100 mmHg, and 1.11 (95% CI, 1.00-1.24) for SBP ≥ 150 mmHg compared with SBP 120-130 mmHg. For non-frail older adults, a tendency toward higher risk among those with SBP ≥ 130 mmHg was observed. The analyses towards cardiovascular mortality showed similar results. CONCLUSION: Our results suggest the presence of effect modification by frailty indicating a possible negative effect for elevated SBP in non-frail older adults and a U-shaped relationship of SBP in frail older adults with respect to mortality even after adjusting for diastolic blood pressure.

Levels and profiles of unintentionally produced persistent organic pollutants in surface soils from Shanxi province, China.

Six species of unintentionally produced persistent organic pollutions comprised of polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, polychlorinated naphthalenes, hexachlorobenzene and pentachlorobenzene in soils collected from Shanxi province, China were determined. The sum toxic equivalent ranged from 0.14 to 2.20 with an average of 0.94 pg TEQ/g. Polychlorinated dibenzo-p-dioxins/furans contributed the most toxic proportion to the total toxic equivalent. CB-126 was the most toxic contributor to polychlorinated biphenyls. CN66/67 and CN73 are the dominant toxic congeners to polychlorinated naphthalenes. From the patterns, it was speculated that thermal related industries were possible sources of unintentionally produced persistent organic pollutions.

[A new perspective of survival data on clinical epidemiology: introduction of competitive risk model].

Competing risks occur frequently in the analysis of survival data that should be dealt with competing risk models. Competing risk is an event whose occurrence precludes the occurrence of the primary event of interest. Previous commonly used Kaplan-Meier method tends to overestimate the cumulative survival functions, while the traditional Cox proportional hazards model falsely evaluates the effects of covariates on the hazard related to the occurrence of the event. There are few domestic reports mentioning the concept, application and methodology of competing risk model as well as the implementation procedures or resolution of model conditions and parameters. The current work aims to explain the core concept and methodology of the competing risk model and to illustrate the process of analysis on cumulative incidence rate, using both the cause-specific hazard function model and the sub-distribution hazard function model. Software macro code in SAS 9.4 is also provided to assist clinical researchers to further understand the application of the model so to properly analyze the survival data.

[Application of SAS macro to evaluated multiplicative and additive interaction in logistic and Cox regression in clinical practices].

Conditional logistic regression analysis and unconditional logistic regression analysis are commonly used in case control study, but Cox proportional hazard model is often used in survival data analysis. Most literature only refer to main effect model, however, generalized linear model differs from general linear model, and the interaction was composed of multiplicative interaction and additive interaction. The former is only statistical significant, but the latter has biological significance. In this paper, macros was written by using SAS 9.4 and the contrast ratio, attributable proportion due to interaction and synergy index were calculated while calculating the items of logistic and Cox regression interactions, and the confidence intervals of Wald, delta and profile likelihood were used to evaluate additive interaction for the reference in big data analysis in clinical epidemiology and in analysis of genetic multiplicative and additive interactions.

Hydatid disease of bone.

Twenty cases of hydatid disease of bone revealed by X-ray examination are presented. The vertebrae were involved in 12 of the cases, the pelvis in four, both the vertebrae and the pelvis in one, the ribs in two and the femur in one. These cases constituted 1.3% of all cases of hydatid disease observed in our hospital during the period 1957-80. The features of the disease are discussed in terms of plain radiographs, myelography and tomography. Tomographic studies have proved of value in differentiating the disorder from other diseases.

Protein-DNA interaction within one cloned chloroplast DNA replication origin of Chlamydomonas.

A partially purified algal protein mixture which supports in vitro DNA replication consists of soluble proteins and proteins extracted from thylakoid membrane. The membrane extract is essential for the specific initiation of replication at a displacement loop (D-loop) site previously mapped by electron microscopy. D-loop site and its flanking sequences have been cloned and sequenced. In this study, fragment-retention assays using various subclones of the sequenced region indicate that some proteins in the membrane extract bind strongly and specifically with a 494 bp restriction fragment which partially overlaps the D-loop site. Protein gel analyses of the protein-DNA complex identify three DNA-binding polypeptides with apparent molecular weights of 18, 24 and 26 kDA, respectively. Treatment with chloramphenicol, an inhibitor of chloroplast protein synthesis, for 1 h has no obvious effect on the contents of the 24 or 26 kDa polypeptides but significantly reduces the content of the 18 kDa polypeptide in the membrane extract.

