[Genetic analysis of carriers of complex chromosome rearrangement].

OBJECTIVE: To comprehensively analyze the numbers of involved chromosomes and breakpoints and the clinical phenotypes of the patients with complex chromosome rearrangement (CCR). METHODS: We selected 23 745 patients with abnormal fertility seeking medical care in the Center of Reproductive Medicine of Peking University Third Hospital from 2011 to 2015, and analyzed their peripheral blood chromosomal karyotypes using G-banding, C-banding and fluorescence in situ hybridization (FISH). RESULTS: A total of 28 CCR carriers (0.118%) were detected among the 23 745 patients with abnormal fertility, including 18 males mainly with azoospermia or oligoasthenospermia and 10 females mainly with infertility, recurrent abortion, embryo termination and abnormal birth. Of the 28 cases of CCR, tripartite rearrangement was found in 9 (32.14%), double translocation in 7 (25%) and special translocation in 12 (42.86%). CCR carrier-related chromosomes were all involved but chromosomes 12 and 19, while 2 and 5 were involved most frequently. CONCLUSION: At present, the incidence of CCR is low. CCR carriers with normal phenotypes are often found because of reproductive problems, and their low chance of having a normal baby necessitates the use of preimplantation genetic test to improve the rate of live birth. Due to the diversity of the chromosomes and breakpoints involved in CCR, it is crucial to give each CCR carrier precise genetic counseling.

[Genetic analysis of a case with mosaicism complex structural aberration of chromosome 18].

OBJECTIVE: To determine the karyotype of a patient with mosaicism complex structural aberration of chromosome 18. METHODS: A male patient with a 2-year history of infertility presented at the Center of Reproductive Medicine of the Third Hospital of Peking University in October 2019 was selected as the study subject. Clinical data of the patient was collected. Peripheral blood sample was taken for chromosomal karyotyping, copy number variation (CNV) analysis and fluorescence in situ hybridization (FISH) assay. Semen sample was taken for single sperm CNV analysis. RESULTS: The patient was found to have a karyotype of mos 47,XY,del(18)(q21q23),+r(18)(q21q23)[84]/46,XY,del(18)(q21q23)[9]/48,XY,del(18)(q21q23),+r(18)(q21q23)×2[6]/47,XY,del(18)(q21q23),+r(18)(q21q23×2)[1].ish 47,XY,del(18)(q21q23),+r(18)(q21q23)[84]/46,XY,del(18)(q21q23)[9]/48,XY,del(18)(q21q23),+r(18)(q21q23)×2[6]/47,XY,del(18)(q21q23),+r(18)(q21q23×2)[1]del(18)(q21q23)(D18Z1+,18p+,18q+,WCP18+),r(18)(q21q23)(WCP18+),r(18)(q21q23×2)(WCP18+). No pathogenic CNV was identified. Sequencing of 20 single sperms showed that 1 sperm was normal, 1 had yielded no result, 9 had harbored del(18q), 7 had harbored dup(18q)×2, and 2 had harbored dup(18q)×3. The dup/del fragments had both spanned approximately 33 Mb. CONCLUSION: It is rare for carriers of complex structural and numerical abnormalities of chromosome 18 to have a normal phenotype. Based on the accurate cytogenetic and molecular analyses and the single sperm CNV analysis, the influence of the aberrant karyotype on the gametogenesis may be evaluated.

Variants in genes related to inflammation and endothelial function can increase the risk for carotid atherosclerosis in southwestern China.

