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Background: Genetic analysis is commonly performed in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency., Study Objective: The objective of the study was to describe comprehensive CYP21A2 mutation analysis in a large cohort of CAH patients., Methods: Targeted CYP21A2 mutation analysis was performed in 213 patients and 232 parents from 182 unrelated families. Complete exons of CYP21A2 were sequenced in patients in whom positive mutations were not identified by targeted mutation analysis. Copy number variation and deletions were determined using Southern blot analysis and PCR methods. Genotype was correlated with phenotype., Results: In our heterogeneous U.S. cohort, targeted CYP21A2 mutation analysis did not identify mutations on one allele in 19 probands (10.4%). Sequencing identified six novel mutations (p.Gln262fs, IVS8+1G>A, IVS9-1G>A, p.R408H, p.Gly424fs, p.R426P) and nine previously reported rare mutations. The majority of patients (79%) were compound heterozygotes and 69% of nonclassic (NC) patients were compound heterozygous for a classic and a NC mutation. Duplicated CYP21A2 haplotypes, de novo mutations and uniparental disomy were present in 2.7% of probands and 1.9 and 0.9% of patients from informative families, respectively. Genotype accurately predicted phenotype in 90.5, 85.1, and 97.8% of patients with salt-wasting, simple virilizing, and NC mutations, respectively., Conclusions: Extensive genetic analysis beyond targeted CYP21A2 mutational detection is often required to accurately determine genotype in patients with CAH due to the high frequency of complex genetic variation.

Single-strand conformation polymorphism (SSCP) analysis detects mutations based on the fact that single-nucleotide changes in DNA sequences alter the mobility of single-stranded DNA in nondenaturing gels. Four methods for detecting mutations based on SSCP are described here. (1) Traditional SSCP analysis is technically easy and can be used for screening large numbers of samples. SSCP-hybrid methods detect mutations based on either an SSCP effect or an altered component independent of the SSCP effect. (2) Dideoxy fingerprinting (ddF) involves PCR amplification of the target and creation of a set of dideoxy-terminated strands with the mutation. (3) Bi-directional dideoxy fingerprinting (Bi-ddF) involves production of two sets of dideoxy-terminated strands that are generated from two different primers. (4) Restriction endonuclease fingerprinting (REF) involves cleavage of the amplified target with five to six groups of restriction endonucleases.

The [detection of virtually all mutations]-SSCP (DOVAM-S) is a highly sensitive variant of single strand conformation polymorphism (SSCP). Mutations in the factor IX gene were used to find a set of five SSCP conditions that detects virtually all mutations. A blinded analysis of the factor IX gene in patients with hemophilia B detected 82 of 82 unique mutations. Since the method was developed and tested on the factor IX gene, it is possible that the conditions selected work more efficiently in the factor IX gene than in other genes. To test the general applicability of the conditions under which DOVAM-S detected all mutations in this gene, blinded analyses were performed in the human factor VIII and ataxia-telangiectasia (ATM) genes. Segments were amplified individually, combined into groups of 16 to 18 amplified segments and electrophoresed in five different nondenaturing conditions of varying matrices, buffers, temperatures and additives. Blinded analyses were performed in 92 samples from patients with hemophilia A (factor VIII gene) and 19 samples from A-T patients (ATM gene). Combined with an earlier blinded analysis in the factor IX gene, all of the 250 mutations and polymorphisms (180 of which are unique) were detected in both analyses. For two, three and four joint conditions, the average detection frequency ranged from 77%-97%, 91%-100% and 95%-100%, respectively. For each of the genes, one mutation may have been missed if only four conditions were used. With DOVAM-S, approximately 500 kb of autosomal sequence can be scanned in five gels with virtually 100% detection of mutations within the scanned region. The detection of 180 out of 180 unique sequence changes implies that DOVAM-S detects at least 96.5% (P = 0.03) of mutations. Blinded analyses that detect 400 unique sequence changes are required to determine that a scanning method detects at least 98.5% of mutations.

Dideoxy fingerprinting (ddF) was used as a tool to search for a generic set of conditions with sufficient power to detect virtually all mutations. For each condition tested, a very large sample of mutation-containing, single-stranded segments (about 1500) were analyzed with ddF. Correlation coefficients identified pairs of conditions in which single-strand conformation polymorphism (SSCP) mobilities were poorly correlated. The data strongly suggest that tertiary structure (e.g., base-sugar and sugar-sugar interactions) rather than secondary structure is the predominant determinant of mobility shifts by SSCP. Five conditions were selected with sufficient redundancy to detect all the mutations. The sensitivity of detection of virtually all mutations-SSCP (DOVAM-S) was determined by blinded analyses on samples containing additional mutations scattered throughout the eight exons and splice junctions in the factor IX gene. The factor IX gene sequence (2.5 kb) was scanned in one lane by 15 PCR-amplified segments (125 kb of sequence scanned per gel). All of the 84 single-base substitutions were detected in the blinded analyses, the first consisting of 50 hemizygous mutant and wild-type (WT) samples and the second consisting of 50 heterozygous mutant and WT samples. DOVAM-S is estimated to be five times faster than fluorescent DNA sequencing for the detection of virtually all mutations when the five conditions are applied.

