Fluorescent Polymerase Chain Reaction Analysis Research Articles

Celocentesis for early prenatal diagnosis of hemoglobinopathy

Celocentesis is an invasive technique that can provide prenatal diagnosis of single-gene disorders, from as early as 7 weeks' gestation. The objective of this study was to examine the safety of celocentesis. In this prospective study, celocentesis was performed for prenatal diagnosis of hemoglobinopathy in 402 singleton pregnancies in which both parents were carriers of β-thalassemia or sickle cell disease trait. We assessed procedure-related maternal discomfort or pain, success of sampling and obtaining results, pregnancy outcome and postnatal follow-up. First, celocentesis was carried out at a median gestational age of 8.6 (range, 6.9-9.9) weeks and celomic fluid was successfully aspirated in 99.8% of cases. Second, 67% of women had no or only mild discomfort, 18% had moderate discomfort, 12% had mild-to-moderate pain and 3% had severe pain. Third, prenatal diagnosis from analysis of the celomic fluid was successful in 93.8% cases, and in the last 121 cases, it was always successful. Fourth, in all cases of successful sampling and analysis of celomic fluid, the diagnosis was concordant with results obtained from additional prenatal or postnatal testing. Fifth, in addition to diagnosis of hemoglobinopathy, quantitative fluorescence polymerase chain reaction analysis, which was performed to evaluate maternal contamination using several markers for chromosomes X, Y, 21, 18 and 13, led to the accurate diagnosis of chromosomal aneuploidy. Sixth, in all cases of an affected fetus diagnosed by celocentesis in which the parents chose termination of pregnancy, this was carried out < 10 weeks' gestation. Seventh, in 97.1% (298/307) of the continuing pregnancies there was live birth, in seven (2.3%) there was miscarriage and in two (0.7%) there was loss to follow-up. Eighth, fetal abnormalities were diagnosed in three (1%) cases, including unilateral transverse amputation of the forearm, unilateral moderate hydronephrosis and small-bowel duplication. All neonates were examined by a pediatrician and were found to be phenotypically normal, except for the three cases with a prenatally diagnosed defect. Celocentesis can be used for early prenatal diagnosis of genetic abnormalities, and the procedure-related risk of pregnancy complications appears to be low. Copyright © 2020 ISUOG. Published by John Wiley & Sons Ltd.

Molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 8 or r(8)(::p11.22→q11.21::) in an 18-year-old female with short stature, obesity, attention deficit hyperactivity disorder, and intellectual disability

ObjectiveWe present molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome (sSMC) derived from chromosome 8. Materials and MethodsAn 18-year-old female presented with short stature, obesity, developmental delay, speech delay, dyslexia, attention deficit hyperactivity disorder, and intellectual disability. Cytogenetic analysis of the peripheral blood revealed a karyotype of 47,XX,+mar[22]/46,XX[18]. Array comparative genomic hybridization and metaphase fluorescence in situ hybridization analyses were performed on the peripheral blood to determine the origin and mosaicism of the sSMC, and quantitative fluorescent polymerase chain reaction was used to exclude uniparental disomy. ResultsArray comparative genomic hybridization analysis of the blood revealed a result of arr 8p11.22q11.21 (39,136,065-49,725,726)×2.80 (Log2 ratio=0.49), consistent with 70–80% mosaicism, encompassing 33 OMIM genes including GOLGA7, AGPAT6, NKX6-3, KAT6A, and FNTA. The sSMC(8) was r(8)(::p11.22→q11.21::). Metaphase fluorescence in situ hybridization analysis using the probes of RP11-754D24 (8p11.21) and RP11-769N21 (8q11.21) showed the sSMC(8) in 12/27 of cultured lymphocytes. Quantitative fluorescent polymerase chain reaction analysis excluded uniparental disomy 8. ConclusionMosaic sSMC(8) derived from r(8)(::p11.22→q11.21::) can be associated with obesity, intellectual disability, and attention deficit hyperactivity disorder.