Aim: To investigate the potential association between polymorphisms in genes involved in endothelial function, inflammation and carotid atherosclerosis. Methods: This was a three-center, population-based sectional survey conducted in Sichuan province of southwestern China. We randomly selected 8 different communities in Sichuan, and the residents in each community volunteered to participate in the survey by face-to-face questionnaire. A total of 2,377 residents with high stroke risk population in the 8 communities were included. Carotid atherosclerosis was evaluated by carotid ultrasound, and the 19 single nucleotide polymorphisms (SNPs) in 10 endothelial function as well as inflammation relevant genes were measured in the high stroke risk population. Carotid atherosclerosis was defined by the presence of carotid plaque or any carotid stenosis ≥15% or mean intima-media thickness (IMT) > 0.9 mm. Generalized multifactor dimensionality reduction (GMDR) approach was used to analyze gene-gene interactions among the 19 SNPs. Results: Among the 2,377 subjects with high stroke risk, 1,028 subjects had carotid atherosclerosis (43.2%), of which 852 (35.8%) cases had carotid plaque, 295 (12.4%) cases had ≥15% carotid stenosis, whereas 445 (18.7%) had mean IMT > 0.9 mm. Multivariate logistic regression revealed that IL1A rs1609682 TT and HABP2 rs7923349 TT served as independent risk factors for carotid atherosclerosis (OR, 1.45, 95% CI: 1.034-2.032, p = 0.031, and OR, 1.829, 95% CI: 1.228-2.723, p = 0.003). GMDR analysis indicated that there was a significant gene-gene interaction found among IL1A rs1609682, ITGA2 rs1991013, and HABP2 rs7923349. After adjusting the covariates, the high-risk interactive genotypes in the 3 variants were significantly associated with a significantly higher risk for carotid atherosclerosis (OR, 2.08, 95% CI: 1.257-5.98, p < 0.001). Conclusion: The prevalence of carotid atherosclerosis was observed to be extremely high in the high-risk stroke population in southwestern China. There were associations observed between the specific variants in inflammation and endothelial function relevant genes and carotid atherosclerosis. The high-risk interactive genotypes among IL1A rs1609682, ITGA2 rs1991013, and HABP2 rs7923349 significantly increased the risk of carotid atherosclerosis. These results are expected to provide novel strategies for the prevention of carotid atherosclerosis. The gene-gene interactive analysis used in this study may be very helpful to elucidate complex genetic risk factors for carotid atherosclerosis.

CYP metabolic pathway related gene polymorphism increases the risk of embolic and atherothrombotic stroke and vulnerable carotid plaque in southeast China.

OBJECTIVE: To investigate the association of CYP metabolic pathway-related genetic polymorphisms with the susceptibility to ischemic stroke and stability of carotid plaque in southeast China. METHODS: We consecutively enrolled 294 acute ischemic stroke patients with carotid plaque and 282 controls from Wenling First People's Hospital. The patients were divided into the carotid vulnerable plaque group and stable plaque group according to the results of carotid B-mode ultrasonography. Polymorphisms of CYP3A5 (G6986A, rs776746), CYP2C9*2 (C430T, rs1799853), CYP2C9*3 (A1075C, rs1057910), and EPHX2 (G860A, rs751141) were determined using polymerase chain reaction and mass spectrometry analysis. RESULTS: EPHX2 GG may reduce the susceptibility to ischemic stroke (OR = 0.520, 95% CI: 0.288 ∼ 0.940, P = 0.030) and AA+AG may increase the risk for ischemic stroke (OR = 1.748, 95% CI: 1.001 ∼ 3.052, P = 0.050). The distribution of CYP3A5 genotypes showed significant differences between the vulnerable plaque and stable plaque groups (P = 0.026). Multivariate logistic regression analysis found that CYP3A5 GG could reduce the risk of vulnerable plaques (OR = 0.405, 95% CI: 0.178 ∼ 0.920, P = 0.031). CONCLUSION: EPHX2 G860A polymorphism may reduce the stroke susceptibility, while other SNPs of CYP genes are not associated with ischemic stroke in southeast China. Furthermore CYP3A5 polymorphism was related with carotid plaque instability.

[Six-sequence-tagged site (STS) versus eight-STS scheme for detection of Y chromosome microdeletions].

OBJECTIVE: To compare the six-sequence-tagged site (STS) with the eight-STS scheme in the detection of Y chromosome microdeletions. METHODS: Using real-time quantitative PCR, we compared the results of the six-STS (sY84, sY86, sY127, sY134, sY254, sY255) scheme with those of the eight-STS (sY84, sY86, sY127, sY134, sY254, sY255, sY145, sY152) scheme in detecting Y chromosome microdeletions. RESULTS: No statistically significant difference was found in the detection rate of the deletion of the azoospermia factor (AZF) regions between the six-STS and eight-STS methods (9.34% ï¼»575/6177ï¼½ vs 8.85% ï¼»542/6122ï¼½, P > 0.05). CONCLUSION: Though the eight-STS scheme increased the detection of AZFd, its detection rate of the AZF region deletion was not significantly different from that of the six-STS method. From the perspectives of experimental operation, economic cost and clinical strategy guidance, the six-STS is better than the eight-STS scheme for the detection of Y chromosome microdeletions.

[Genetic analysis of a case with mosaicism of a small supernumerary marker chromosome derived from idic(15)].

