A Fetus with Maternal Uniparental Disomy on Chromosome 20: Case Report with Genetic Analysis and Prenatal Diagnosis.

BACKGROUND: A fetus with increased copy number of chromosome 20 was identified by NIPT. Here we utilize several genetic tests and analyses to illuminate the etiology of such aneuploidy. METHODS: Amniotic fluid cells were extracted from pregnant woman and sent for karyotype and chromosomal microarray analysis (CMA). Trio pedigree analysis was conducted with Chromosome Analysis Suite and uniparental disomy (UPD)-tool software. RESULTS: CMA identified consistent results, which were 2 regions of homozygosity: arr[GRCh37]20p12.2q11.1 (11265096_26266313)hmz and arr[GRCh37]20q11.21q13.2(29510306_54430467)hmz. The trio pedigree analysis discovered that the fetal chromosome 20 was the entire maternal UPD mosaic with isodisomy and heterodisomy. CONCLUSIONS: When a large segment of chromosome is homozygous, appropriate genetic tests are required to find the potential mechanisms for UPD formation.

[Application of various genetic techniques for the diagnosis of Prader-Willi syndrome].

OBJECTIVE: To discuss the advantages and technical limitations of various molecular genetic techniques in the diagnosis of two infants featuring all-round developmental retardation. METHODS: The two patients were initially screened by using chromosomal microarray analysis (CMA). For patient 1, his parents were also subjected to CMA analysis, and the data was analyzed by using ChAS and UPD-tool software. For patient 2, methylation-specific PCR (MS-PCR) was carried out. RESULTS: Patient 1 was diagnosed with maternal uniparental disomy (UPD) type Prader-Willi syndrome (PWS) by CMA and UPD-tool family analysis. His chromosomes 15 were of maternal UPD with homology/heterology. Patient 2 was diagnosed with deletion type PWS by combined CMA and MS-PCR. CONCLUSION: Correct selection of laboratory methods based on the advantages and limitations of various molecular techniques can help with diagnosis of genomic imprinting disorders and enable better treatment and prognosis through early intervention.

[Association of GPER gene polymorphism with social function of children with attention deficit hyperactivity disorder].

OBJECTIVE: To assess the association of G protein-coupled estrogen receptor(GPER) gene polymorphism with social function of children with attention deficit hyperactivity disorder (ADHD). METHODS: The social function of 135 children with ADHD were assessed by Weiss Functional Impairment Scale-Parent form (WFIRS-P). The coding region of GPER gene of all patients was subjected to Sanger sequencing. The association of polymorphisms with the social function of the ADHD children was analyzed. RESULTS: In the case group, the social function scores of Learning and School and Risky Activities of boys were significantly higher than those of girls (t=2.704, P=0.008; t=2.289, P=0.027). No significant difference was found in the genotypic frequencies of the c.-9T/C and c.789G/A loci between different genders. But the learning and school scores of those with a TC genotype for the c.-9T to C locus were significantly higher than those with a TT genotype (t= 2.159, P=0.033). CONCLUSION: For children with ADHD, the social function of Learning and School of those with a TC genotype of the GPER gene c.-9T/C locus is more severely damaged compared with those with a TT genotype.

[Genetic analysis of a fetus with partial 18p tetraploidy syndrome].

OBJECTIVE: To analyze a fetus with abnormal cardiac ultrasound by using various techniques and explore its genotype-phenotype correlation. METHODS: Lymphocytes derived from umbilical cord blood sample were subjected to G-banding analysis. Short tandem repeats quantitative fluorescence PCR (STR-QF-PCR) was used for analysis of fetal DNA as an auxiliary test. Low-coverage whole genome sequencing (WGS) was used to detect chromosomal deletion/duplication which exceeded 100 kb in size. RESULTS: The karyotype of the fetus was 47,XN,+mar. As detected by STR-QF-PCR, the copy number of GATA178F11 locus on chromosome 18 was 4, and the duplicated fragment was derived from the mother. WGS suggested that the fetus to be 46,XN,dup(18p11.21p11.32).seq [GRCh37/hg19](10 001-15 378 887)× 4, with the duplicated fragment spanning approximately 15.38 Mb. CONCLUSION: The cardiac malformation of the fetus may be attributed to the partial duplication of chromosome 18p. Combined cytogenetic and molecular methods can facilitate prenatal detection of genetic abnormalities.

