The Rapid Evaluation of Down Syndrome With Quantitative Fluorescence Polymerase Chain Reaction (QF-PCR): A Pilot Study Among the Population in Eastern Uttar Pradesh, India.

Background and objective Down syndrome (DS) is characterized by the presence of an additional chromosome; it is a typical chromosomal disorder causing intellectual disability in individuals. The diagnostic process for DS often involves conventional karyotyping, which can be time-consuming. Trisomy 21 and other chromosomal abnormalities may now be quickly and accurately diagnosed using quantitative fluorescence polymerase chain reaction (QF-PCR). In light of this, this study aimed to investigate chromosomal abnormalities in DS using conventional karyotyping and QF-PCR among the population in eastern Uttar Pradesh, India. Methods Blood samples from 40 individuals with clinically diagnosed DS were collected. Conventional karyotyping involved standard cytogenetic techniques, while QF-PCR utilized DNA extraction and analysis with chromosome-specific short tandem repeat (STR) markers. Results Various distinct physical characteristics were observed in the DS individuals, such as mongoloid slant and low-set ears. Karyotyping and QF-PCR analyses revealed different chromosomal configurations associated with DS trisomy 21, with additional chromosomal abnormalities found in some individuals, including partial monosomy 18 and mosaic trisomy 21. However, in a few cases, neither karyotyping nor QF-PCR revealed any abnormalities. Conclusions The study demonstrated that QF-PCR is a reliable and rapid method for diagnosing DS, providing results within 24 hours. This approach allows for the simultaneous diagnosis of a large number of samples and reduces the time required to obtain results. In the diagnostic procedure for DS, we believe QF-PCR will prove to be a useful tool. Furthermore, therapeutic interventions based on their clinical traits and molecular karyotyping can enhance the quality of life of people with DS.

Post-mortem rapid aneuploidy testing for holoprosencephaly.

BACKGROUND: Abortion and fetal death are common in fetuses with holoprosencephaly, so genetic examinations often have to be made in a post-mortem setting. The efficiency of the conventional karyotyping using cultured fibroblasts in these situations is limited due to frequent culture failure. In the current study, archived cases of holoprosencephaly, where post-mortem genetic evaluation was requested and sufficient frozen material was available, were reevaluated using the quantitative fluorescence polymerase chain reaction (QF-PCR) technique. METHODS: Testing for aneuploidies of chromosomes 13, 15, 16, 18, 21, 22, X, and Y with the QF-PCR technique was carried out on DNA isolated from archived frozen chorionic villi in seven cases of holoprosencephaly. RESULTS: QF-PCR was successful in all seven cases. Two cases of trisomy 13, two cases of triploidy, and one case of trisomy 18 was found meaning a 71% diagnostic yield. The success rate of QF-PCR (100%, 7/7) was superior compared to conventional karyotyping (43%, 3/7). CONCLUSIONS: Rapid aneuploidy testing using the QF-PCR technique is a simple, reliable, time- and cost-effective method sufficient to conclude the etiologic investigation in the majority of holoprosencephaly cases post-mortem.

Molecular biology techniques in real life - case study introducing medical students to QF-PCR technique used in medical diagnostics.

QF-PCR is a widely used molecular biology method. To name just a few of its uses, it is considered to be useful in paternity tests, identification tests or prenatal diagnostics. Therefore, there is a good chance that medical faculty students would come into contact with this technology - directly or indirectly - during their professional work. The following article proposes a teaching classes scenario conducted in the problem-based learning manner, which aims to familiarize students with the QF-PCR technique. In addition, other modern methods of molecular genetics are among topics that students can learn during the problem-based learning modules. The classes are divided into three parts. In the first part, students learn about the possible usage of QF-PCR in paternity tests. The second part focuses on learning about the advantages and limitations of QF-PCR in prenatal diagnosis. Learning activities in the last part are designed to show the limitations of the diagnostic properties of the method - students analyze the case study, in which QF-PCR must be replaced by other modern methods of molecular genetics. By analyzing three independent stories, students learn about usage, advantages and limitations of QF-PCR, and additionally gain knowledge in basic, pre-clinical and clinical sciences. This course is designated as an elective course for final year medical students who have completed either: a basic genetics course, a molecular genetics course, a biochemistry course or a molecular biology course. The focus of the classes is to draw students' attention to the possible application and rapid development of molecular biology techniques, which is the base for modern therapeutic and diagnostic strategies.