OBJECTIVE: To determine the origin of a mosaicism small supernumerary marker chromosome (sSMC) by cytogenetic and molecular analysis. METHODS: Karyotype analysis, fluorescence in situ hybridization (FISH) and SNP-array were carried out. RESULTS: The karyotype of the patient was mos47,XX,+mar[45]/48,XX,+2mar[3]/46,XX[52]; the SNP-array result was arr[hg19]15q11.1q11.2 (20 161 372-24 314 675)×3, and the repeat fragment was about 4.15 Mb. FISH showed that approximately 50% of the cells have contained a sSMC with double D15Z1 probe site segments derived from abnormal idic(15). This sSMC did not contain SNRPN and PML probe fragments of Prader-Willi syndrome/Angelman syndrome. CONCLUSION: When the patient's karyotype and phenotype are inconsistent, cytogenetic and molecular biology technologies should be combined to clarify the karyotype and gene location, so as to provide more accurate genetic consultation for the follow-up treatments.

[Genetic analysis of an individual with a fragile site at 16q22].

OBJECTIVE: To analyze a patient with infertility and a fragile site found at 16q22 by using cytogenetic methods. METHODS: Peripheral blood sample was taken from the patient and subjected to chromosomal karyotyping and single nucleotide polymorphism microarray (SNP-array) analysis. RESULTS: The patient was found to be a mosaicism for a fragile site at 16q22, which has a variable morphology and cannot be induced by folic acid treatment. No abnormality was found by SNP-array analysis. CONCLUSION: A rare fragile site, which can be induced without folic acid treatment, has been identified at 16q22. The strategy of assisted reproduction for such individuals is yet to be explored.

Preceding Antithrombotic Treatment is Associated With Acute Ischemic Stroke Severity and Functional Outcome at 90 Days Among Patients With Atrial Fibrillation.

BACKGROUND: Antithrombotic therapies are known to prevent ischemic stroke (IS) for patients with atrial fibrillation (AF), but are often underused in clinical practice. The aim of present study was to investigate the prevalence of patients with acute IS with known history of AF who were not receiving antithrombotic treatment before stroke and to evaluate the association of preceding antithrombotic treatment with stroke severity and outcomes at 90 days after admission. MATERIALS AND METHODS: This was a retrospective, multi-center, observational study of 748 patients with acute IS and known history of AF admitted to 6 participating hospitals between March 2016 and October 2017. The primary outcome was stroke severity at admission as assessed using National Institutes of Health Stroke Scale (NIHSS) score. The secondary outcome was functional outcome at 90 days after admission as measured by modified Rankin Scale (mRS) score. RESULTS: A total of 748 patients, 54 (7.2%) were receiving therapeutic warfarin (international normalized ratio [INR] ≥ 2) and 100 (13.4%) had subtherapeutic warfarin anticoagulation (INR < 2), 340 (45.5%) were receiving antiplatelet treatment, and 254 (34.0%) were not receiving any antithrombotic treatment prior to stroke. Compared with no antithrombotic treatment, therapeutic warfarin (OR: 0.64; 95% CI: 0.52-0.82; P = .022), and antiplatelet therapy only (OR: 0.89; 95% CI: 0.76-0.96; P = .041) were associated with lower odds ratio of moderate or severe stroke (NIHSS ≥ 16). Patients receiving preceding therapeutic warfarin (OR: 1.32; 95% CI: 1.22-3.57; P = .025), antiplatelet therapy only (OR: 1.13; 95% CI: 1.07-2.59; P = .043), and subtherapeutic warfarin with INR 1.5 to 1.99 (OR: 1.15; 95% CI: 1.10-2.66; P = .042) had higher odds ratio of better functional outcome (mRS ≤ 2) at 90 days. CONCLUSIONS: Among patients with AF who had experienced an acute IS, inadequate therapeutic warfarin preceding the stroke was very prevalent in China. Therapeutic warfarin was associated with less severe stroke and better functional outcome at 90 days.

Matrix metalloproteinase-9 gene polymorphisms are associated with ischemic stroke severity and early neurologic deterioration in patients with atrial fibrillation.