Molecular cytogenetic characterization of an inv dup(15) chromosome presenting as a small supernumerary marker chromosome associated with the inv dup(15) syndrome

ObjectiveTo present molecular cytogenetic characterization of an inverted duplication of proximal chromosome 15 [inv dup(15)] presenting as a small supernumerary marker chromosome (sSMC) associated with the inv dup(15) syndrome. Case ReportA 35-year-old woman underwent amniocentesis because of advanced maternal age at 27 weeks of gestation, which revealed an sSMC that was confirmed by fluorescence in situ hybridization (FISH) to be derived from chromosome 15. Prenatal ultrasound findings were unremarkable. A 3434-g male baby was delivered at term with no phenotypic abnormalities. The cord blood analysis revealed a bisatellited dicentric inv dup(15). When followed up at 21 years of age, the proband manifested hypotonia, ataxic gait, developmental delay, intellectual disability, epilepsy, poor speech, and autism consistent with the inv dup(15) syndrome. Array comparative genomic hybridization of the peripheral blood revealed arr 15q11.1q13.2 (20,686,219–30,390,043) × 4, 15q13.2q13.3 (30,390,043–32,445,226) × 3. Conventional cytogenetic analysis of the peripheral blood revealed a karyotype of 47,XY,+inv dup(15)(pter→q13::q13→pter). Quantitative fluorescent polymerase chain reaction analysis showed a maternal origin of the inv dup(15) chromosome. FISH analysis confirmed an inv dup(15) chromosome. ConclusionMolecular cytogenetic techniques are useful for rapid diagnosis of an inv dup(15) chromosome associated with the inv dup(15) syndrome.

Correlation between thyroglobulin gene polymorphisms and autoimmune thyroid disease.

The aim of the present study was to detect thyroglobulin (Tg) gene polymorphisms in a Han Chinese population from the Northern regions of Henan province, China, and to study the correlation between Tg gene polymorphisms and autoimmune thyroid disease (AITD). A total of 270 patients with AITD and 135 healthy controls were enrolled. Genomic DNA was extracted and fluorescence polymerase chain reaction analysis was performed; high‑resolution melting curve analysis (HRMA) was used to detect single‑nucleotide polymorphisms (SNPs) in exons 10, 12 and 33 of the Tg gene. SNPs were then correlated with AITD. Han people from the Northern regions of Henan displayed four Tg exon SNPs: E10SNP24 T/G, E10SNP158 T/C, E12SNP A/G and E33SNP C/T. Several allele and genotype frequencies differed between the AITD group and the healthy control group (Tg E10SNP: Allele T, P<0.01; allele G, P<0.01; and Tg genotype GG, P<0.01; genotype TG, P<0.01. Tg E12SNP: Allele A, P<0.01; allele G, P<0.01; Tg genotype GG, P<0.01; genotype AG, P<0.01). A statistically significant difference in the frequency of selected Tg SNPs haplotypes was also present between AITD patients and healthy controls (P<0.05). There was no significant difference in haplotypes between various types of AITD (hypothyroidism, hyperthyroidism and Hashimoto's disease). The Tg SNP frequency distribution was significantly different between Han populations of the Northern regions of Henan province and the Xi'an regions of Shaanxi province. The results of the present study suggested that specific Tg gene alleles or genotypes were correlated with AITD; specific Tg SNP haplotypes were associated with hypothyroidism, hyperthyroidism and Hashimoto's disease, and the Tg SNP frequency distribution differed depending on the geographical location of the Han Chinese populations.

De novo unbalanced translocation resulting in monosomy for distal 5p (5p14.1 → pter) and 14q (14q32.31 → qter) associated with fetal nuchal edema, microcephaly, intrauterine growth restriction, and single umbilical artery: Prenatal diagnosis and molecular cytogenetic characterization

ObjectiveTo present prenatal diagnosis of partial monosomy 5p (5p14.1 → pter) and partial monosomy 14q (14q32.31 → qter). Materials and MethodsA 33-year-old woman underwent amniocentesis at 20 weeks of gestation because of abnormal fetal ultrasound. Amniocentesis revealed a dicentric chromosome of dic(5;14). Level II ultrasound at 23 weeks of gestation revealed a fetus with intrauterine growth restriction, microcephaly, nuchal edema, a single umbilical artery, and fetal biometry equivalent to 19 weeks. At 23 weeks of gestation, she requested repeated amniocentesis. Whole-genome array comparative genomic hybridization on uncultured amniocytes was performed. Quantitative fluorescent polymerase chain reaction analysis was performed on uncultured cord blood and parental blood. A fetus was delivered with microcephaly, low-set ears, hypertelorism, depressed nasal bridge, increased nuchal fold, and a single umbilical artery. ResultsThe fetal karyotype was 45,XX,dic(5;14)(p14.1;q32.31)dn. Whole-genome array comparative genomic hybridization analysis on uncultured amniocytes detected arr 5p15.33p14.1 (36,238-28,798,509)×1 and arr 14q32.31q32.33 (101,508,967-107,349,540)×1. Quantitative fluorescent polymerase chain reaction assays showed that the aberrant dic(5;14) was from paternal origin. ConclusionConcomitant occurrence of monosomy for distal 5p and distal 14q my present nuchal edema, microcephaly, IUGR, and single umbilical artery on prenatal ultrasound.

