Jon Sherlock

Assessment of diagnostic quantitative fluorescent multiplex polymerase chain reaction assays performed on single cells

We have refined polymerase chain reaction (PCR) assays for the detection of sickle cell anaemia, the delta F 508 deletion causing cystic fibrosis, and the IVS1–110 mutation leading to beta‐thalassaemia, allowing them to be successfully performed upon single cells using fluorescent primers. We have also assessed the possibility of detecting aneuploidies of chromosomes 13, 18 and 21 using a quantitative fluorescent polymerase chain reaction (QF‐PCR) with primers flanking polymorphic short tandem repeat (STR) markers. Trisomies were readily diagnosed by the detection of tri‐allelic patterns. However some heterozygote normal and trisomic diallelic patterns did not produce the expected ratios of amplified PCR products due to preferential DNA sequence amplification. Total allelic drop out (ADO) did not occur with any of the cells tested. Multiplex QF‐PCR assays can be performed on a single cell in under 6 h and simultaneously provide diagnosis of single gene defects, sex determination and an indication of selected chromosome aneuploidy.

Rapid detection of aneuploidies by microsatellite and the quantitative fluorescent polymerase chain reaction

Several studies have been performed to assess the diagnostic value of using small tandem repeat (STR) markers and quantitative fluorescent polymerase chain reaction (QF‐PCR) assays for the rapid detection of aneuploidies involving chromosomes 21, 18, 13 (Mansfield, 1993; Pertl et al., 1994, 1996; Adinolfi et al., 1995a). The results of these investigations have documented the diagnostic advantages of this approach to perform prenatal tests using amniotic and chorionic samples, or fetal nucleated cells retrieved from peripheral maternal blood or endocervical samples. The use of two or more STR markers for each autosome facilitates the diagnosis of aneuploidies, while avoiding the need to employ internal non‐polymorphic markers. Multiplex quantitative fluorescent analyses can be performed in about six hours from the collection of the samples and, although targeted to specific abnormalities, they can exclude the presence of the most frequent chromosomal disorders.

Strategies for preimplantation genetic diagnosis of single gene disorders by DNA amplification

Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis for ensuring the genetic health of offspring born to families affected by inherited disease. This paper sets out to review current protocols for the diagnosis of single gene defects in human preimplantation embryos. These methods, which depend on DNA amplification using PCR, are subject to a vareity of pitfalls, such as allele dropout (ADO), contamination and reduced amplification efficiency. Advances in single cell DNA amplification, such as improved multiplex PCR protocols, fluorescent‐PCR and whole genome amplification (WGA), can be applied to address some of these problems. Different PGD strategies are discussed in the context of their clinical application. Copyright

Rapid detection of trisomies 21 and 18 and sexing by quantitative fluorescent multiplex PCR

Abstract Aneuploidies involving chromosomes 21, 18, 13, X and Y account for over 95% of all chromosomal abnormalities in live-born infants. Prenatal diagnosis of these disorders is usually accomplished by cytogenetic analysis of amniotic or chorionic cells but this is a lengthy procedure requiring great technical expertise.In this paper, we assess the diagnostic value of using a quantitative fluorescent polymerase chain reaction (PCR) suitable for the simultaneous and rapid diagnosis of trisomies 21 and 18 together with the detection of DNA sequences derived from the X and Y chromosomes. Samples of DNA, extracted from amniotic fluid, fetal blood or tissues, and peripheral blood from normal adults were investigated by quantitative fluorescent PCR amplification of polymorphic small tandem repeats (STRs) specific for two loci on each of chromosomes 21 and 18. Quantitative analysis of the amplification products allowed the diagnosis of trisomies 21 and 18, while sexing was performed simultaneously using PCR amplification of DNA sequences derived from the chromosomes X and Y. These results indicate the advantages of using two sets of STR markers for the detection of chromosome 21 trisomies and confirmed the usefulness of quantitative fluorescent multiplex PCR for the rapid prenatal diagnosis of selected chromosomal abnormalities.