Sequential application of copy number variation sequencing and quantitative fluorescence polymerase chain reaction in genetic analysis of miscarriage and stillbirth.

BACKGROUND: Copy number variation sequencing (CNV-seq) could detect most chromosomal abnormalities except polyploidy, and quantitative fluorescence polymerase chain reaction (QF-PCR) is a supplementary method to CNV-seq in triploid detection. This study aimed to evaluate the feasibility of sequential application of CNV-seq and QF-PCR in genetic analysis of miscarriage and stillbirth. METHODS: A total of 261 fetal specimens were analyzed by CNV-seq, and QF-PCR was only further performed for samples with normal female karyotype identified by CNV-seq. Cost and turnaround time (TAT) was analyzed for sequential detection strategy. Subgroup analysis and logistic regression were carried out to evaluate the relationship between clinical characteristics (maternal age, gestational age, and number of pregnancy losses) and the occurrence of chromosomal abnormalities. RESULTS: Abnormal results were obtained in 120 of 261 (45.98%) cases. Aneuploidy was the most common abnormality (37.55%), followed by triploidy (4.98%) and pathogenic copy number variations (pCNVs) (3.45%). CNV-seq could detect the triploidy with male karyotype, and QF-PCR could further identify the remaining triploidy with female karyotype. In this study, we found more male triploidies than female triploidies. With the same ability in chromosomal abnormalities detection, the cost of sequential strategy decreased by 17.35% compared with combined strategy. In subgroup analysis, significant difference was found in the frequency of total chromosomal abnormalities between early abortion group and late abortion group. Results of logistic regression showed a trend that pregnant women with advanced age, first-time abortion, and abortion earlier than 12 weeks were more likely to detect chromosomal aberrations in their products of conception. CONCLUSION: Sequential application of CNV-seq and QF-PCR is an economic and practical strategy to identify chromosomal abnormalities in fetal tissue.

Chromosomal Abnormalities in Early Pregnancy Losses: A Study of 900 Samples.

Chromosomal abnormalities are the most common causes of early pregnancy losses (EPLs). In this study, we aimed to evaluate the incidence and spectrum of chromosomal abnormalities in EPLs and correlate them with different clinical characteristics. We performed Quantitative Fluorescent PCR (QF-PCR), followed by subtelomeric Multiplex Ligation Probe Amplification (MLPA) analysis to detect chromosomal abnormalities in 900 products of conceptions (POCs) from EPLs collected over a period of 10 years. Chromosomal abnormalities were present in 56.25% of uncontaminated EPLs, with significantly higher incidence in women ≥36 years (71.37%, p<0.0001) in comparison to women ≤30 years of age (43.40%). Trisomies were also more common in women ≥36 years (79.68%, p<0.0001) than in those ≤30 years of age (48.70%). In contrast, triploidy and monosomies were more prevalent in women ≤30 years of age (26.09%, p<0.0001 and 16.52%, p=0.0066 respectively) than in women ≥36 years of age (6.42% and 6.42% respectively). Trisomy 16 was more common in women ≤30 (39.29%, p=0.0009) than in those ≥36 years of age (16.78%), while trisomy 22 was predominant among women ≥36 (23.49%, p=0.013), and was not present in the group of women ≤30 years of age. The frequency of chromosomal abnormalities in POCs from women with sporadic (61.19%) was higher than in those with recurrent EPLs (55.21%). This difference, however, was not statistically significant (p=0.164). Although some differences in the chromosomal aneuploidy rates among women with different ABO blood groups, as well as among 6-8 and 9-11 gestational week EPLs were observed, further larger studies are required to confirm these findings. In conclusion, our study enriches the knowledge about chromosomal abnormalities as a cause of EPLs and confirms the higher incidence of foetal chromosomal abnormalities in EPLs in women of older reproductive age. Furthermore, it shows that using QF-PCR and MLPA methodologies, a high detection rate of chromosomal abnormalities in EPLs can be reached.

Cytogenetic Analysis of Sporadic First-Trimester Miscarriage Specimens Using Karyotyping and QF-PCR: A Retrospective Romanian Cohort Study.