Non-invasive prediction of preeclampsia using the maternal plasma cell-free DNA profile and clinical risk factors.

Background: Preeclampsia (PE) is a pregnancy complication defined by new onset hypertension and proteinuria or other maternal organ damage after 20 weeks of gestation. Although non-invasive prenatal testing (NIPT) has been widely used to detect fetal chromosomal abnormalities during pregnancy, its performance in combination with maternal risk factors to screen for PE has not been extensively validated. Our aim was to develop and validate classifiers that predict early- or late-onset PE using the maternal plasma cell-free DNA (cfDNA) profile and clinical risk factors. Methods: We retrospectively collected and analyzed NIPT data of 2,727 pregnant women aged 24-45 years from four hospitals in China, which had previously been used to screen for fetal aneuploidy at 12 + 0 ~ 22 + 6 weeks of gestation. According to the diagnostic criteria for PE and the time of diagnosis (34 weeks of gestation), a total of 143 early-, 580 late-onset PE samples and 2,004 healthy controls were included. The wilcoxon rank sum test was used to identify the cfDNA profile for PE prediction. The Fisher's exact test and Mann-Whitney U-test were used to compare categorical and continuous variables of clinical risk factors between PE samples and healthy controls, respectively. Machine learning methods were performed to develop and validate PE classifiers based on the cfDNA profile and clinical risk factors. Results: By using NIPT data to analyze cfDNA coverages in promoter regions, we found the cfDNA profile, which was differential cfDNA coverages in gene promoter regions between PE and healthy controls, could be used to predict early- and late-onset PE. Maternal age, body mass index, parity, past medical histories and method of conception were significantly differential between PE and healthy pregnant women. With a false positive rate of 10%, the classifiers based on the combination of the cfDNA profile and clinical risk factors predicted early- and late-onset PE in four datasets with an average accuracy of 89 and 80% and an average sensitivity of 63 and 48%, respectively. Conclusion: Incorporating cfDNA profiles in classifiers might reduce performance variations in PE models based only on clinical risk factors, potentially expanding the application of NIPT in PE screening in the future.

Application of various genetic analysis techniques for detecting two rare cases of 9p duplication mosaicism during prenatal diagnosis.

BACKGROUND: The identification of genetic mosaicism and the genetic counseling needed following its discovery have been challenging problems in the field of prenatal diagnosis. Herein, we describe the clinical phenotypes and various prenatal diagnostic processes used for two rare cases of 9p duplication mosaicism and review the prior literature in the field to evaluate the merits of different methods for diagnosing mosaic 9p duplication. METHODS: We recorded ultrasound examinations, reported the screening and diagnosis pathways, and analyzed the mosaic levels of the two cases of 9p duplication using karyotype analysis, chromosomal microarray analysis (CMA), and fluorescence in situ hybridization analysis (FISH). RESULTS: Case 1 had a normal clinical phenotype for tetrasomy 9p mosaicism, and Case 2 showed multiple malformations caused by both trisomy 9 and trisomy 9p mosaicism. Both cases were initially suspected after non-invasive prenatal screening (NIPT) based on cell-free DNA. The mosaic ratio of 9p duplication found via karyotyping was lower than what was discovered by CMA and FISH, in both cases. Contrary to previous findings, the mosaic level of trisomy 9 found by karyotype analysis was greater than what was found by CMA, in terms of complex mosaicism involving trisomy 9 and trisomy 9p, in Case 2. CONCLUSION: NIPT can indicate 9p duplication mosaicism during prenatal screening. Different strengths and limitations existed in terms of diagnosing mosaic 9p duplication by karyotype analysis, CMA, and FISH. The combined use of various methods may be capable of more accurately determining break-points and mosaic levels of 9p duplication during prenatal diagnosis.

Health economic evaluation of noninvasive prenatal testing and serum screening for down syndrome.