OBJECTIVES: The mechanisms of ischemic stroke severity and early neurologic deterioration (END) are not fully understood. The aim of the present study was to investigate the association of six variants in MMP-9 gene with ischemic stroke severity and the risk for END in ischemic stroke (IS) patients with atrial fibrillation (AF). METHODS: This was a multi-center, prospective, observational study of 615 acute IS patients with AF admitted to six participating hospitals between June 2016 and October 2017. Ischemic stroke severity was assessed using the National Institutes of Health Stroke Scale (NIHSS) score on admission. END was defined as an increase of four or more points in NIHSS within 10 days of admission. Six variants of MMP-9 gene were examined using mass spectrometry. RESULTS: Among the 615 enrolled patients, 112 (18.2%) patients presented with moderate or severe stroke (NIHSS score ≥16), and 108 (17.6%) patients suffered from END within 10 days of admission. Multiple logistic analysis showed that prestroke antiplatelet therapy, prestroke anticoagulant therapy, rs3918242 CT/TT, and rs3787268 AG/GG were independent predictors for stroke severity. Cox proportional hazard regression revealed that diabetes mellitus, prestroke antiplatelet therapy, prestroke anticoagulant therapy, rs1056628 AC/CC, and rs3918242 CT/TT were independently associated with the risk of END. CONCLUSIONS: The incidence of moderate or severe stroke and END was very common in acute IS patients with AF. MMP-9 polymorphisms were independently associated with severe stroke and higher risk of END, and prestroke antithrombotic treatment was associated with less severe stroke and lower risk of END in patients with AF.

[Screening of common deaf genes in pregnant women and prevention of deafness at birth].

OBJECTIVE: To determine the carrier rate for common mutations causing deafness among pregnant women in order to prevent births of deaf children. METHODS: For 893 pregnant women, 2 mL peripheral venous blood was taken and DNA was extracted. A deafness DNA microarray screening was applied to such samples, and DNA sequencing was applied to husbands of women with positive screening results. RESULTS: A total of 40 carriers were detected, with the overall mutation rate being 4.48%. Among such carriers, GJB2 235delC was the most common heterozygous mutation (18 cases) and the mutation rate was 2.02%. GJB2 299A-T heterozygous mutation was detected in 7 cases with a mutation rate of 0.78%. IVS7-2A to G heterozygous mutation was detected in 9 cases with a mutation rate of 1.02%. There were 2 cases carrying GJB3 heterozygous mutation and 2 cases of mitochondrial 12S rRNA heterozygous mutation, with a mutation rate of 0.22%. IVS7-2A>G with GJB3 538C>T double heterozygous mutation was detected in 1 case, and IVS7-2A>G with GJB2 299A-T double heterozygous mutation was detected in another case, with the mutation rate of each being 0.11%. DNA sequencing has failed to find presence of mutations in the same gene in the husbands. The results of neonatal hearing follow-up were all normal. CONCLUSION: Applications of the deaf genes screening in pregnant women may play prove to be valuable for the early detection for neonatal deafness.

Ischemic stroke risk in a southeastern Chinese population: Insights from 5-lipoxygenase activating protein and phosphodiesterase 4D single-nucleotide polymorphisms.

BACKGROUND/PURPOSE: Through a genome-wide linkage scan, an Icelandic genetic research group identified two new genes associated with ischemic stroke: the 5-lipoxygenase activating protein (ALOX5AP) gene and the phosphodiesterase 4D (PDE4D) gene. Because they regulate arterial inflammation and are closely related to atherosclerosis and plaque instability, these two mutated genes have become a research hotspot. The purpose of this study was to investigate the association between the risk of ischemic stroke and single-nucleotide polymorphisms (SNPs) in the ALOX5AP and PDE4D genes in a southeastern Chinese population. METHODS: A total of 459 patients with stroke and 462 control individuals were recruited in the study. Four ALOX5AP SNPs (SG13S32, SG13S42, SG13S89, and SG13S114), and three PDE4D SNPs (SNP83, SNP87, and SNP45) were studied. SNP genotypes were determined by polymerase chain reaction amplification followed by allele-specific primer extension, with detection by matrix-assisted laser desorption/ionization time-of-flight. Data were coded and entered in SPSS Windows (version 16.0). Odds ratios and 95% confidence intervals were calculated using multivariate logistic regression analysis. Generalized multifactor dimensionality reduction (GMDR) analysis was applied to detect gene-gene interactions. RESULTS: No statistically significant differences were found in the SNP genotype frequencies between cases and controls for the seven SNPs studied. GMDR analysis revealed no evidence of interactions between these seven polymorphic sites and an increased stroke risk. In addition, no association between different stroke types and the control group was detected. Results showed that only the ALOX5AP gene, and specifically the rs9551963 and rs4769060 genotypes, exhibited significantly different distributions between the stroke and control groups in female participants. CONCLUSION: No association was found between SNPs of ALOX5AP or PDE4D and the risk of overall ischemic stroke in a southeastern Chinese population. Interactions between these two genes were not risk factors for cerebral infarction. In atherothrombotic and small-artery disease subtypes, none of the seven SNPs was associated with any stroke risk; however, the ALOX5AP gene might be related to ischemic stroke incidence in females.

