First trimester prenatal diagnosis of adrenoleukodystrophy by determination of very long chain fatty acid levels and by linkage analysis to a DNA probe

Summary A first trimester prenatal diagnosis of adrenoleukodystrophy has been done on chorionic villi biopsy in the pregnancy of a carrier woman. Two different approaches allowed one to determine that the male fetus was affected: the linkage analysis of DNA from chorionic villi using the highly polymorphic probe St 14 and the determination of very long chain fatty acid levels in cultured chorionic villi.     

Genomic carrier detection and prenatal diagnosis of haemophilia A in families at risk using the polymerase chain reaction (PCR).

The polymerase chain reaction (PCR) was applied in genomic analysis of families at risk for haemophilia A using the intragenic Bel I and Hind III polymorphism in introns 18 and 19, respectively, of factor VIII gene. For the latter the primers derived from exon 19 and 20 sequences allowed to amplify the whole intron 19 resulting in a 730 bp fragment. Hind III restriction of this fragment provides polymorphic fragments of 250 bp or 160 bp and 90 bp respectively. An also occurring 480 bp fragment can be used as internal control to circumvent misdiagnosis due to incomplete or failure of restriction. The Hind III polymorphism was successfully used in prenatal diagnosis of an affected male in the first trimenon of pregnancy. Fetal sexing was also performed by PCR technique using Y specific primers.     

A case of first trimester prenatal diagnosis of 21-hydroxylase deficiency with human complement C4 cDNA probe

Early prenatal diagnosis of 21-hydroxylase (21-OHase) deficiency would enable treatment to be done to protect the fetus from masculinization and/or life-threatening adrenal crisis at birth. We report here the prenatal diagnosis of 21-OHase deficiency with human complement component C4 cDNA to probe DNA from chorionic villi at 10 weeks of gestation. Southern analysis with human C4 cDNA identified TaqI restriction fragment length polymorphisms (RFLPs) in the family. Family analysis with these RELPs showed that the fetus was not affected at greater than 99% probability, because the frequency of recombination between the 21-OHase B gene and the C4 gene would be extremely low.     

Infantile hypophosphatasia: localization within chromosome region 1p36.1-34 and prenatal diagnosis using linked DNA markers.

Linkage analysis was performed on data from Manitoba Mennonite families identified by a proband with infantile hypophosphatasia (HOPS), an autosomal recessive disorder characterized by defective skeletal mineralization. Southern blot analysis of Msp-I-digested DNA from HOPS nuclear families probed with a 2.55-kb liver/bone/kidney alkaline phosphatase (ALPL) cDNA revealed two previously undescribed RFLPs at 2.4/2.3 kb and 2.0/1.9 kb. Maximum combined lod score equals 13.25 at theta = 0. This establishes very close linkage between ALPL and HOPS and allows for the regional assignment of the HOPS gene to chromosome 1p36.1-34. Prenatal RFLP studies in an informative Mennonite family correctly predicted an unaffected fetus following chorionic villus sampling at 12 wk gestation. In addition in our Mennonite population, a nonrandom association exists between the polymorphic ALPL alleles and HOPS. These results suggest that strong linkage disequilibrium exists between HOPS and the ALPL markers. This will allow for improved carrier assignment in this high-risk population. Preliminary analysis suggests approximately 1/25 Manitoba Mennonites are HOPS carriers.     

DNA fingerprints of sheep using an M13 probe

The bacteriophage M13 DNA was used to detect hypervariable minisatellites in several families of Booroola sheep as well as Merino and Suffolk sheep. Digestion of sheep DNA gave rise to three to eight fragments with different restriction enzymes demonstrating considerable polymorphism between the different breeds. The length of informative DNA fragments varied in size from 6 to 20kb. The DNA fingerprints generated were individual specific and allowed for differentiation between closely related animals. The pattern obtained with sheep DNA was different from that observed with humans and other vertebrates in the proportion of high molecular weight DNA fragments present. Pedigree analysis of DNA patterns of dams and their offspring for several sets of twins and triplets showed a clear distinction between individuals and failed to reveal the presence of monozygosity.     

Porcine malignant hyperthermia carrier detection and chromosomal assignment using a linked probe

In pigs, the gene for glucosephosphate isomerase (GPI) is linked to the halothane (HAL) gene which is responsible for malignant hyperthermia (MH). A single copy DNA probe, designated GPI8R, has been isolated from a pig genomic library using a porcine GPI cDNA probe. This probe detects, as was the case for the cDNA probe, a five allele polymorphism in SacI and PvuII digested pig DNA. Family studies show that this polymorphism is linked to the HAL locus and hence can be used in carrier detection. In situ hybridization with GPI8R assigned the GPI locus to bands p12-q22 of chromosome 6. We conclude that the HAL linkage group resides on chromosome 6.     

