Blood transfusion in clinical medicine

DNA in amounts representative of hundreds of eukaryotic genomes was extended on silanized surfaces by dynamic molecular combing. The precise measurement of hybridized DNA probes was achieved directly without requiring normalization. This approach was validated with the high-resolution mapping of cosmid contigs on a yeast artificial chromosome (YAC) within yeast genomic DNA. It was extended to human genomic DNA for precise measurements ranging from 7 to 150 kilobases, of gaps within a contig, and of microdeletions in the tuberous sclerosis 2 gene on patients' DNA. The simplicity, reproducibility, and precision of this approach makes it a powerful tool for a variety of genomic studies.

https://science.sciencemag.org/content/sci/277/5331/1518.full.pdf

Dynamic Molecular Combing: Stretching the Whole Human Genome for High-Resolution Studies

Over the past decade, the human identity testing community has settled on a set of core short tandem repeat (STR) loci that are widely used for DNA typing applications. A variety of commercial kits enable robust amplification of these core STR loci. A brief history is presented regarding the selection of core autosomal and Y-chromosomal STR markers. The physical location of each STR locus in the human genome is delineated and allele ranges and variants observed in human populations are summarized as are mutation rates observed from parentage testing. Internet resources for additional information on core STR loci are reviewed. Additional topics are also discussed, including potential linkage of STR loci to genetic disease-causing genes, probabilistic predictions of sample ethnicity, and desirable characteristics for additional STR loci that may be added in the future to the current core loci. These core STR loci, which form the basis for DNA databases worldwide, will continue to play an important role in forensic science for many years to come.

https://www.sjsu.edu/people/steven.lee/courses/JS111FLUOR/s0/Butler2006JFS_coreSTRreview.pdf

Genetics and genomics of core short tandem repeat loci used in human identity testing.

Even when conditions are optimal, the maximum chance of a clinically recognized pregnancy occurring in a given menstrual cycle is 30-40%. Increasing evidence points to preclinical pregnancy loss rather than failure of conception as the principal cause for the relatively low fecundity observed in humans. While sensitive assays for hCG have provided a glimpse of the events occurring between implantation and the missed menstrual period, new cytogenetic techniques have further opened this 'black box', providing novel insights into the causes of early pregnancy wastage. In this article, the evidence and causes of preclinical or 'occult' pregnancy are reviewed, and the implications for the infertile patient are addressed.

Conception to ongoing pregnancy: the ‘black box’ of early pregnancy loss

Epigenetic silencing can mimic genetic mutation by abolishing expression of a gene. We hypothesized that an epimutation could occur in any gene as a germline event that predisposes to disease and looked for examples in tumor suppressor genes in individuals with cancer. Here we report two individuals with soma-wide, allele-specific and mosaic hypermethylation of the DNA mismatch repair gene MLH1. Both individuals lack evidence of genetic mutation in any mismatch repair gene but have had multiple primary tumors that show mismatch repair deficiency, and both meet clinical criteria for hereditary nonpolyposis colorectal cancer. The epimutation was also present in spermatozoa of one of the individuals, indicating a germline defect and the potential for transmission to offspring. Germline epimutation provides a mechanism for phenocopying of genetic disease. The mosaicism and nonmendelian inheritance that are characteristic of epigenetic states could produce patterns of disease risk that resemble those of polygenic or complex traits.

/pdf/germline-epimutation-of-mlh1-in-individuals-with-multiple-4oljkkb7dp.pdf

Germline epimutation of MLH1 in individuals with multiple cancers

Rapid molecular method for prenatal detection of Down's syndrome.

The purpose of this study was to develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma. Maternal DNA extracted from plasma samples of pregnant women at term and newborn DNA isolated from cord blood were used to genotype 12 mother/child pairs at nine different polymorphic short tandem repeat loci. Multiplex fluorescent PCR was used to detect fetus-specific alleles in the corresponding maternal plasma samples. Fetus-specific alleles were found in all maternal plasma samples studied. Using these polymorphic repeat sequences, every mother/child pair was informative in at least four of nine loci. Paternally inherited fetal alleles were detected in 84% of informative short tandem repeats. This approach may have implications for non-invasive prenatal diagnosis. Compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma, our proposed technique can be applied to both female and male fetuses.

Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats

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. QF-PCR can be exploited to analyse DNA present in single or clumps of cells and thus to perform prenatal diagnoses on maternal peripheral blood or transcervical cell samples and on preimplantation embryos.

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

We report the results of the first major study of applying quantitative fluorescence polymerase chain reaction (QF-PCR) assays for the detection of major chromosome numerical disorders. The QF-PCR tests were performed on a total of 247 chorionic villus samples, which were analysed blind, without any knowledge of the results obtained using conventional cytogenetic analysis.
The aims of this investigation were to evaluate the detection power and accuracy of this approach by testing a large number of fetal samples and to assess the diagnostic value of each of the chromosome specific small tandem repeat (STR) markers used. In addition, we introduced three more markers specific for chromosomes 13, 18, and X to allow an accurate analysis of samples homozygous for a particular STR. Fluorescent labelled primers were used to amplify 12 STRs specific for chromosomes 21, 18, 13, X, and the amylogenin-like DNA sequence AMXY, expressed on the X and Y chromosomes.
In this blind study of 247 fetal samples, 222 were correctly diagnosed by QF-PCR as normal for each of the five chromosomes investigated; 20 were diagnosed by QF-PCR as trisomic for chromosomes 21, 18, or 13, in agreement with the cytogenetic tests. Only one false negative result was observed, probably owing to the mishandling of the sample, which had been transferred through three laboratories before being analysed by QF-PCR. The 247 samples also included four cases of mosaicism or translocation; one case of mosaic trisomy 21 was detected by QF-PCR and the other cases were not identified by QF-PCR.
The results of this investigation provide clear evidence that the QF-PCR assays are powerful adjuncts to conventional cytogenetic techniques and can be applied for the rapid and accurate prenatal diagnosis of the most frequent aneuploidies.


Keywords: prenatal diagnosis; aneuploidies; quantitative fluorescence PCR

https://jmg.bmj.com/content/jmedgenet/36/4/300.full.pdf

Barbara Pertl

Papers

Rapid molecular method for prenatal detection of Down's syndrome.

Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats

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

Rapid detection of chromosome aneuploidies by quantitative fluorescence PCR: first application on 247 chorionic villus samples

Fetal DNA in maternal plasma: emerging clinical applications.