A Unique Patient Presenting With Concomitant Klinefelter Syndrome, Alport Syndrome, and Craniopharyngioma

A 31-year-old Caucasian male was referred for panhypopituitarism resulting from a surgically removed craniopharyngioma. The patient had been previously submitted to kidney transplantation for end-stage renal disease from X-linked Alport syndrome (ATS). Subsequent quantitative fluorescent polymerase chain reaction analysis indicated a 47,XXY karyotype consistent with Klinefelter syndrome (KS). The relevance of this unique case stems from several issues: 1) KS was an unexpected finding because of a previous diagnosis of hypogonadotropic hypogonadism resulting from craniopharyngioma; 2) the discovery of a de novo p.G406S substitution causing ATS; and 3) the multifactor origin of severe sexual dysfunction. This is the first description of the co-occurrence of KS, ATS, and craniopharyngioma.

Prenatal diagnosis and molecular cytogenetic characterization of a derivative chromosome der(18;18)(q10;q10)del(18)(q11.1q12.1)del(18)(q22.1q22.3) presenting as apparent isochromosome 18q in a fetus with holoprosencephaly

Objective To present prenatal diagnosis and molecular cytogenetic characterization of a derivative chromosome der(18;18)(q10;q10)del(18)(q11.1q12.1)del(18)(q22.1q22.3). Materials, Methods, and Results A 32-year-old woman was referred for genetic counseling of prenatally detected isochromosome 18q [i(18q)]. She had undergone amniocentesis at 19 gestational weeks because of a trisomy 18 risk of 1/39 derived from abnormally low levels of maternal serum unconjugated estriol, inhibin A, α-fetoprotein, and total β-human chorionic gonadotropin. Amniocentesis revealed a karyotype of 46,XX,i(18)(q10). Parental karyotypes were normal. Prenatal ultrasound showed alobar holoprosencephaly. Repeated amniocentesis was requested and performed at 21 gestational weeks. Array-comparative genomic hybridization analyses revealed a 14-Mb deletion of 18p11.32-p11.21, a 37.8-Mb duplication of 18q12.1-q22.1, and a 6.9-Mb duplication of 18q22.3-q23. Metaphase fluorescence in situ hybridization study showed the absence of an 18q12.1-specific probe signal in one arm and the absence of an 18q22.2-specific probe signal in the other arm of the derivative chromosome. Quantitative fluorescent polymerase chain reaction analysis determined a paternal origin of the derivative chromosome. The cytogenetic result was 46,XX,der(18;18)(q10;q10)del(18)(q11.1q12.1)del(18)(q22.1q22.3). The fetus postnatally manifested cebocephaly. Conclusion Concomitant monosomy 18p and trisomy 18q can be associated with holoprosencephaly and abnormal maternal serum screening results. Array-comparative genomic hybridization, fluorescence in situ hybridization, and quantitative fluorescent polymerase chain reaction are useful in genetic counseling of prenatally detected isochromosomes by providing information on the origin and genetic components of the isochromosome.

Prenatal detection of full monosomy 21 in a fetus with increased nuchal translucency: Molecular cytogenetic analysis and review of the literature

Full monosomy 21 is an extremely rare chromosomal disorder. A 38-year-old woman attended a first trimester scan. Ultrasound (U/S) imaging of the fetus at 12 weeks of gestation showed features of increased nuchal translucency measurement (12 mm). Chorionic villi sampling (CVS) was performed after genetic counseling. At 16 weeks of gestation the fetus showed U/S characteristics of severe intrauterine growth restriction, generalized edema and hydrothorax. Cytogenetic examination was performed using quantitative fluorescent polymerase chain reaction analysis, standard Giesma banding and fluorescent in situ hybridization analysis. Non-mosaic full monosomy 21 was detected and the parents opted to terminate the pregnancy. Microsatellite analysis demonstrated maternal origin of the single chromosome. This case represents one of the few cases of prenatally diagnosed full monosomy 21 confirmed only by CVS, in which the parental origin of the single chromosome was determined.