Electrochemical detection of mismatched DNA using a MutS probe

A direct and label-free electrochemical biosensor for the detection of the protein–mismatched DNA interaction was designed using immobilized N-terminal histidine tagged Escherichia coli. MutS on a Ni-NTA coated Au electrode. General electrochemical methods, cyclic voltammetry (CV), electrochemical quartz crystal microbalance (EQCM) and impedance spectroscopy, were used to ascertain the binding affinity of mismatched DNAs to the MutS probe. The direct results of CV and impedance clearly reveal that the interaction of MutS with the CC heteroduplex was much stronger than that with AT homoduplex, which was not differentiated in previous results (GT > CT > CC ≈ AT) of a gel mobility shift assay. The EQCM technique was also able to quantitatively analyze MutS affinity to heteroduplexes.     

A DNA marker closely linked to the factor IX (haemophilia B) gene

Summary We have isolated a DNA segment, pX58dIIIc, from an X-chromosome library which identifies an SstI restriction fragment length polymorphism (RFLP) at locus DXS99. Linkage analysis in six informative families has shown that the DXS99 locus lies close to the factor IX gene (F9). No recombination was detected between these loci in 39 informative meioses (Z=9.79, =0.0). Therefore, DXS99 will be useful as a DNA marker for the assessment of carrier status in families with haemophilia B where intragenic markers are not informative. Heterozygosity at DXS99 is approximately 50% and, in conjunction with the RFLPs at F9, 90% of females at risk for being haemophilia B carriers should be diagnosed.     

Direct detection of Salmonella spp. in estuaries by using a DNA probe.

A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.     

Direct detection of Salmonella spp. in estuaries by using a DNA probe

A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.     

Detection of carriers and prenatal diagnosis for fucosidosis in Calabria

Summary A significant proportion of patients affected with fucosidosis have Italian ancestors. We assayed for -fucosidase activity purified mononuclear cells and/or leukocytes obtained from 64 members of two large pedigrees from Calabria, in which seven children had been diagnosed as affected with fucosidosis. Of these 64 individuals, 22 were diagnosed as carriers, while the values for the remainder were within normal limits, indicating a clear bimodal distribution among individuals at risk.These data confirm that carrier detection for fucosidosis requires the measurement of -L-fucosidase activity in purified mononuclear cells from peripheral blood. In addition, this program has made prenatal identification of an affected fetus possible.     

Application of fetal DNA detection in maternal plasma: a prenatal diagnosis unit experience.

Non-invasive prenatal diagnosis tests based on the analysis of fetal DNA in maternal plasma have potential to be a safer alternative to invasive methods. So far, different studies have shown mainly fetal sex, fetal RhD, and quantitative variations of fetal DNA during gestation with fetal chromosomal anomalies or gestations at risk for preeclampsia. The objective of our research was to evaluate the use of fetal DNA in maternal plasma for clinical application. In our study, we have established the methodology needed for the analysis of fetal DNA. Different methods were used, according to the requirements of the assay. We have used quantitative fluorescent polymerase chain reaction (QF-PCR) to perform fetal sex detection with 90% sensitivity. The same technique permitted the detection of fetal DNA from the 10th week of gestation to hours after delivery. We have successfully carried out the diagnosis of two inherited disorders, cystic fibrosis (conventional PCR and restriction analysis) and Huntington disease (QF-PCR). Ninety percent of the cases studied for fetal RhD by real-time PCR were correctly diagnosed. The detection of fetal DNA sequences is a reality and could reduce the risk of invasive techniques for certain fetal disorders in the near future.     

First trimester prenatal diagnosis of 21-hydroxylase deficiency by linkage analysis to HLA-DNA probes and by 17-hydroxyprogesterone determination

Summary The close genetic linkage between the gene for congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency and HLA genes allowed us to use the polymorphism of this system as a marker of the disease. HLA genotyping can be performed by using restriction enzyme fragments hybridized with specific probes instead of serologic methods. In seven pregnancies at risk for 21-OH deficiency, a first trimester prenatal diagnosis has been performed by determining the fetal genotype by linkage analysis of DNA from chorionic villi using HLA class I and class II probes. In four of these pregnancies, determination of 17-OH progesterone in first trimester amniotic fluid afforded a complementary approach to the diagnosis.     

A complex of single-strand binding protein and M13 DNA as hybridization probe.

Single-stranded DNA was complexed to the single-strand binding protein (SSB) of Escherichia coli in a mass ratio of 30:1. The protein moiety of this complex can be labelled by a number of methods of which we have chosen radio-iodination and biotinylation as examples. The SSB-M13 DNA complexes, labelled to high specific activities, were used as probes in hybridization experiments in which 1.6 X 10(-18) moles of immobilized target DNA were detected. The stability of the hybrids was not severely decreased by the binding of SSB. Analysis of hybrids by electron microscopy showed that complexing of DNA with SSB could be used to allow its subsequent identification in the hybrids.     