The 18-kD protein that binds to the chloroplast DNA replicative origin is an iron-sulfur protein related to a subunit of NADH dehydrogenase.

From a high-salt extract of the purified thylakoid membrane, an 18-kD protein was detected. This protein was translated by the chloroplast ribosomes and could form a stable DNA-protein complex with a cloned chloroplast DNA replicative origin [Nie, Z.Q., Chang, D.Y., and Wu, M. (1987) Mol. Gen. Genet. 209, 265-269]. In this paper, the 18-kD protein is linked to frxB, a chloroplast-encoded, ferredoxin-type, iron-sulfur protein, by N-terminal microsequencing of the purified protein and computer analysis. The identification is further supported empirically by the fact that the electron paramagnetic resonance spectra of the protein indicate the presence of iron-sulfur clusters. A polyclonal antibody raised against a synthetic pentadecameric peptide with amino acid sequence corresponds to the highly conserved region of the frxB protein and reacts strongly and specifically with the 18-kD protein band in protein gel blot analyses. The 18-kD iron-sulfur protein is found to be related to a subunit of the respiratory chain NADH dehydrogenase by its cross-reaction with a polyclonal antibody raised against highly purified NADH-ubiquinone oxidoreductase, a key enzyme of the respiratory chain. These data are consistent with chlororespiration, and, thus, possible implication of chlororespiration in regulating the initiation of chloroplast DNA replication is discussed.

Purification and characterization of a gamma-like DNA polymerase from Chlamydomonas reinhardtii.

A crude in vitro system which initiates chloroplast DNA synthesis near the D-loop site mapped by electron microscopy [Wu, M., Lou, J. K., Chang, D. Y., Chang, C. H., & Nie, Z. Q. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6761-6765] consists of soluble proteins and proteins extracted from purified thylakoid membrane. In this paper, a DNA polymerase activity was purified to near homogeneity from the soluble protein fraction of this in vitro system by sequential chromatographic separations on heparin-agarose, DEAE-cellulose, and single-stranded DNA-agarose columns and sedimentation in a glycerol gradient. In the glycerol gradient, the enzyme activity sedimented at a position corresponding to a 110-kDa protein. Electrophoretic analysis of the highly purified fraction on SDS-polyacrylamide gel revealed a major polypeptide band with an apparent molecular mass of approximately 116 kDa. In situ DNA polymerase activity assay shows that the DNA polymerization function is associated with the 116-kDa band and an 80-kDa band which could be a subunit of the enzyme. Polymerization activity is inhibited by N-ethylmaleimide, ethidium bromide, and dideoxycytosine triphosphate and is relatively resistant to aphidicolin. Poly(dA).(dT)10 and gapped double-stranded DNA are preferred templates. The purified enzyme contains no exonuclease activity and can initiate DNA replication in a supercoiled plasmid DNA template containing the chloroplast DNA replication origin.

Structure and function of a chloroplast DNA replication origin of Chlamydomonas reinhardtii.

Chloroplast DNA replication in Chlamydomonas reinhardtii is initiated by the formation of a displacement loop (D-loop) at a specific site. One D-loop site with its flanking sequence was cloned in recombinant plasmids SC3-1 and R-13. The sequence of the chloroplast DNA insert in SC3-1, which includes the 0.42-kilobase (kb) D-loop region, as well as 0.2 kb to the 5' end and 0.43 kb to the 3' end of the D-loop region, was determined. The sequence is A+T-rich and contains four large stem-loop stuctures. An open reading frame potentially coding for a polypeptide of 136 amino acids was detected in the D-loop region. One stem-loop structure and two back-to-back prokaryotic-type promoters were mapped within the open reading frame. The 5.5-kb EcoRI fragment cloned in R-13 contains the 1.05-kb SC3-1 insert and its flanking regions. A yeast autonomously replicating (ARS) sequence and an ARC sequence, which promotes autonomous replication in Chlamydomonas, have been mapped within the flanking regions [Vallet, J.-M. & Rochaix, J.-D. (1985) Curr. Genet. 9, 321-324]. Both R-13 and SC3-1 were active as templates in a crude algal preparation that supports DNA synthesis. In this in vitro system, chloroplast DNA synthesis initiated near the D-loop site.