Prenatal diagnosis of Down syndrome using cell-free fetal DNA in amniotic fluid by quantitative fluorescent polymersase chain reaction.

BACKGROUND: Amniotic fluid (AF) supernatant contains cell-free fetal DNA (cffDNA) fragments. This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis, which could be combined with simple quantitative fluorescent polymerase chain reaction (QF-PCR) to provide an ancillary method for the prenatal diagnosis of trisomy 21 syndrome. METHODS: AF supernatant samples were obtained from 27 women carrying euploid fetuses and 28 women carrying aneuploid fetuses with known cytogenetic karyotypes. Peripheral blood samples of the parents were collected at the same time. Short tandem repeat (STR) fragments on chromosome 21 were amplified by QF-PCR. Fetal condition and the parental source of the extra chromosome could be determined by the STR peaks. RESULTS: The sensitivity of the assay for the aneuploid was 93% (26/28; confidence interval, CI: 77%-98%) and the specificity was 100% (26/26; CI: 88%-100%). The determination rate of the origin of the extra chromosome was 69%. The sensitivity and the specificity of the assay in the euploid were 100% (27/27). CONCLUSIONS: Trisomy 21 can be prenatally diagnosed by the QF-PCR method in AF supernatant. This karyotype analysis method greatly reduces the requirement for the specimen size. It will be a benefit for early amniocentesis and could avoid pregnancy complications. The method may become an ancillary method for prenatal diagnosis of trisomy 21.

Interaction between ALOX5AP and CYP3A5 gene variants significantly increases the risk for cerebral infarctions in Chinese.

In this study, we investigated associations between susceptibility genes and cerebral infarctions in a Chinese population, and whether gene-gene interactions increase the risk of cerebral infarctions. Overall, 292 patients with cerebral infarctions and 259 healthy control individuals were included. Eight variants in five candidate genes were examined for the risk of stroke, including the SG13S32 (rs9551963), SG13S42 (rs4769060), SG13S89 (rs4769874), and SG13S114 (rs10507391) variants of the 5-lipoxygenase activating protein (ALOX5AP) gene, the G860A (rs751141) variant of the soluble epoxide hydrolase (EPHX2) gene, the A1075C (rs1057910) variant of the CYP2C9*2 gene, the C430T (rs1799853) variant of the CYP2C9*3 gene, and the A6986G (rs776746) variant of the CYP3A5 gene. Gene-gene interactions were explored using generalized multifactor dimensionality reduction methods. There were no statistically significant differences in the frequencies of the genotypes of the eight candidate genes. The generalized multifactor dimensionality reduction analysis showed a significant gene-gene interaction between SG13S114 and A6986G, with scores of 10 for cross-validation consistency and 9 for the sign test (P=0.0107). These gene-gene interactions predicted a significantly higher risk of cerebral infarction (adjusted for age, hypertension, and diabetes mellitus; odds ratio=1.80495%, confidence interval: 1.180-2.759, P=0.006). A two-loci gene interaction confers a significantly higher risk for cerebral infarction. The combinational analysis used in this study may be helpful in the elucidation of genetic risk factors for common and complex diseases.

Clinical, cytogenetic, and molecular analysis with 46,XX male sex reversal syndrome: case reports.

PURPOSE: To investigate the clinical characteristics of different categories of sex-reversed 46,XX individuals and their relationships with chromosomal karyotype and the SRY gene. METHODS: Chromosome karyotyping for peripheral blood culture and multi-PCR and FISH were performed. RESULTS: Endocrinological data showed that their endocrine hormone levels were similar to that observed for Klinefelter syndrome, with higher FSH and LH levels and lower T levels. Chromosome karyotyping for peripheral blood culture revealed 46, XX complement for 11 males. Molecular studies showed that there were locus deletions at SY84, SY86, SY127, SY134, SY254 and SY255 in AZF on chromosome Y in 9 cases, with the SRY gene present at the terminus of the X chromosome short arm. In one case, besides 6 locus deletions in AZF, there was also SRY gene deletion. In another case, there were locus deletions only at SY254 and SY255, with SY84, SY86, SY127 SY134 loci and SRY present. CONCLUSIONS: The majority (10/11) of 46,XX males were SRY positive, with the SRY gene translocated into the terminus of the X chromosome short arm. These patients were caused mainly by an X/Y chromosomal inter-change during paternal meiosis, leading to the differentiation of primary gonads into testes. Only a single patient (1/11) was SRY-negative, in which there might be some unknown downstream genes involved in sex determination.