It is well known that first-trimester miscarriages are associated with chromosome abnormalities, with numerical chromosome abnormalities being the ones most commonly detected. Conventional karyotyping is still considered the gold standard in the analysis of products of conception, despite the extended use of molecular genetic techniques. However, conventional karyotyping is a laborious and time-consuming method, with a limited resolution of 5-10 Mb and hampered by maternal cell contamination and culture failure. The aim of our study was to assess the type and frequency of chromosomal abnormalities detected by conventional karyotyping in specimens of sporadic first-trimester miscarriages in a Romanian cohort, using QF-PCR to exclude maternal cell contamination. Long-term cultures were established and standard protocols were applied for cell harvesting, slide preparation, and GTG banding. All samples with 46,XX karyotype were tested for maternal cell contamination by QF-PCR, comparing multiple microsatellite markers in maternal blood with cell culture and tissue samples. Out of the initial 311 specimens collected from patients with sporadic first-trimester miscarriages, a total of 230 samples were successfully analyzed after the exclusion of 81 specimens based on unsuitable sampling, culture failure, or QF-PCR-proven maternal cell contamination. Chromosome abnormalities were detected in 135 cases (58.7%), with the most common type being single autosomal trisomy (71/135-52.6%), followed by monosomy (monosomy X being the only one detected, 24/135-17.8%), and polyploidy (23/135-17.0%). The subgroup analysis based on maternal age showed a statistically significant higher rate of single trisomy for women aged 35 years or older (40.3%) compared to the young maternal age group (26.1%) (p = 0.029). In conclusion, the combination of conventional karyotyping and QF-PCR can lead to an increased chromosome abnormality detection rate in first-trimester miscarriages. Our study provides reliable information for the genetic counseling of patients with first-trimester miscarriages, and further large-scale studies using different genetic techniques are required.

Clinical and molecular cytogenetic findings and pregnancy outcomes of fetuses with isochromosome Y.

BACKGROUND: The mosaic forms and clinical phenotypes of fetuses with isochromosome Y are difficult to predict. Therefore, we summarized the cases of nine fetuses with isochromosome Y identified in prenatal diagnosis with a combination of molecular cytogenetic techniques, providing clinical evidence for prenatal genetic counseling. METHODS: The prenatal diagnosis and pregnancy outcomes of nine fetuses with isochromosome Y were obtained by a  retrospective analysis. Isochromosome Y was identified prenatally by different approaches, such as conventional karyotyping, chromosomal microarray analysis (CMA), quantitative fluorescent polymerase chain reaction (QF-PCR) and fluorescence in situ hybridization (FISH). RESULTS: Seven idic(Y) fetuses and two i(Y) fetuses were identified. One fetus was complete for i(Y)(p10), and the rest with 45,X had mosaic forms. A break and fusion locus was identified in Yp11.3 in one fetus, in Yq11.22 in six fetuses and in Yp10 in two fetuses. The CMA results suggested that different deletions and duplications were found on the Y chromosome. The deletion fragments ranged from 4.7 Mb to the entire Y chromosome, and the duplication fragments ranged from 10.4 to 18.0 Mb. QF-PCR analysis suggested that the AZF region was intact in one fetus, four fetuses had AZFb+c+d deletion, one fetus had AZFa+b+c+d deletion, and one fetus had AZFc+d deletion. Finally, four healthy male neonates were delivered successfully, but the parents of the remaining five fetuses, including three healthy and two unhealthy fetuses, chose to terminate their pregnancies. CONCLUSION: The fetus and neonate phenotype of prenatally detected isochromosome Y usually is that of a normally developed male, ascertained in the absence of other indicators of a fetal structural anomaly. Our study provides clinical reference materials for risk assessment and permits better prenatally counseling and preparation of parents facing the birth of isochromosome Y fetuses.

Non-Invasive Prenatal Screening: The First Report of Pentasomy X Detected by Plasma Cell-Free DNA and Karyotype Analysis.

Pentasomy X is a sex chromosome anomaly caused by the presence of three extra X chromosomes in females (49,XXXXX instead of 46,XX) and is probably due to a nondisjunction during the meiosis. So far, only five cases prenatally diagnosed were described. The main features in 49,XXXXX karyotype include severe intellectual disability with delayed speech development, short stature, facial dysmorphisms, osseous and articular abnormalities, congenital heart malformations, and skeletal and limb abnormalities. Prenatal diagnosis is often difficult due to the lack of a clear echographic sign like nuchal translucency (NT), and mostly cases were postnatally described. We report the first case of a 49,XXXXX female that was detected by non-invasive prenatal screening (NIPS), quantitative fluorescence polymerase chain reaction (QF-PCR) and a fetal karyotype.