Noninvasive prenatal diagnosis of fetal trisomy 18 and trisomy 13 by maternal plasma DNA sequencing

Massively parallel sequencing of DNA molecules in the plasma of pregnant women has been shown to allow accurate and noninvasive prenatal detection of fetal trisomy 21. However, whether the sequencing approach is as accurate for the noninvasive prenatal diagnosis of trisomy 13 and 18 is unclear due to the lack of data from a large sample set. We studied 392 pregnancies, among which 25 involved a trisomy 13 fetus and 37 involved a trisomy 18 fetus, by massively parallel sequencing. By using our previously reported standard z-score approach, we demonstrated that this approach could identify 36.0% and 73.0% of trisomy 13 and 18 at specificities of 92.4% and 97.2%, respectively. We aimed to improve the detection of trisomy 13 and 18 by using a non-repeat-masked reference human genome instead of a repeat-masked one to increase the number of aligned sequence reads for each sample. We then applied a bioinformatics approach to correct GC content bias in the sequencing data. With these measures, we detected all (25 out of 25) trisomy 13 fetuses at a specificity of 98.9% (261 out of 264 non-trisomy 13 cases), and 91.9% (34 out of 37) of the trisomy 18 fetuses at 98.0% specificity (247 out of 252 non-trisomy 18 cases). These data indicate that with appropriate bioinformatics analysis, noninvasive prenatal diagnosis of trisomy 13 and trisomy 18 by maternal plasma DNA sequencing is achievable.     

The use of cloned Y chromosome-specific DNA probes for fetal sex determination in first trimester prenatal diagnosis

Summary Prenatal diagnosis by chorion biopsy in the first trimester of pregnancy has advantages over second trimester amniocentesis because diagnosis can be achieved at 9–12 weeks gestation, reducing prenatal anxiety and avoiding the trauma of late abortion. DNA can be prepared from chorionic villus biopsies in sufficient quantity and purity for use in prenatal diagnosis systems using specific DNA probes hybridised to restriction endonuclease digests.DNA probes derived from the Y chromosome have been used to determine fetal sex. The use of such probes means that the chromosomal sex of the fetus can be identified more quickly than by chromosome preparation and more accurately than by sex chromatin staining, and has the additional advantage that the same DNA preparation can be used for other diagnostic tests. A dot hybridisation method has been successfully used to provide even more rapid results than conventional hybridisation to Southern blots of restriction endonuclease digests.There is a risk that Y chromosome-specific DNA probes for sex determination may be subject to error if the parents have extreme Y chromosome variants such as a small or non-fluorescent Y or a Y autosome chromosome translocation. The precise extent to which such chromosome variants may lead to error has been investigated. Even extreme Y chromosome variants totally lacking fluorescence were identified as male by the cloned probes used. However, Y autosome translocations carried by females could cause error if not identified in the parents. The value of the probes has been confirmed provided that parental chromosomes and DNA are examined in parallel with the chorionic biopsy material     

A DNA probe for specific detection of Mycoplasma pulmonis

Mycoplasma pulmonis was specifically detected by using a 2.3 kilobase pair (kbp) cloned DNA fragment derived from M. pulmonis m 53 as a probe. This probe recognized 2.3-kbp DNA fragments of three M. pulmonis strains in Southern hybridization, while it did not hybridize with the DNA of M. arthritidis or M. neurolyticum. Determination of the sensitivity of the probe by dot hybridization revealed that 10 ng of M. pulmonis DNA was detected by a biotinylated probe and 1 ng of M. pulmonis DNA was detected by a radioactive probe.     

Carrier detection and prenatal diagnosis of cystic fibrosis using an intragenic TA-repeat polymorphism

Summary We have analysed the segregation of a TA-repeat polymorphism in intron 17b of the cystic fibrosis transmembrane conductance regulator gene responsible for cystic fibrosis (CF) in 23 French CF families non-informative for the F508 mutation (i.e. with at least one parent not carrying F508) or closely linked DNA markers. At least 13 different alleles ranging from 7 to 45 repeats were observed and the detected heterozygosity was 89%. Of the 23 families studied, 19 were fully informative for prenatal diagnosis or carrier detection, 3 were partially informative and one was not informative. In 6 families, prenatal diagnosis for CF or carrier detection in siblings of CF cases were performed using this polymorphism.     

First experiences in application of RFLP analysis for carrier detection in preparation of prenatal diagnosis of hemophilia A in the GDR

The application of the intragenic probe F8 e16-19 of factor VIII gene is reported for carrier detection in preparation of prenatal diagnosis of hemophilia A in a family at risk. The proband's mother is heterozygous for the Bcl I polymorphism and prenatal diagnosis can be offered to this family.