Genetic markers of male infertility: Y chromosome microdeletions and cystic fibrosis transmembrane conductance gene mutations.

Today, approximately 15% of couples have reduced fertility. In most cases the reason is male infertility, usually of genetic origin. Thus, in the context of research in genes involved in reproduction and sex determination, genetic defects in gametogenesis are being extensively studied. The most frequent pathogenic causes of male infertility are Y chromosomal microdeletions and obstructive azoospermia due to congenital absence of the vas deferens (CAVD) in the presence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We have investigated the most common CFTR gene alterations in Croatian men with CAVD, using Roche research prototype assays. Results revealed that the 5T variant was present in 27% of the subjects. The F508 deletion was found in 21% of the subjects. It was the most frequent mutation, although its incidence was much lower than among patients with cystic fibrosis. The prevalence of microdeletions in the azoospermia factor region (AZF) of the Y chromosome in Croatia was 4.5%. This is the first report of Y microdeletions in the Croatian population. Genetic counseling of all couples with the diagnosis of male infertility is recommended before intrauterine insemination, in vitro fertilization, and intracytoplasmic sperm injection, and should also include AZF and CFTR genotyping. Couples requesting assisted reproductive treatment should be offered molecular analysis of the CFTR gene, if male infertility due to obstructive azoospermia is the underlying cause. Also, men with severe oligozoospermia or non-obstructive azoospermia seeking assisted reproductive treatment should be screened for deletions in the Y chromosome.

Issues in implementing prenatal screening for cystic fibrosis: results of a working conference.

PURPOSE: To summarize a conference convened to examine how cystic fibrosis screening might appropriately be introduced into routine prenatal practice. METHODS: Participants included experts from various relevant disciplines. Systematic reviews and data from individual trials were presented; issues were identified and discussed. RESULTS: Judged by published criteria, prenatal cystic fibrosis screening is suitable for introduction. Screening can be performed cost-effectively by identifying racial/ethnic groups at sufficient risk and then using either of two models for delivering laboratory services. Validated educational materials exist. Ethical issues are not unique. CONCLUSIONS: Once adequate facilities for patient and provider education, testing, counseling, quality control, and monitoring are in place, individual programs can begin prenatal screening for cystic fibrosis.

Molecular analysis of northwestern Mexican patients with cystic fibrosis: screening of 10 known mutations. Mutations in brief no. 185. Online.

We have analyzed 45 unrelated Northwestern Mexican patients with Cystic Fibrosis for 10 known CF mutations (DF508, G542X, G551D. R553X, W1282X, NI303K, R334W, R347H, S549R, and R1162X). Screening was performed on exons 7, 10, 11, 19, 20 and 21 using standard methods such as polymerase chain reactions, reverse dot blot hybridization (non-radioactive), and restriction enzyme digestion. The analysis for these ten mutations permitted the identification of only two mutations in 37.7% of CF chromosomes in this sample. The major mutation, delta F508, accounts for 34.4% of CF chromosomes. Of the 45 CF patients 9 (20.0%) were homozygous delta F508 deletion, 11 (24.4%) were heterozygous for the delta F508 mutation and an unknown mutation. One additional mutation G542X was also found in 3 chromosomes in our population (3.3%). Two patients were documented to be a compound heterozygote for DF508/G542X, and other one heterozygous for G542X and an unknown mutation. Therefore 62.2% of chromosomes remain uncharacterized.

Analysis of 16 cystic fibrosis mutations in Mexican patients.

We carried out molecular analysis of 80 chromosomes from 40 unrelated Mexican patients with a diagnosis of cystic fibrosis. The study was performed in two PCR steps: a preliminary one to identify mutation delta F508, the most frequent cause of cystic fibrosis worldwide, and the second a reverse dot-blot with allele-specific oligonucleotide probes to detect 15 additional common mutations in the Caucasian population. A frequency of 45% for delta F508 was found, making it the most common in our sample of Mexican patients. Another five mutations (G542X, 3,849 + 10 kb C-->T, N1303K, SN549N, and 621 + 1 G-->T) were detected, and those accounted for 11.25%. The remaining mutations (43.75%) were undetectable with the methodology used.

Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma.

Pancreatic adenocarcinomas are known to have a high incidence of K-ras gene mutations. Differential diagnosis of pancreatic cancer and chronic pancreatitis sometimes presents a clinical dilemma. We recently developed a highly sensitive and specific polymerase chain reaction capable of detecting 3-30 copies of mutant K-ras genes harboring codon 12 single base changes in the presence of 300,000 normal copies. Mutant ras genes were detected in DNA purified from pancreatic juice from all 6 cases of pancreatic adenocarcinoma and 1 case of intraductal papillary neoplasms of the pancreas. In 2 of 6 other cases with pancreatic adenocarcinoma, circulating metastatic cells were detected in DNA purified from peripheral blood. Activated ras genes were not found in pancreatic juice of three control cases (chronic pancreatitis and choledocholithiasis) or in the peripheral blood of two patients with insulinomas. Notable conclusions of this study are that there can be significant levels of shed tumor cells in peripheral blood and an even higher number in pancreatic juice. In addition, two different K-ras mutations were found in some patients.

High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity.

The Thermus aquaticus DNA polymerase I (Taq Pol I) gene was cloned into a plasmid expression vector that utilizes the strong bacteriophage lambda PL promoter. A truncated form of Taq Pol I was also constructed. The two constructs made it possible to compare the full-length 832-amino-acid Taq Pol I and a deletion derivative encoding a 544-amino-acid translation product, the Stoffel fragment. Upon heat induction, the 832-amino-acid construct produced 1-2% of total protein as Taq Pol I. The induced 544-amino-acid construct produced 3% of total protein as Stoffel fragment. Enzyme purification included cell lysis, heat treatment followed by Polymin P precipitation of nucleic acids, phenyl sepharose column chromatography, and heparin-Sepharose column chromatography. For full-length 94-kD Taq Pol I, yield was 3.26 x 10(7) units of activity from 165 grams wet weight cell paste. For the 61-kD Taq Pol I Stoffel fragment, the yield was 1.03 x 10(6) units of activity from 15.6 grams wet weight cell paste. The two enzymes have maximal activity at 75 degrees C to 80 degrees C, 2-4 mM MgCl2 and 10-55 mM KCl. The nature of the substrate determines the precise conditions for maximal enzyme activity. For both proteins, MgCl2 is the preferred cofactor compared to MnCl2, CoCl2, and NiCl2. The full-length Taq Pol I has an activity half-life of 9 min at 97.5 degrees C. The Stoffel fragment has a half-life of 21 min at 97.5 degrees C. Taq Pol I contains a polymerization-dependent 5' to 3' exonuclease activity whereas the Stoffel fragment, deleted for the 5' to 3' exonuclease domain, does not possess that activity. A comparison is made among thermostable DNA polymerases that have been characterized; specific activities of 292,000 units/mg for Taq Pol I and 369,000 units/mg for the Stoffel fragment are the highest reported.

Prenatal diagnosis of alpha-thalassemia by polymerase chain reaction and dual restriction enzyme analysis.

Asian couples at risk for a fetus with homozygous alpha-thalassemia (hydrops fetalis) are often identified by their low erythrocyte mean corpuscular volume (MCV) and normal hemoglobin electrophoresis when little time remains to test their genotypes by restriction enzyme analysis. DNA analysis is performed directly on chorionic villi or amniocytes remaining after an aliquot is used to establish a backup cell culture. The polymerase chain reaction (PCR) protocol quickly determines whether the fetus has hydrops fetalis without waiting for cultured cells to grow. Previously, growing cultured fetal cells to obtain more fetal material to establish unambiguously the fetal genotype with two independent restriction enzyme digests absorbed a significant portion of the time remaining to complete prenatal diagnosis. A dual restriction enzyme digestion protocol was development using a 3' zeta-globin probe to clearly distinguish the most common alpha-thalassemia deletions that represent nearly all the alpha-thalassemia haplotypes in Southeast Asia.

Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes.

The analysis of DNA for the presence of particular mutations or polymorphisms can be readily accomplished by differential hybridization with sequence-specific oligonucleotide probes. The in vitro DNA amplification technique, the polymerase chain reaction (PCR), has facilitated the use of these probes by greatly increasing the number of copies of target DNA in the sample prior to hybridization. In a conventional assay with immobilized PCR product and labeled oligonucleotide probes, each probe requires a separate hybridization. Here we describe a method by which one can simultaneously screen a sample for all known allelic variants at an amplified locus. In this format, the oligonucleotides are given homopolymer tails with terminal deoxyribonucleotidyltransferase, spotted onto a nylon membrane, and covalently bound by UV irradiation. Due to their long length, the tails are preferentially bound to the nylon, leaving the oligonucleotide probe free to hybridize. The target segment of the DNA sample to be tested is PCR-amplified with biotinylated primers and then hybridized to the membrane containing the immobilized oligonucleotides under stringent conditions. Hybridization is detected nonradioactively by binding of streptavidin-horseradish peroxidase to the biotinylated DNA, followed by a simple colorimetric reaction. This technique has been applied to HLA-DQA genotyping (six types) and to the detection of Mediterranean beta-thalassemia mutations (nine alleles).

Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus.

The thermostable properties of the DNA polymerase activity from Thermus aquaticus (Taq) have contributed greatly to the yield, specificity, automation, and utility of the polymerase chain reaction method for amplifying DNA. We report the cloning and expression of Taq DNA polymerase in Escherichia coli. From a lambda gt11:Taq library we identified a Taq DNA fragment encoding an epitope of Taq DNA polymerase via antibody probing. The fusion protein from the lambda gt11:Taq candidate selected an antibody from an anti-Taq polymerase polyclonal antiserum which reacted with Taq polymerase on Western blots. We used the lambda gt11 clone to identify Taq polymerase clones from a lambda Ch35:Taq library. The complete Taq DNA polymerase gene has 2499 base pairs. From the predicted 832-amino acid sequence of the Taq DNA polymerase gene, Taq DNA polymerase has significant similarity to E. coli DNA polymerase I. We subcloned and expressed appropriate portions of the insert from a lambda Ch35 library candidate to yield thermostable, active, truncated, or full-length forms of the protein in E. coli under control of the lac promoter.

Rapid prenatal diagnosis of beta thalassemia using DNA amplification and nonradioactive probes.

We used in vitro DNA amplification by the polymerase chain reaction and nonradioactive probes for prenatal diagnosis of beta thalassemia in Chinese from the Guangdong province. Exact molecular diagnoses were made in all 20 fetuses studied over a 6-month period. We conclude that this method of prenatal diagnosis for beta thalassemia is a viable approach in many parts of the world where this disease is common.

Diagnosis of sickle cell anemia and beta-thalassemia with enzymatically amplified DNA and nonradioactive allele-specific oligonucleotide probes.

We have developed a simple and rapid nonradioactive method for detecting genetic variation and have applied it to the diagnosis of sickle cell anemia and beta-thalassemia. The procedure involves the selective amplification of a segment of the human beta-globin gene with oligonucleotide primers and a thermostable DNA polymerase, followed by hybridization of the amplified DNA with allele-specific oligonucleotide probes covalently labeled with horseradish peroxidase. The hybridized probes were detected with a simple colorimetric assay. We demonstrated the usefulness of this method in a retrospective analysis of two pregnancies at risk for beta-thalassemia and one at risk for sickle cell anemia, as well as in an analysis of nine DNA samples simulating three family sets.

Amplification and analysis of DNA sequences in single human sperm and diploid cells.

The use of the polymerase chain reaction for analysing DNA sequences in individual diploid cells and human sperm shows that two genetic loci can be co-amplified from a single sperm, which may allow the analysis of previously inaccessible genetic phenomena.

A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions.