Stepwise molecular-genetic examination in aborted fetuses.

OBJECTIVE: The evaluation of quantitative fluorescence PCR (QF-PCR) and single nucleotide polymorphism array (SNP array) analysis for the identification of chromosomal abnormalities in products of conception (POC). MATERIALS AND METHODS: A total of 1,094 POC samples were processed at Gennet in the years 2018-2020. Chromosomal aneuploidies were tested by QF-PCR using a Omnibor set (STR markers 13, 18, 21, X a Y), SAB-I set (STR markers 2, 7, 15, 16, 22), SAB-II set (from November 2019, STR markers 4, 6, 14) followed by SNP array analysis (Illumina) on samples with a negative QF-PCR result. All POC samples were tested for maternal contamination. RESULTS: After exclusion of maternal contamination (32% samples) the total number of 742 POC samples were tested by QF-PCR. Chromosomal aneuploidies were found in 273 POC samples (36.8%). Then, 469 QF-PCR negative POC samples were tested by SNP array analysis. Normal female/male profile was confirmed in 402 samples (85.7%) and chromosomal aneuploidies and chromosomal aberrations (deletion/duplication > 10 Mb) in 51 samples (10.9%). Microdeletion/microduplication was found in 16 POC samples (3.4%), two were classified as pathogenic variants and 14 as variants of unknown significance. In a group of women > 35 years of age, statistically significant increase of the chromosomal abnormalities was confirmed. No statistically significant difference between the in vitro fertilization group and the group of spontaneous conception was found. CONCLUSION: The application of the molecular work-up based on the stepwise use of QF-PCR and SNP array clarifies the cause of the abortion in 43% POC samples. The overall detection rate in the I. trimester was 50.4%.

Detection of partial deletion and mosaicism using quantitative fluorescent polymerase chain reaction: Case reports and a review of the literature.

BACKGROUND: Aneuploidy of chromosomes 13, 18, 21, X, and Y can be detected by the quantitative fluorescence polymerase chain reaction (QF-PCR) performed with short tandem repeat (STR) markers. Although QF-PCR is designed to detect whole chromosome trisomy, the partial deletion or mosaic of chromosomes may also be detected. METHODS: Partial deletion or mosaic of chromosomes in three cases was detected by QF-PCR. Karyotyping and chromosome microarray analysis(CMA) were performed. We further reviewed the clinical utility of QF-PCR in detecting mosaicisms and deletions/duplications. RESULTS: QF-PCR demonstrated structurally abnormal 21, X, and Y chromosomes in primary amniotic cells. QF-PCR results in these three cases showed abnormal peak height/peak area, which could not be interpreted according to the kit instructions. QF-PCR results suggested that there were partial deletions or mosaicism, which were confirmed by karyotyping and CMA. CONCLUSION: In addition to detecting trisomies of whole chromosomes, QF-PCR can also detect deletion and mosaicism of chromosomes 13, 18, 21, X, and Y, which could suggest the presence of copy number variants (CNVs). Additional testing with genetic technologies, such as karyotyping or microarrays, is recommended when an uninformative pattern is suspected.

Detección rápida de aneuploidías para la definición de incompatibilidad con la vida extrauterina independiente / Rapid detection of aneuploidies for definition of incompatibility with independent extrauterine life

OBJETIVO: Analizar la implementación de la prueba rápida de reacción en cadena de la polimerasa cuantitativa y fluorescente (QF-PCR) para la detección de aneuploidías. MÉTODO: Se incluyeron todas las pacientes que se realizaron una QF-PCR entre septiembre de 2017 y mayo de 2021. En todos los casos se consignaron los datos clínicos, ecográficos y de laboratorio, y se efectuó un seguimiento de quienes se realizaron además cariograma y su resultado fue normal. RESULTADOS: Se realizaron 213 procedimientos invasivos genéticos prenatales, siendo 72 para detección rápida de aneuploidía mediante QF-PCR. El promedio de edad de las madres con QF-PCR fue de 37 años y 48 pacientes (67%) tenían menos de 15 semanas de gestación. La QF-PCR demostró aneuploidía de los cromosomas 18, 13 y de triploidía en 21 de 49 casos informados como anormales. De los 22 casos sin sugerencia de alteración, 17 accedieron a proseguir el estudio con cariotipo, que resultó anormal en 6 casos. Hubo 4 casos de discordancia entre la QF-PCR y el cariotipo, que pudo afectar el manejo clínico de la gestación. En 25/72 casos (34,7%) la aneuploidía era letal. CONCLUSIONES: Considerando la necesidad de tener un diagnóstico rápido, pero también completo y que permita un consejo genético apropiado, debería integrarse la QF-PCR a un protocolo de diagnóstico que considere variables clínicas y ecográficas.