BACKGROUND: Down syndrome (DS), also known as trisomy 21 (T21), is the most common genetic disorder associated with intellectual disability. There are two methods commonly used for prenatal testing of DS: serum screening (SS) for biomarkers in maternal serum and noninvasive prenatal testing (NIPT) for aneuploidy by cell-free DNA (cfDNA) in maternal plasma. However, cost-effectiveness analyses of these two methods are mostly based on data derived from simulations with various models, with theoretical values calculated. In this study, we statistically analyzed clinical DS screening data and pregnancy outcomes during the follow-up of pregnant women in Zhuhai City, China. The economics of the two mainstream prenatal DS screening methods was evaluated from a public health perspective. METHODS: A retrospective analysis was performed on the data of 17,363 pregnant women who received SS and NIPT during gestation in Zhuhai from 2018 to 2019, and a cost-effectiveness analysis was performed with four screening strategies. In strategy I, all pregnant women received SS, and those with T21 risk ≥1/270 had invasive prenatal diagnosis (IPD). In strategy II, all pregnant women received SS, those with T21 risk ≥ 1/270 had IPD, and those with 1/270 > T21 risk ≥ 1/1,000 had NIPT; then, women at high risk based on NIPT also had IPD. In strategy III, all pregnant women received SS, and those with T21 risk ≥1,000 had NIPT; then, women at high risk based on NIPT results had IPD. In strategy IV, all pregnant women received NIPT and those at high risk based on NIPT results had IPD. Finally, to assess the cost and effectiveness of DS screening, the total costs were calculated as the sum of screening and diagnosis as well as the direct and indirect economic burden during the average life cycle of DS patients. RESULTS: A total of 22 of the 17,363 (1/789) pregnant women had DS, of which only one woman was over 35 years of age. SS detected 1,024 cases at high risk of T21 (≥1/270), 8 cases were true positive, with a positive predictive value of 0.78% and a detection rate of 36.4%. NIPT detected 27 cases at high risk of T21 (Z ≥ 3) and 22 cases of DS, with a positive predictive value of 81.5% and a detection rate of 100%. Strategy I had the largest total cost of 65.54 million CNY, strategy II and III had similar total costs of 40 million CNY, and strategy IV had the lowest total cost of 14.91 million CNY. By comparison, the screening strategy with NIPT alone had the highest health economic value for DS. CONCLUSIONS: SS was greatly affected by nuchal translucency and the accuracy of gestational age measured by ultrasonography. Unstandardized ultrasonography was an important reason for the low DS detection rate with SS. The influence of interfering factors on NIPT was much lower than in SS. NIPT can be used as an alternative to SS and as a primary screening strategy of prenatal DS screening for secondary prevention and control of birth defects. NIPT greatly decreased the frequency of IPD and the miscarriages associated with IPD, saved the limited medical and health resources, and greatly increased DS detection rate. Therefore, NIPT has great social and economic benefits.

Prenatal diagnosis of a 4.5-Mb deletion at chromosome 4q35.1q35.2: Case report and literature review.

OBJECTIVE: We present a genetic analysis of an asymptomatic family with a 4q terminal deletion; we also review other similar published studies and discuss the genotype-phenotype correlation. METHODS: A karyotype analysis was performed on the amniotic fluid cells of a woman at 24 weeks of pregnancy and peripheral blood lymphocytes from both parents and their older son with the conventional G-banding technique. Chromosomal microarray analysis (CMA) testing was carried out for both parents and the fetus to analyze copy number variation (CNV) in the whole genome. RESULTS: The results showed no abnormalities in the karyotypes of the father and older son, and the karyotypes of the mother and fetus were 46,XX,del(4)(q35.1) and 46,XY,del(4)(q35.1), respectively. CMA results showed a partial deletion at the 4q terminus in both the fetus and mother. The deletion region of the fetus was arr[GRCh37] 4q35.1q35.2(186,431,008_190,957,460) × 1; the loss size of the CNV was approximately 4.5 Mb and involved 14 protein-coding genes, namely, CYP4V2, F11, FAM149A, FAT1, FRG1, FRG2, KLKB1, MTNR1A, PDLIM3, SORBS2, TLR3, TRIML1, TRIML2, and ZFP42. No variation on chromosome 4 was detected in the father's CMA results. CONCLUSION: Deletion of the 4q subtelomeric region is a familial variation. The arr[GRCh37] 4q35.1q35.2(186,431,008_190,957,460) region single-copy deletion did not cause obvious congenital defects or mental retardation. The application of high-resolution genetic testing technology combined with the analysis of public genetic database information can more clearly elucidate the genotype-phenotype correlation of the disease and provide support for both prenatal and postnatal genetic counseling.

