Population-specific screening by mutation analysis for diseases frequent in Ashkenazi Jews.

We describe a partially automated DNA mutation assay for detecting the most frequent mutations in the alpha-subunit of beta-hexosaminidase A, the acid beta-glucosidase and the cystic fibrosis transmembrane conductance regulator genes for the Ashkenazi Jewish population. The assay detects carriers for Tay-Sachs disease, Gaucher disease, and cystic fibrosis with sensitivities of at least 92%, 96%, and 97%, respectively. Among 1,364 young adults of Ashkenazic ancestry in the Dor Yeshurim community who were tested, 52 were Tay-Sachs carriers, 110 were Gaucher carriers, and 62 were cystic fibrosis carriers. Ten individuals were carriers for two diseases, and three unsuspected cases were diagnosed with Gaucher disease based on mutation test results. In addition to Tay-Sachs mutation data, results for hexosaminidase A activity were also available. All of 1,254 samples normal by enzyme quantitation were also negative for the three alpha-subunit mutations tested, and all of 43 samples with 'inconclusive' enzyme results were negative by DNA. Only 52 of 67 samples positive by enzyme assay were also positive for one of the three mutations tested for Tay-Sachs disease. The data suggest a high degree of false positivity inherent in enzyme identification of carriers. There are no correlative methods to assess the sensitivity of Gaucher and CF carrier testing. The results show that population screening can be carried out efficiently by DNA analysis, with the accrual of carrier information for three separate diseases conducted as a single test. Furthermore, the DNA method for Tay-Sachs screening appears to exceed the specificity of hexosaminidase A enzyme testing.

A robotics-assisted procedure for large scale cystic fibrosis mutation analysis.

We describe a convenient, efficient, semiautomated protocol for assaying large numbers of DNA samples for over 20 mutations causing cystic fibrosis. The protocol uses the following: (1) a programmable robotic workstation to perform rapid pipetting and dot-blotting operations, (2) an allele-specific oligonucleotide hybridization in a single water bath without correcting for G+C content of oligonucleotides, and (3) a combinatorial system that allows direct determination of the genotype for more frequent mutations. We have used this system routinely for 16 months for carrier detection and for diagnosis of cystic fibrosis. The method can be readily applied to any combination of allele-specific oligonucleotide assays whether for multiple alleles at one locus or for a few alleles at multiple loci.

Experience of an academic reference laboratory using automation for analysis of cystic fibrosis mutations.

OBJECTIVE: To examine the use of partial automation for molecular analysis of cystic fibrosis and to evaluate the diagnostic experience gained. DESIGN: Twenty-four cystic fibrosis mutations, with cumulative mutation detection of 89% in North American whites and of 97% in the Ashkenazim, were tested by multiplex amplification and allele-specific oligonucleotide hybridization. SETTING: A university-based DNA diagnostic laboratory. SUBJECTS: More than 700 serial specimens were analyzed for cystic fibrosis mutations over a 5-month period. The study included 377 individuals tested for carrier status, of which 288 had no family history for cystic fibrosis; prenatal diagnosis for 17 fetuses at a one in four risk and eight pregnancies at lower risk; fetal or parental samples for 33 pregnancies with fetal ultrasound abnormalities; 40 individuals diagnosed with cystic fibrosis; and 87 individuals with a possible diagnosis of the disease. RESULTS: Automation has permitted increasing numbers of mutations while decreasing personnel time and cost. Mutation testing identified 10 carriers with no family history for cystic fibrosis, four couples at a one in four risk, and five affected fetuses, one ascertained by abnormal fetal ultrasound. Mutation analysis also identified two mutant copies of the gene in 26 of 40 individuals with a clinical diagnosis of cystic fibrosis, and in two of 87 patients with possible cystic fibrosis. CONCLUSION: This partially automated, direct mutation analysis provides DNA diagnostic laboratories with the capacity to process a larger number of samples at lower cost with greater sensitivity for mutation detection. As pilot screening programs are reported, it is appropriate to reevaluate recommendations regarding population-based carrier screening for cystic fibrosis.