OBJECTIVE: To analyze the performance of QF-PCR test for the detection of aneuploidies. METHOD: All patients who underwent QF-PCR from September 2017 to May 2021, were included. Clinical, ultrasound and laboratory data were recorded in all cases, as well as follow-up of the cases, including those performing karyotype and the result was normal. RESULTS: 213 prenatal genetic invasive procedures were performed in the study period, 72 for rapid detection of aneuploidy by QF-PCR. 48 patients (67%) were less than 15 weeks at the time of ultrasound diagnosis. The QF-PCR test demonstrated aneuploidy of chromosomes 18, 13, and triploidy in 21/49 cases reported as abnormal. Of the cases without suggestion of alteration (22), 17 agreed to continue the study with a karyotype, which was abnormal in 6 cases. There were 4 cases of discrepancy between QF-PCR and karyotype, which could affect the clinical management of pregnancy. 25/72 cases (34. 7%) corresponded to lethal aneuploidy. CONCLUSIONS: Our results justify the use of QF-PCR. Considering the need to have a rapid diagnosis, but also complete and that allows appropriate genetic counseling, it is that QF-PCR should be integrated into a protocol that considers clinical and ultrasound variables.

QF-PCR: a valuable first-line prenatal and postnatal test for common aneuploidies in South Africa.

Quantitative fluorescence-polymerase chain reaction (QF-PCR) is useful for the detection of aneuploidies involving chromosomes 13, 18, 21, X and Y. Due to the rapid turn-around time and reduced cost compared to traditional karyotyping, QF-PCR has been used as an alternative test for both pre- and postnatal aneuploidy detection in Johannesburg, South Africa since 2001. An internal review of 13,396 aneuploidy tests processed using QF-PCR between January 2015 and December 2019 was performed, and the results showed that the majority (~ 88%) of cases were postnatal tests, with prenatal samples accounting for only ~ 12% of cases. The most common aneuploidies detected were Trisomy 21 (20.6%), Trisomy 18 (3.7%) and Trisomy 13 (2.4%), while sex chromosome aneuploidies were only detected in < 1% of cases. The average percentage of positive cases over the 5-year period was 32.1% for postnatal samples and 11.3% for prenatal samples. QF-PCR testing of the common aneuploidies is being used appropriately, and the high percentage of positive cases demonstrates the value of QF-PCR as prenatal and postnatal tests, particularly in limited resource settings. The higher proportion of positive postnatal cases suggests that referrals are clinically appropriate. However, there is under- and uneven utilization of genetic services in many provinces in South Africa, and the state of prenatal genetic services is poor, as reflected by the low number of prenatal referrals. These results demonstrate the need for programs which will improve the genetic knowledge of referring doctors and the general public, thereby improving the broader utilisation of QF-PCR aneuploidy diagnostic testing, so that patients receive appropriate diagnoses and subsequent management.

Combined diagnosis of QF-PCR and CNV-Seq in fetal chromosomal abnormalities: A new perspective on prenatal diagnosis.