[A community-based genetic screening of large-scale population and prenatal diagnosis for alpha and beta thalassemia in Zhuhai city of Guangdong province].

OBJECTIVE: To describe a community-based model for prevention and control of severe alpha and beta thalassemias in Zhuhai city of Guangdong province. METHODS: Couples for premarital medical examination or regular healthcare examination in pregnancy were enrolled in this prospective screening program, which was supported by the two-level network composed of 6 local hospitals for testing thalassemias and follow-up for genetic counseling. A conventional heterozygote screening strategy was used to determine alpha and beta thalassemia traits in women and their partners according to the standard procedures of hematological phenotype analysis. Then confirmative diagnosis of alpha and beta thalassemia was performed on those couples suspected at-risk for severe thalassemia by using the PCR-based molecular diagnostic assays. The couples at-risk for severe thalassemia were counseled and offered prenatal diagnosis and termination of pregnancy in case of an affected fetus. RESULTS: During the period between January 1998 and December 2005, the screened records included 85522 young females and their partners for premarital screening and 10439 pregnant women for prenatal screening, with 71.38% coverage of total population recorded in this city for premarital screening. Six thousands five hundreds and sixty-three individuals in total were found to be the carriers of thalassemias, with 4312 for alpha thalassemia (4.5%) and 2251 for beta thalassemia (2.3%), respectively. One hundred and forty-eight couples were diagnosed to be at-risk for thalassemias, including 103 for alpha thalassemia and 45 for beta thalassemia, respectively. Successful prenatal diagnosis was made for 142 (98 for alpha thalassemia and 44 for beta thalassemia) out of 148 (95.9%) pregnancies at-risk for severe thalassemias. Twenty-three cases of hydrops fetalis, 4 of Hb H diseases and 14 of beta thalassemia were identified. All 41 pregnancies with affected fetuses were voluntarily terminated. Thus, this has led to a marked decrease of severe thalassemia syndrome since the program started. CONCLUSION: We presented the first community-based prospective screening program in China for control of alpha and beta thalassemia in Zhuhai city with a population of 1.29 million through premarital or prenatal screening. This model could be used for control of thalassemias and other hemoglobinopathies in other regions of China and also in other developing countries.

[Correlative study between angiotensin-converting enzyme gene polymorphism and hepatorenal syndrome].

OBJECTIVE: To investigate the relationship between insertion/deletion (I/D) polymorphism of angiotensin-converting enzyme (ACE) gene and uncompensated cirrhosis of liver with hepatorenal syndrome (HRS). METHODS: ACE I/D polymorphism was detected by polymerase chain reaction amplification of DNA fragment in 56 patients of uncompensated liver cirrhosis with HRS, and 60 healthy individuals served as the controls. At the same time, alanine aminotransferase, aspartate transaminase, serum creatinine (SCr), blood urea nitrogen (BUN) and glomerular filtration rate (GFR) etc. were measured in all the subjects, and the difference between these variables among different genotypes was noted. RESULTS: There was no significant difference in genotypes and allele frequency between the HRS group and controls(all P>0.05). The I allele frequency was higher than the D allele in all the subjects (all P<0.01). But in the control group, there was no significant difference in the genotype frequency among three genomic groups, while the II genotype frequency was higher than the one of ID and DD (all P<0.05). SCr and BUN of the II genotype were higher in the HRS group than that of ID and DD(both P<0.05) and GFR of the II genotype was lower than the one of ID and DD in the HRS group(P<0.05). CONCLUSION: There is relationship between ACE gene polymorphism and the incidence of uncompensated liver cirrhosis with HRS. II genotype may be the genetic factor of vulnerability to HRS patients with uncompensated cirrhosis of liver. The degree of kidney failure in II genotype population is more serious than in ID and DD individuals with uncompensated liver cirrhosis complicated by HRS.