Rapid detection of chromosomes X and Y aneuploidies by quantitative fluorescent PCR

Quantitative fluorescent polymerase chain reaction (QF‐PCR) assays and small tandem repeat (STR) markers have been successfully employed for the rapid detection of major numerical aneuploidies affecting human autosomes. So far, the analysis of chromosomes X and Y disorders has been hampered by the rarity of highly polymorphic markers which could distinguish normal female homozygous PCR patterns from those seen in patients with Turners syndrome. A new marker (X22) of the X/Y chromosomes has been identified which maps in the Xq/Yq pseudoautosomal region PAR2; used together with the HPRT it allows the rapid diagnosis of numerical aneuploidies of the sex chromosomes. Blood samples from normal male and female subjects and from patients with X and Y chromosome disorders (45,X and 47,XXY) have been tested by QF‐PCR with the X22 polymorphic pentanucleotide (12 alleles) together with the HPRT and P39 markers. The samples were also tested by multiplex QF‐PCR with STRs specific for chromosomes 21,18,13 and amelogenin (AMXY). Tested by QF‐PCR, all samples from normal females were heterozygous for either the X22 or the HPRT marker with fluorescent peak ratios near 1:1, thus allowing a correct, rapid diagnosis of their chromosome complement. Turners patients (45,X) showed only one X22 and one HPRT fluorescent peak, thus documenting the presence of a single X chromosome. Turners patients with mosaicism showed a major fluorescent peak for the X22 and HPRT markers and a minor peak revealing the presence of a second minor population of cells. Two 47,XXY cases could also be diagnosed. Multiplex analyses can be performed using simultaneously STR markers for chromosomes 21,18,13 X and Y. The diagnostic value of a third X‐linked marker (P39) was also investigated. These results suggest that rapid diagnosis of major numerical anomalies of the X and Y chromosomes can be performed using QF‐PCR with a new highly polymorphic X‐linked marker, X22, which maps in the Xq/Yq pseudoautosomal region PAR 2. Multiplex QF‐PCR tests—using the X22 STR in association with HPRT and, in rare cases, a third P39 marker—allow the rapid diagnosis of major aneuploidies affecting chromosomes 21, 18, 13, X and Y. The X22 marker can also be employed for the detection of fetal cells present in maternal peripheral blood or the endocervical canal. Copyright

Quantitative fluorescence polymerase chain reaction for the rapid prenatal detection of common aneuploidies and fetal sex

OBJECTIVE We have developed a quantitative fluorescence multiplex polymerase chain reaction assay for the rapid detection of sex and aneuploidies involving chromosomes 21, 18, and 13. STUDY DESIGN Samples of deoxyribonucleic acid (n = 85) extracted from amniotic fluid, fetal tissues, and blood were investigated by multiplex polymerase chain reaction amplification of polymorphic small tandem repeat markers specific for chromosomes 21, 18, 13, and X. RESULTS Quantitative analysis of the polymerase chain reaction products allowed us to distinguish between normal samples and samples with autosomal trisomies while sexing was performed simultaneously. From 85 samples only three produced unsatisfactory results with one of the two chromosome 13-specific markers. In these three cases the amplification of the other chromosome 13 marker always resulted in a correct normal pattern. CONCLUSION Quantitative fluorescence multiplex polymerase chain reaction is a reliable and rapid method that allows prenatal diagnosis of the major numeric chromosomal abnormalities to be performed within 24 hours.