Analysis of an origin of DNA replication located at the L terminus of the genome of pseudorabies virus.

We have localized an origin of DNA replication at the L terminus of the pseudorabies virus genome. This origin differs in location as well as in general structure from the origins of replication of other herpesviruses that have been identified. The 600 leftmost nucleotides of the genome that were found to include origin function have been analyzed. This sequence is composed of an 82-bp palindrome whose center of symmetry is separated by 352 unique bp (UL2). Within the UL2, a sequence that fits the consensus sequence of the NF1 binding site, as well as one that has partial homology to the binding site of UL9 of herpes simplex virus, is present. Using truncated fragments of DNA, sequences essential for minimal origin function were delimited to within a fragment that includes the terminal 104 bp of the left end of the genome. Within these 104 bp, two elements essential to origin function have been identified. One of these elements is present within the terminal 64 bp of the L component (within one of the palindromic arms). The other is present within the 22 bp of the UL2 adjacent to this palindromic arm. Other auxiliary elements, although not essential for origin function, contribute to more efficient replication. The NF1 and UL9 binding site homologies were found to be nonessential to origin function.

Structural organization of the termini of the L and S components of the genome of pseudorabies virus.

The sequences of several hundred nucleotides around the junctions between the L and S components in concatemeric DNA and in mature virion DNA were ascertained. The two ends of the mature genome (which are joined in concatemeric DNA) show no sequence homology. Several directly repeated elements are present near both ends of the genome. Furthermore, the last 82 nucleotides at the left end of the L component (and of the genome) are repeated in inverted form (inverted repeat within the L component [IRL]) approximately 350 to 600 nucleotides downstream (depending on the virus isolate) bracketing the UL2 component. A comparison between the sequences at the right and left ends of the L component of the genome showed patchy homology, probably representing a vestigial inverted repeat bracketing the L component (IRL). Furthermore, less than 5% of the genomes have an L component that is in the orientation opposite to that of most of the viral genomes, indicating that the vestigial IRL that brackets the UL sequence may be sufficient to mediate inversion of the L component in some of the genomes. On the other hand, the UL2 component, which is bracketed by a perfect IRL, does not invert to a greater extent than does the L component (if it inverts at all). Analysis of the nucleotide sequence at the concatemeric junction of three different pseudorabies virus isolates showed almost complete sequence conservation. The sequence and organization of the repeated elements in the different isolates were almost identical, despite their different histories and origins. The high degree of conservation of these repeated elements implies that they may fulfill an essential function in the life cycle of the virus.

Nucleotide sequences at recombinational junctions present in pseudorabies virus variants with an invertible L component.

The genome of pseudorabies virus (PrV) consists of two components--a noninvertible long (L) and an invertible short (S) component. The S component is bracketed by inverted repeats. In some variant strains of PrV (which have a selective growth advantage in certain cell lines), a sequence normally present at the left end of the L component has been translocated to the right end of the L component next to the inverted repeat. Consequently, these strains have acquired a genome with an L component that is bracketed by inverted repeats and that also inverts. We have determined the restriction maps and have analyzed the nucleotide sequences of those parts of the genome of three strains with invertible L components that contain the translocated segment of DNA. The results were as follows. The translocated fragments were derived in all cases from the extreme left end of the L component only. The sizes of the translocated fragments were similar, ranging from 1.3 to 1.4 kilobase pairs. The junction between the L and S components in these strains was the same as that in standard viral concatemeric DNA. The translocation of sequences from the left end of the genome next to the inverted repeats was always accompanied by a deletion of sequences from the right end of the L component. The sizes of the deleted fragments varied considerably, ranging from 0.8 to 2.3 kilobase pairs. Sequence homology at the points of recombination, i.e., at the junction between the right end and the left end of the L component, existed sometimes but not always. A model depicting how a virus with a class 2 genome (such as PrV) may acquire a genome with characteristics of a class 3 genome (such as herpes simplex virus) is presented.