Tumor suppressor gene CDKN2 (also called MTS1, CDK4I and p16INK4) is located in 9p21 and deleted homozygously in a high percentage of tumor cell lines. We have examined the sequence of CDKN2 in 154 tumor cell lines that are not homozygously deleted for CDKN2. Overall, 18% (27/154) of the cell lines carried mutations in CDKN2. These mutations were found in cell lines derived from melanoma, bladder, lung and prostate cancers, as well as sarcomas of various origin. The spectrum of the CDKN2 mutations found in melanoma cell lines indicated a major role for ultraviolet light in generating the mutations, suggesting the mutations occurred in vivo. The frequency of loss of heterozygosity in 9p21 in this set of lines is only slightly higher than the background rate of aneuploidy, suggesting that a second 9p21 tumor suppressor gene, if it exists, must lie near CDKN2.

The rapid pace of gene discovery has led to new opportunities for clinical diagnosis using molecular genetic technologies. Recent achievements include the culmination of the 10-year search for the Huntington's disease gene, the identification of predisposing genes for certain familial colon cancers, and the characterization of potential genetic risk indicators for Alzheimer's disease, hypertension, and coronary heart disease. These advances, coupled with the previous discoveries of important disease genes (e.g. those for cystic fibrosis, Duchenne muscular dystrophy, and fragile X syndrome) have quickly expanded the capacity of genetic analysis, allowing the design of enhanced and novel approaches for diagnostic testing. The transfer of molecular technology to the area of clinical genetic analysis, although associated with many potential benefits, has raised some concern regarding the possible misuse of genetic tests and information, particularly with regard to presymptomatic diagnosis of disease and population screening.

Archival autopsy studies of sickle cell disease have often been hampered by inadequate documentation of the genotype. Although the polymerase chain reaction (PCR) has been applied to the prenatal diagnosis of sickle cell disease, its use has not been reported in archival studies of sickle cell disease. In this study, DNAs from formalin-fixed, paraffin-embedded archival tissues were amplified by PCR and analyzed by dot-blot hybridization using allele-specific oligonucleotides. These S and C genotypes for 9 of 10 archival specimens studied blindly were correctly identified by PCR. The tenth specimen consistently failed to amplify by PCR, yielding no result. These data demonstrate the utility of PCR for retrospective identification of the genotype of sickle cell disease. This application of PCR will significantly expand the number of autopsy cases suitable for retrospective studies of the morbidity and mortality of sickle cell disease.

Cystic fibrosis (CF) is the most common severe genetic disease of Caucasians. Recent work by several laboratories has resulted in the identification of the CF gene and its major mutation. These findings have greatly facilitated carrier detection and prenatal diagnosis for members of families with a history of CF. This review briefly considers the historical background of CF testing, the basic features of the CF gene, and the methodologies for CF gene analysis. Examples of the application of recent technologies for analysis of CF families are discussed. In addition to family analysis, CF carrier detection in the general population is becoming more feasible. The possibility of carrier screening for CF has been the topic of much discussion recently with arguments presented both for and against. Some basic issues pertaining to population testing are presented.

Mouse embryonal carcinoma cells are refractory to infection by wild-type polyoma virus, the infection process apparently being blocked at a stage after adsorption and penetration but before early protein synthesis. Polyoma virus mutants capable of productive infection of mouse embryonal carcinoma cells have been isolated and these mutants all have DNA sequence alterations in a noncoding region near the origin of replication of the viral genome. PyF101 and PyF441 are two mutants selected for their ability to infect the embryonal carcinoma cell line F9. Here we show that these PyF mutants do not rescue replication of wild-type polyoma during a mixed infection of F9 cells. The mutant and wild-type DNAs were distinguished on the basis of restriction fragments obtained by digestion with Msp I or BstNI, and no wild-type DNA was detected in F9 cells coinfected with wild-type polyoma and with either PyF101 or PyF441. The mutant viruses do not appear to inhibit wild-type replication during a mixed infection because both mutant and wild-type DNAs can replicate efficiently in coinfected 3T6 cells which are permissive for both mutant and wild-type viruses. A double mutant having the PyF101 mutation and the ts-25E temperature-sensitive mutation in polyoma large tumor antigen was constructed and found to be temperature-sensitive for replication in F9 cells. This double mutant, designated PyFts-1, can be rescued in F9 cells at the restrictive temperature by coinfection with PyF441. These results suggest that the PyF mutations affect two processes in F9 cells, one involving expression of polyoma early genes and a second involving viral DNA replication.