Detection of trophoblast cells in transcervical samples collected by lavage or cytobrush

Objective To compare two methods of obtaining fetal cells from the endocervical canal for prenatal diagnosis during the first trimester: lavage with physiologic saline, and the endocervical passage of a cytobrush. Methods Fetal cells were identified morphologically using conventional and immunohistochemical staining. Y-specific sequences were detected using polymerase chain reaction (PCR) and fluorescent in situ hybridization. The presence of fetal DNA was also demonstrated by PCR amplification of a polymorphic short tandem repeat marker. Results Syncytiotrophoblast cells were identified in four of 11 lavage samples and in one of 11 brush samples. Sex determination using fluorescent in situ hybridization was achieved in all 11 lavage samples and in nine of 11 cytobrush samples; no Y sequences were located in two brush samples from male embryos. Sex determination was achieved successfully in all 22 samples using PCR of X-and Y-specific DNA sequences. Small tandem repeat pattern analysis indicated the presence of fetal DNA in five of 11 lavage samples and in one of eight cytobrush samples, which were informative because the maternal and fetal small tandem repeat patterns differed. Conclusion Although lavage retrieves more trophoblast cells than does brushing, both methods are potentially suitable for molecular prenatal diagnosis.

A successful strategy for preimplantation genetic diagnosis of myotonic dystrophy using multiplex fluorescent PCR

The most common form of inherited muscular dystrophy in adults is myotonic dystrophy (DM), an autosomal‐dominant disease caused by the expansion of an unstable CTG repeat sequence in the 3′ untranslated region of the myotonin protein kinase (DMPK) gene. Expanded (mutant) CTG repeat sequences are refractory to conventional PCR, but alleles with a number of repeats within the normal range can be readily amplified and detected. Preimplantation genetic diagnosis (PGD) of DM has been successfully applied. However, a misdiagnosis using the reported protocol was recently documented. Two new PGD protocols for DM have been developed which utilise multiplex fluorescent PCR. Ideally a linked polymorphic marker, APOC2, is amplified in addition to the normal DMPK alleles, thus providing a back‐up diagnostic result. However, the two couples reported in the present study were not fully informative at the APOC2 locus and so an unlinked short tandem repeat (STR) marker, D21S1414, was substituted. The highly polymorphic nature of the D21S1414, DMPK and APOC2 loci means that a very simple genetic fingerprint can be generated by analyses of these loci. This allows most DNA contaminants to be detected. Contamination is a significant problem for PGD and is the primary reason for the inclusion of D21S1414 and APOC2 in this protocol. This paper reports the first clinical experience and pregnancies following PGD for DM using a multiplex fluorescent PCR protocol. Copyright

Fetal cells in transcervical samples at an early stage of gestation

AbstractSeveral investigations are in progress with the aim of performing prenatal diagnosis of inherited disorders by noninvasive or minimally invasive techniques. The most important approaches are based on the detection of fetal nucleated cells in maternal blood, the analysis of fetal DNA present in maternal plasma, and the identification and isolation of fetal trophoblastic cellular elements shed into the uterine cavity and the endocervical canal. In this review, we discuss the methods that have been employed for the collection of the transcervical samples at an early stage of gestation and the techniques used for the identification of fetal cells. We also report the results of using endocervical cells for the detection of fetal chromosomal disorders by fluorescent in-situ hybridization and for performing prenatal diagnosis of fetal Rh(D) phenotypes. Recent investigations have also shown that — after the isolation of trophoblastic cells from maternal contaminants by micromanipulation — transcervical samples can be employed for the prenatal diagnosis of single gene defects, such as those causing thalassemia and sickle cell anemia. Although the present results are promising, further investigations are required to demonstrate the feasibility of performing accurate diagnosis of fetal diseases by this minimally invasive approach in all transcervical samples retrieved at an early stage of gestation.

Prenatal detection of Hb mutations using transcervical cells

Prenatal diagnoses were performed on six selected pairs of parents known to be carriers of Hb mutations by testing transcervical cells (TCCs) retrieved, prior to chorionic villus sampling (CVS), by aspiration of the cervical mucus from the pregnant mothers at 10–12 weeks of gestation. A concordance between the results of testing chorionic villus cells and isolated clumps of trophoblastic cellular elements was observed in four of the six cases.