Role of glycoproteins of pseudorabies virus in eliciting neutralizing antibodies.

The experiments described in this paper were designed to assess the role of the various virus glycoproteins of pseudorabies virus (PrV) in eliciting the production of neutralizing antibodies during the normal course of infection of swine. They also address the question of the degree of antigenic variation within each glycoprotein between different virus isolates. The results show the following: Antigenic variation between strains of PrV isolated from different geographic areas are readily detectable; antigenic differences between strains isolated from the same geographic area are less common. No antigenic drift in glycoprotein gII was observed. Glycoprotein gIII and, to some extent, also glycoprotein gI showed a high level of antigenic drift. The neutralizing activity of pooled convalescent sera of swine is not directed against glycoprotein gI. A large part of the neutralizing activity of pooled convalescent sera of swine is directed against glycoprotein gIII.

Correlation between stimulation of host cell DNA synthesis by human cytomegalovirus and lack of expression of a subset of early virus genes.

The stimulation of host cell DNA synthesis was studied in permissive human embryonic lung (HEL) cells and in nonpermissive rabbit kidney (RK) cells infected with human cytomegalovirus (HCMV). Host cell DNA synthesis was induced by HCMV infection in resting cells of both types. In permissive cultures the stimulation of cellular DNA synthesis was detectable mainly in those cells which had not become productively infected and in which virus antigens were not detectable. In abortively infected RK cells, on the other hand, stimulation of host cell DNA synthesis and the expression of virus antigens were detected in the same cells. Infection of actively growing permissive HEL cells resulted in a shutdown of cellular DNA synthesis beginning approximately 10 hr postinfection. Shutdown of cellular DNA synthesis also occurred when the infected cells were treated with phosphonoacetic acid and was thus classified as an "early" virus function. In actively growing, abortively infected RK cells, on the other hand, host cell DNA synthesis was not affected, indicating that the early virus function(s) responsible for inhibition of cellular DNA synthesis was not expressed in these cells. Virus-encoded DNA polymerase activity, another early virus gene function, was also not detected in these abortively infected cultures. In RK cells the cellular DNA synthesized as a result of infection was capable of undergoing at least one further round of replication, indicating that the HCMV gene expression which occurred in abortively infected RK cells was not lethal for these cells.

Nature of the block in the expression of some early virus genes in cells abortively infected with human cytomegalovirus.

Certain functions normally expressed during the early phase of productive human cytomegalovirus (HCMV) infection in human embryonic lung (HEL) cells are not expressed in abortively infected rabbit kidney (RK) cells. To determine the stage at which infection is blocked in RK cells, HCMV transcripts synthesized by these abortively infected cultures were compared to those synthesized during early stages of infection in productively infected HEL cells. The rate of accumulation of HCMV-specific RNA was approximately six- to eightfold lower in RK than in HEL cells during the first 24 hr postinfection. Although the rate of RNA accumulation did not decrease thereafter in RK cells, it did increase significantly in HEL cells and was dependent on the replication of virus DNA. Transcripts obtained from HEL cells 24 hr postinfection (either in the absence or presence of phosphonoacetic acid) and from abortively infected RK cells were analyzed by the Southern technique. Most of the "early" transcripts which accumulated in infected permissive cells were also present in the infected nonpermissive cells at 24 hr postinfection. However, some of the early transcripts (complementary to approximately 7% of the genome) were significantly underrepresented in RK cells. Furthermore, transcripts originating from one region of the genome, which were abundantly represented as cytoplasmic RNA both in productively and nonproductively infected cells, and which were polysome-associated in HEL cells, were found associated with polysomes in RK cells in very low amounts only. Northern blot analysis of the total and polysomal RNA from infected HEL cells and RK cells confirmed these findings. These results show that differences between productively infected HEL and nonproductively infected RK cells exist at the level of accumulation of some early transcripts, as well as at the level of association of some of these transcripts with polysomes.