Diagnosis and prediction of parental origin of triploidies by fetal nuchal translucency and maternal serum free β‐hCG and PAPP‐A at 11–14 weeks of gestation

The study objective was to determine the parental origin of triploidy in relation to findings from early risk assessment in a combined screening program between 2004 and the end of 2006. Triploidy was diagnosed in six chorion villus samples and two samples from missed abortions. After informed consent, quantitative fluorescence polymerase chain reaction analysis was performed on the five cases where we received blood from both parents and tissue from fetuses. In four cases the origin of the triploidy was paternal and in one maternal, in accordance with previous findings in type I and type II triploidies. Finding triploidy is possible by risk assessment (ultrasound and double test), and thereby women may have the opportunity for early termination of pregnancy.

Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Manure Using Whatman FTA Card Technology and Lightcycler Real-Time PCR

A modified forensic DNA extraction and real-time fluorescent polymerase chain reaction assay has been evaluated for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine fecal samples using primers and fluorescent resonance energy transfer (FRET) probes targeting the IS900 gene sequence of MAP. DNA was successfully extracted from manure samples by utilizing the Whatman FTA card technology, which allows for simple processing and storage of samples at room temperature. The FTA cards were washed and subjected to a Chelex-100 incubation to remove any remaining polymerase chain reaction (PCR) inhibitors and to elute the DNA from the FTA card. This isolated DNA was then subjected to direct real time fluorescent PCR analysis. Detection of MAP DNA from bovine fecal samples spiked with known concentrations of viable MAP cells was obtained. The detection limits of the assay was consistently found to be between 10(2) and 10(4) colony forming units [CFU]/g, with some samples containing as low as 10 CFU/g, yielding positive assay results. This cost-efficient assay allows reporting of results as early as 4 h after fecal collection, which can be particularly useful in highthroughput herd screening.

First description of somatic mosaicism in MYH9 disorders

MYH9 disorders are characterized by giant platelets, thrombocytopenia, and Dohle body-like cytoplasmic granulocyte inclusion bodies that result from mutations in MYH9, which encodes non-muscle myosin heavy chain-A (NMMHCA). These disorders are known to be transmitted in an autosomal dominant manner, although about 20% of cases are considered to be sporadic. We report here the first case of a MYH9 disorder because of somatic mosaicism. The patient was the father of a male with typical May-Hegglin anomaly. The father had normal platelet counts, however, both normal-sized and giant platelets were observed on his peripheral blood smears. In addition, 14% of neutrophils contained inclusion bodies and the rest showed a normal morphology. Quantitative fluorescent polymerase chain reaction analysis showed that only 6% of DNA from peripheral blood leucocytes harboured the mutation. The mosaicism was demonstrated at a similar rate in different tissues, buccal mucosa cells and hair bulb cells, implying that the mutation had occurred before gastrulation. Mosaicism might account for some de novo mutations in MYH9 disorders.

Identification of a high frequency of mutation at exon 8 of the ATP7B gene in a Chinese population with Wilson disease by fluorescent PCR.

Wilson disease (WD) is an autosomal recessive disorder of copper transport. Mutation analysis has led to the discovery of more than 100 mutations at ATP7B, and most of them are population specific. To verify the high frequency of mutation at exon 8 of ATP7B in Chinese patients with WD and to establish a DNA diagnostic method for WD. University medical centers. Screening for mutations at exon 8 of ATP7B by fluorescent polymerase chain reaction analysis and restriction analysis was conducted in 106 unrelated Chinese patients with WD and in 55 individuals from 10 Chinese families with WD. Five homozygotes and 32 heterozygotes were identified. Sequence analysis showed a missense mutation (2273G-->T) and a nonsense mutation (2250C-->G) together at exon 8. The rate of gene mutation in 106 patients was 35% (5% homozygous and 30% heterozygous). Samples of DNA from 55 individuals from 10 Chinese families with WD were examined by fluorescent polymerase chain reaction. We found that 13 siblings were carriers (24%). A high frequency of mutation at exon 8 of the ATP7B gene exists in the Chinese population, and fluorescent polymerase chain reaction analysis may be an effective and accurate assay in detection of the WD gene.