Specific, end-labeled DNA fragments can be simply and rapidly prepared using the polymerase chain reaction (PCR). Such fragments are suitable for use in DNase I protection footprint assays, chemical sequencing reactions, and for the production and analysis of paused RNA polymerase transcription complexes. Moreover, a general means of introducing a specific mutation at any position along the length of such PCR-generated fragments is described. These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA. A rapid means of removing excess oligonucleotide primers from completed PCRs is also described.

Analysis of RAS gene mutations in acute myeloid leukemia by polymerase chain reaction and oligonucleotide probes.

In vitro DNA amplification followed by oligonucleotide dot blot analysis were used to study RAS gene mutations in acute myeloid leukemia (AML). Fifty-two presentation AML DNAs were screened for mutations in codons 12, 13, and 61 of NRAS and in codons 12 and 61 of KRAS and HRAS. Fourteen (27%) contained mutations--all in NRAS and predominantly in codon 12. The most common amino acid substitution identified was of glycine by aspartic acid at codon 12 (7/18), with a G----A transition being the most common base change (11/18). No particular correlation was observed between disease subtype and the incidence or type of NRAS mutation. In DNA samples from four patients, 2 NRAS mutations were found to coexist. NIH 3T3 focus-formation assays revealed that in each case the mutations were present in different NRAS alleles. We also report the absence of a mutated RAS gene in relapse DNAs of four patients in which a RAS oncogene had been detected at presentation. These observations suggest that RAS mutations arise as part of the evolution of neoplastic transformation.

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.

A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.

Characterization of beta-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA.

Direct sequencing of specific regions of genomic DNA became feasible with the invention of the polymerase chain reaction (PCR) which permits amplification of specific regions of DNA. Recently, human mitochondrial DNA was amplified and directly sequenced. Using a thermostable DNA polymerase of T. aquaticus (Saiki, R.K. et al., manuscript in preparation) in the PCR, we have applied a combination of PCR and direct sequence analysis of the amplified product to a human single-copy gene. We studied the genomic DNA of five patients with beta-thalassaemia whose mutant alleles were uncharacterized, and found two previously undescribed mutations, along with three known alleles. One new allele is a frameshift at codons 106-107 and the other is an A-C transversion at the cap site (+1) of the beta-globin gene. This latter is the first natural mutation observed at the cap site and it occurs in a gene which is poorly expressed.

Rapid prenatal diagnosis of sickle cell anemia by a new method of DNA analysis.

We have used a new method of DNA analysis for the rapid prenatal diagnosis of sickle cell anemia in two fetuses at risk for this disease. This method of detecting the sickle gene is a modification of standard restriction-enzyme techniques and requires only a small amount of DNA. The first step involves a 200,000-fold enzymatic amplification of the specific beta-globin DNA sequences that may carry the sickle mutation. This provides a sufficient quantity of DNA for the analysis. Next, a short radiolabeled synthetic DNA sequence homologous to normal beta A-globin gene sequences is hybridized to the amplified target sequences. The hybrid "duplexes" are then digested sequentially with two restriction endonucleases. The presence of beta A- or beta S-globin gene sequences in the amplified target DNA from the patient determines whether the beta A-hybridization probe anneals perfectly or with a single nucleotide mismatch. This difference affects the restriction-enzyme digestion of the DNA and the size of the resulting radiolabeled digestion products, which can be distinguished by electrophoresis followed by autoradiography. This method is sufficiently sensitive and rapid that the prenatal diagnosis of sickle cell anemia can be made on the same day that the fetal DNA is made available. It can also be applied to the diagnosis of hemoglobin C disease.

Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides.

The "polymerase chain reaction" (PCR) procedure for amplifying specific gene sequences has recently been combined with sequence-specific oligonucleotide (SSO) probe hybridization to develop a highly sensitive, rapid, and simple method for analyzing allelic variations in genomic DNA. In the present study we have used PCR/SSO to analyze partially purified DNA extracted from formalin-fixed, paraffin-embedded tissue specimens. We report that this DNA, including samples that were partially degraded, proved to be suitable for analysis by the PCR/SSO procedure.