OBJECTIVE: This study aimed to evaluate the effect of QF-PCR and CNV-seq in diagnosing prenatal fetal chromosomal aberrations, explore the advantages and necessity of multimethod joint diagnosis. METHODS: We chose pregnant women with the indication of fetal chromosome examination in our hospital last year, collected 657 cases of amniotic fluid for QF-PCR and CNV-seq analyzes. RESULTS: While detecting aneuploidy, the coincidence rate of QF-PCR and CNV-seq was 100% (56/56). For all 46 chromosomes, 523 cases (79.60%, 523/657) coincided precisely, 128 cases (19.48%, 128/657) showed abnormality with CNV-seq, 8 cases (1.22%, 8/657) revealed abnormality by QF-PCR. In serological Down's syndrome screening, 328 cases showed a high risk of trisomy 21, of which CNV-seq and QF-PCR were consistent in 4 cases (1.22%, 4/328), CNV-seq found 87 cases of CNVs in 78 samples except for chromosomal aneuploidy abnormalities, among these, 18 cases (20.69%, 18/87) were polymorphic, 7 cases (8.05%, 7/87) might cause disease, 13 cases (14.94%, 13/87) caused disease explicitly, 21 cases (24.14%, 21/87) were possibly benign, 17 cases (19.54%, 17/87) were explicitly benign, and the classification of 11 cases (12.64%, 11/87) was unclear. CONCLUSION: QF-PCR and CNV-seq were highly consistent in diagnosing chromosomal aneuploidy. The high risk of serological Down's screening might not only due to the aneuploidy of chromosomes 21, 18, and NTD, but also the microdeletion or microduplication of all 46 chromosomes. So using CNV-seq combined with QF-PCR could effectively reduce the risk of missed diagnosis.

The Contribution of QF-PCR and Pathology Studies in the Diagnosis of Diandric Triploidy/Partial Mole.

OBJECTIVE: the aim of our study was to assess the contribution of quantitative fluorescent polymerase chain reaction (QF-PCR) and pathology studies in the diagnosis of diandric triploidies/partial hydatidiform moles. METHODS: this study included all fet al triploidies diagnosed by QF-PCR in chorionic villi or amniotic fluid in the 2 centers of BCNatal in which a maternal saliva sample was used to establish its parental origin. Pathology studies were performed in products of conception and concordance between a partial hydatidiform mole diagnosis and the finding of a diandric triploidy was assessed. RESULTS: among 46 fetal triploidies, found in 13 ongoing pregnancies and in 33 miscarriages, there were 26 (56%) diandric triploidies. Concordant molecular (diandric triploidy) and pathology results (partial mole) were achieved in 14 cases (54%), while in 6 cases (23%) pathology studies were normal, and in the remaining 6 cases (23%) pathology studies could not be performed because miscarriage was managed medically. CONCLUSIONS: diandric triploidy is associated with partial hydatidiform mole and its diagnosis is crucial to prevent the development of persistent trophoblastic disease. QF-PCR analysis in chorionic villi or amniotic fluid provides a more accurate diagnosis of the parental origin of triploidy than the classical pathology studies.

Copy number variation sequencing combined with quantitative fluorescence polymerase chain reaction in clinical application of pregnancy loss.

PURPOSE: In this study, we evaluated the feasibility of the combining CNV-seq and quantitative fluorescence polymerase chain reaction (QF-PCR) for miscarriage analysis in clinical practice. METHODS: Over a 35-month period, a total of 389 fetal specimens including 356 chorionic villi and 33 fetal muscle tissues were analyzed by CNV-seq and QF-PCR. Relationships between the risk factors (e.g., advanced maternal age, abnormal pregnancy history, and gestational age) and incidence of these chromosomal abnormalities were further analyzed by subgroup. RESULTS: Clinically significant chromosomal abnormalities were identified in 58.95% cases. Aneuploidy was the most common abnormality (46.84%), followed by polyploidy (8.16%) and structural chromosome anomalies (3.95%). In sub-group analysis, significant differences were found in the total frequency of chromosomal abnormalities between the early abortion and the late abortion group, as well as in the distribution of chromosomal abnormalities between the advanced and the younger maternal age group. Meanwhile, the results of the logistic regression analysis identified a trend suggesting that the percentage of fetal chromosomal abnormalities is significantly higher in advanced maternal age, lesser gestational age, and lesser number of prior miscarriages. CONCLUSION: Our study suggests that CNV-seq and QF-PCR are efficient and reliable technologies in the fetal chromosome analysis of miscarriages and could be used as a routine selection method for the genetic analysis of spontaneous abortion.

Distribution of diandric and digynic triploidy depending on gestational age.

PURPOSE: To establish the distribution of diandric and digynic triploidy depending on gestational age. METHODS: 107 triploid samples tested prospectively in a single genetic department during a four-year period were analyzed for parental origin of triploidy by Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) (n=95) with the use of matching parental samples or by MS-MLPA (n=12), when parental samples were unavailable. Tested pregnancies were divided into three subgroups with regard to the gestational age at spontaneous pregnancy loss: <11 gestational weeks, 11-14 gestational weeks, and >14 gestational weeks. RESULTS: Diandric triploidy constituted overall 44.9% (46.5% in samples miscarried <11 gestational weeks, 64.3% in samples miscarried between 11 and 14 gestational weeks, and 27.8% in pregnancies which survived >14 gestational weeks). CONCLUSIONS: The distribution of diandric and digynic triploidy depends on gestational age. The majority of diandric triploid pregnancies is lost in the first trimester of pregnancy. In the second trimester, diandric cases are at least twice less frequent than digynic ones.