F9 mouse embryonal carcinoma cells are resistant to productive infection by wild-type polyoma virus. Continued passage of F9 cells initially infected with wild-type polyoma virus eventually leads to the selection of polyoma virus mutants that are capable of productive infection of undifferentiated F9 cells. Three mutants, PyF101, PyF111 and PyF441, have been plaque-purified and examined. All three PyF mutant DNAs are altered from the wild-type sequence in the Pvu II-4 fragment that spans 67.6 to 70.2 map units on the polyoma genome. PyF441 has a single base change of A to G at 69.6 map units. PyF101 and PyF111 DNAs also contain this point mutation at 69.6 map units. In addition, PyF101 and PyF111 DNAs have exact tandem duplications of 54 and 31 bp, respectively, of sequences encompassing the point mutation, and both copies of the tandem duplication have the point mutation. Other than these changes, no difference exists in the nucleotide sequences of wild-type and PyF mutant DNAs from the BcI I site at 65.6 map units clockwise through the origin of viral DNA replication to the BgI I site at 72.2 map units. DNA infections of F9 cells with wild-type-mutant hybrid DNAs formed by ligation of heterologous combinations of the small and large DNA fragments generated by double digestion with the restriction enzymes BcII and BGI I show that the DNA sequence changes described above are responsible for the ability of the PyF mutants to infect F9 cells.

In vivo messenger RNA for bacteriophage phiX174 was fractionated in agarose gels into a number of discrete species ranging in size from 0.23 X 10(6) to 2.3 X 10(6) daltons. These RNA species were eluted from the gels and hybridized to specific fragments derived from phiX-174 replicative form DNA by cleavage with restriction enzymes. A map of the orientation of in vivo messenger RNAs with respect to the bacteriophage genetic map was constructed. This map indicated that initiation of messenger RNA occurred before gene B, gene C or D, and probably before gene A and that termination occurred after genes E, F, G, and H. Termination of transcription at any particular site appeared not to be entirely effective such that a number of overlapping transcripts with the same 5'-terminus was observed. Messenger RNA for gene A seemed to be relatively unstable.

A point mutation at nucleotide 5258 in the B enhancer of the polyomavirus host range mutant F441 leads to productive infection of F9 embryonal carcinoma cells, which are refractory to infection by wild-type polyomavirus. Specific oligonucleotides were used to construct mutations in two other potentially important domains within the B enhancer of F441 DNA. One of these domains is the binding site for a factor present in nuclear extracts of F9 cells, and the other is a region that has sequence similarity to putative core sequences observed in a number of different viral enhancers. Mutation within either of these two domains, even in the presence of the F441 mutation, was detrimental to polyomavirus enhancer activity in F9 cells, as determined by both transfection and infection assays.

Although wild-type polyoma virus does not productively infect murine embryonal carcinoma (EC) cells, a number of mutants (PyEC mutants) that do infect undifferentiated EC cells have been isolated. All PyEC mutants have DNA sequence alterations within the enhancer region of the viral genome. This report describes an activity present in nuclear extracts of F9 EC cells which, by "footprint" analyses, binds specifically to a small region of about 20 base pairs (nucleotides 5180-5200) within the subregion of the polyoma enhancer designated as the B or beta element. While no difference in binding of factor was detected between wild-type polyoma enhancer and the enhancers of the PyEC mutants, PyF111 and PyF441, which had been selected for productive infection of F9 cells, definite differences between wild-type and mutants were observed in the digestion patterns of their naked DNAs with either DNAase I or exonuclease III. This difference was restricted to the region around the point mutation (nucleotide 5258) common to these mutant DNAs.

A point mutation in the enhancer of polyomavirus host range mutant, PyEC F441, permits productive infection of the murine embryonal carcinoma cell line, F9. This mutation at nucleotide position 5258 introduces a local conformational change in naked viral DNA. The effect of all four possible nucleotide sequences at position 5258 on local DNA conformation was analyzed by gel electrophoresis of fragments produced by ligation of synthetic oligonucleotides having these sequences. The results indicated that both the wild-type and the F441 sequences introduced local structural polymorphism that can lead to DNA bending. The wild-type sequence had a greater effect on DNA curvature than the F441 sequence. The two other sequences at nucleotide 5258 did not appear to introduce detectable amounts of DNA curvature.

A point mutation at nucleotide 5258 in the enhancer of the polyomavirus host range mutant F441 permits productive infection of F9 embryonal carcinoma cells, which, when undifferentiated, are refractory to infection by wild-type polyomavirus. Synthetic oligonucleotides were used to construct viral genomes containing all four possible nucleotide pairs at nucleotide 5258. While all four of the viruses infected 3T6 cells efficiently, only F441, which has a guanosine in place of the wild-type adenosine in the early strand of DNA at position 5258, was able to infect F9 cells. Transfection assays with enhancer-dependent plasmid constructs expressing the chloramphenicol acetyltransferase gene under the control of the polyomavirus early promoter verified that only the F441 enhancer had any significant activity in F9 cells. DNase I footprinting showed that the F441 mutation creates a strong binding site for purified CCAAT box transcription factor, which is identical to nuclear factor 1. The three other mutations at nucleotide 5258 alter the affinity and the quality of factor binding at this site.

The mouse teratocarcinoma stem cell line, F9, becomes permissive for productive polyoma infection upon treatment with retinoic acid. Through the use of M13-polyoma recombinant single-stranded DNA probes, spliced and unspliced early viral RNA were detected after polyoma infection of retinoic acid-treated and untreated F9 cultures.