Evaluation of clinical application of gap-PCR as a routine method for alpha-thalassemia carrier detection.

OBJECTIVE: To evaluate the feasibility of using gap-PCR for routine screening of alpha-thalassemia in clinical laboratory. METHODS: A total of 382 clinical blood samples randomly collected from the population of Zhuhai city were screened for alpha-thalassemia determinants with hematological and gap-PCR method respectively in a double-blind manner. Parallel analysis with Southern blotting was performed to verify the genotyping results by PCR. RESULTS: Of the 382 samples tested, 3 common alpha-thalassemia genes with genotypes of --(SEA)/alpha alpha, -alpha(3.7)/alpha alpha and -alpha(4.2)/alpha alpha were detected in 21 (5.50%), 7 (1.83%) and 3 (0.79%) cases respectively by gap-PCR, including 7 cases with normal phenotype and 3 case of iron-deficiency anemia. The overall incidence of alpha-thalassemia was 8.12% in the population of Zhuhai city, as determined by gap-PCR, in total agreement with the results by Southern blotting. Only 21 of the 31 alpha-thalassemia cases were identified by hematological analysis (besides 2 cases with alpha-thalassemia phenotype undetermined), which had a false-negative rate of 32.3%. Seven silent alpha-thalassemia and 3 mild alpha-thalassemia cases failed to be detected by hematological analysis, resulting in a rate of 2.62% for failure of detection. CONCLUSION: Gap-PCR method is specific and feasible as a better alternative for alpha-thalassemia screening, especially advantageous in detecting silent carriers in comparison with hematological method.

[Clinical phenotype genotype correlation in children with hemoglobin H disease in Zhuhai area of China].

OBJECTIVE: Alpha-thalassemia is one of the most common monogene disorders in the world. Most frequently, it is caused by deletions of alpha-globin gene (-alpha or --), and less commonly resulted from the non-deletional mutation (alpha(T)alpha). Hemoglobin H (HbH) disease is the most severe type among survivors of alpha-thalassemia. The clinical presentation of children with the disease was highly heterogeneous. The aim of this study was to investigate the effect of alpha-globin genotypes in the children with HbH disease on predicting the phenotypic severity and to define the factors involved in the disease progress. METHODS: Forty-three children with the disease in Zhuhai area of Guangdong, China were examined by using established techniques to detect genotypes of alpha-globin and to determine all hematological parameters. All detailed clinical data of the cases were recorded. Then clinical and hematological findings, and the correlation with genotypes were evaluated. RESULTS: Six alpha-thalassemia mutations were detected and interacted to produce 5 HbH disease genotypes. Of these genotypes, -alpha(3.7)/--(SEA)(60%), -alpha(4.2)/--(SEA) (19%) and alpha(CS)alpha/--(SEA) (12%) HbH diseases were prevalent in the area. Compared with -alpha(3.7)/--(SEA) HbH disease, significantly lower red blood cell (RBC) count, hemoglobin (Hb), mean corpuscular hemoglobin (MCHC) and HbA(2) (P < 0.05, 0.01, 0.01 and 0.01, respectively), and significantly higher mean corpuscular hemoglobin volume (MCV) and HbH levels (both P < 0.01), and more severe clinical phenotypes were found in the HbH disease with alpha(T)alpha/--(SEA) genotype. While the differences were much more significant when compared with -alpha(3.7)/--(SEA) then compared with -alpha(4.2)/--(SEA) not only in the hematological parameters, but also in the severity of clinical phenotypes. In addition, HbH levels showed anegatively correlation with the RBC count (r = -0.39, P < 0.01). CONCLUSION: The phenotypes of HbH disease may be mainly related to the underlying genotypes. The children with alpha(T)alpha/--(SEA) genotype presented with more severe hematological and clinical phenotypes followed by the -alpha(4.2)/--(SEA) and then -alpha(3.7)/--(SEA) genotypes. But phenotypic severity was not simply related to the degree of alpha-globin deficiency. HbH levels were found to exacerbate anemia. These data might provide comprehensive and very valuable and basic information for the management of HbH disease, genetic counseling and prenatal diagnosis.