An experience in prenatal diagnosis via QF-PCR of a female child with a 9.9 Mb pure deletion at 18p11.32-11.22.

Quantitative Fluorescent - Polymerase Chain Reaction (QF-PCR) is a rapid prenatal diagnosis test for 21, 18, 13 and sex chromosomal aneuploidy detection. However, it could not detect partial trisomy or partial monosomy of those chromosomes. Here, we report a 19-month-old Vietnamese female with a 9.9 Mb pure deletion of chromosome 18 at 18p11.32-11.22 confirmed by next generation sequencing. The patient was short statured with facial dysmorphic features as well as motor skill and speech delays. First trimester screening showed high risk of trisomy 21 with only increased nuchal translucency (NT 3.9 mm) by ultrasound as an indication. Prenatal diagnosis by QF-PCR from amniotic DNA revealed normal disomy. Noticeably, two short tandem repeat (STR) markers D18S391 and D18S976 located on 18p exhibited uninformative patterns (one peak). Thus, our case suggested that the combination of both D18S391 and D18S976 markers with uninformative patterns in QF-PCR for prenatal diagnosis and increased NT in the first trimester ultrasound may be a significant indication of 18p monosomy.

Investigation of LOY in Prostate, Pancreatic, and Colorectal Cancers in males: A case-control study.

Background: LOY is associated with ageing and increase the incidence of cancers. Aims: To elucidate the role of LOY in various cancer types, namely, prostate (PRT), pancreatic (PC), and colorectal (CRC) cancers in males. Material and Methods: Fifty CRC patients [mean age = 44.58±11.2 years], fifty PRT [mean age= 60.48± 17.07 years] and fifty PC [mean age = 48.74 ±16.45 years] along with 100 healthy controls [mean age= 54.06 ±15.04 years] were recruited. DNA was isolated from peripheral blood and was subjected to multiplex QF-PCR. The Y/X ratio was calculated from the peak height. Results: The mean Y/X ratio was lower in all patients with cancers (0.875333± 0.086; p value˂ 0.0001) than in controls (1.11 ± 0.071), as well as, in CRC (0.926±0.192; p value˂0.0001), PC (0.85 ± 0.0311; p value˂0.0001) and PRT (0.85±0.122; p value˂0.0001) when calculated separately. Multivariate logistic regression analysis was used to analyze the strength of the presence of cancer prediction using the percentage of LOY and age showed that LOY (p= 0.001) is a better predictor of cancer presence than age (p= 0.359). Conclusion: LOY in blood could be a predictive biomarker in the carcinogenesis of males.

Two Reliable Methodical Approaches for Non-Invasive RHD Genotyping of a Fetus from Maternal Plasma.

Noninvasive fetal RHD genotyping is an important tool for predicting RhD incompatibility between a pregnant woman and a fetus. This study aimed to assess a methodological approach other than the commonly used one for noninvasive fetal RHD genotyping on a representative set of RhD-negative pregnant women. The methodology must be accurate, reliable, and broadly available for implementation into routine clinical practice. A total of 337 RhD-negative pregnant women from the Czech Republic region were tested in this study. The fetal RHD genotype was assessed using two methods: real-time PCR and endpoint quantitative fluorescent (QF) PCR. We used exon-7-specific primers from the RHD gene, along with internal controls. Plasma samples were analyzed and measured in four/two parallel reactions to determine the accuracy of the RHD genotyping. The RHD genotype was verified using DNA analysis from a newborn buccal swab. Both methods showed an excellent ability to predict the RHD genotype. Real-time PCR achieved its greatest accuracy of 98.6% (97.1% sensitivity and 100% specificity (95% CI)) if all four PCRs were positive/negative. The QF PCR method also achieved its greatest accuracy of 99.4% (100% sensitivity and 98.6% specificity (95% CI)) if all the measurements were positive/negative. Both real-time PCR and QF PCR were reliable methods for precisely assessing the fetal RHD allele from the plasma of RhD-negative pregnant women.