Post-transcriptional control of human cytomegalovirus gene expression.

During the immediate-early, early, and late phases of human cytomegalovirus infection in human fibroblasts, transcripts accumulated, respectively, from approximately 20, 75, and 90% of the sequences on the genome. Not all of the sequences which accumulated during the immediate-early and early phases were represented on polysomes, however. Four transcripts synthesized in cycloheximide-treated cells were studied in detail. A 2.2-kb transcript (0.713-0.733 map units) represented 95% of the polysome-associated RNA in cycloheximide-treated cells and was the first to be detected on polysomes at 2 hr postinfection in untreated cells. A second, less abundant, transcript of 5.2 kb (0.670-0.733 map units) was also found on polysomes in cycloheximide-treated cells, and preliminary evidence suggested that this transcript may be spliced during processing. A 3.25-kb transcript (0.190-0.217 map units) was identified also as a minor polysome-associated species of RNA. One transcript of 4.8 kb (0.630-0.670) remained associated with the nucleus and was not processed into mRNA in cycloheximide-treated cells. Differential stability between the various transcripts was observed, the 2.2-kb transcript being the most stable. The results showed that in human cytomegalovirus-infected cells controls exist at the level of transcript accumulation, transport into the cytoplasm, preferential association with polysomes, and relative stability of RNAs.

Patterns of transcription of human cytomegalovirus in permissively infected cells.

The rate of accumulation of cytomegalovirus transcripts in permissively infected human embryonic lung (HEL) cells was analyzed at various times after infection by hybridization of infected cell RNA to undigested or restriction endonuclease-digested cytomegalovirus DNA fixed to nitrocellulose filters. Differences in patterns of transcript accumulation were determined by measuring the abundance levels of RNA which hybridized to different HindIII-, XbaI-, or EcoRI-generated fragments of cytomegalovirus DNA. The results showed that a small but significant amount of cytomegalovirus RNA was detectable within the first 3 h after infection and that the rate of accumulation of these transcripts was static during the first 24 h, but increased thereafter. In general, the viral transcripts accumulating in infected cells could be divided into three classes. Immediate-early RNA (synthesized in the absence of protein synthesis in infected cells) hybridizes predominantly to a very restricted part of the genome and can be identified during the first 2 to 4 h postinfection. Early RNA (synthesized up to about 24 h after infection) originates from most regions of the genome but is characterized by the presence of transcripts which hybridize in great abundance to certain fragments. Late RNA (synthesized after 24 h, i.e., after the onset of viral DNA synthesis) hybridizes in approximately equal abundance to most regions of the viral genome. These results showed that a block in the transition from immediate-early to early RNA did not account for the extended period of time that elapses between the time of infection and the initiation of viral DNA synthesis. Interestingly, despite rapid adsorption and penetration and a static level of accumulation of transcripts in the cultures during the first 24 h, the number of cells that synthesized detectable amounts of viral antigens increased steadily during this time.

Induction of cellular DNA synthesis by defective human cytomegalovirus.

The results presented in this paper show that virus stocks enriched for defective particles are more effective in stimulating cellular DNA synthesis than are stocks relatively free of defective particles. Several lines of evidence support this conclusion: (1) Low levels of UV irradiation enhance the ability of standard virus stocks to stimulate cellular DNA synthesis. (2) Three different stocks derived from individual plaques (which presumably contain very few defective particles) and passaged only twice at low multiplicity, were found to be poor inducers of cellular DNA synthesis. (3) Stocks of virions obtained after serial undiluted passage which were shown to be enriched for defective particles were quite effective in stimulating cellular DNA synthesis.

Physiological state of human embryonic lung cells affects their response to human cytomegalovirus.

Cultures of human embryonic lung (HEL) cells in different physiological states were studied for their susceptibility to infection with human cytomegalovirus (CMV) with respect to production of infectious virus, synthesis of viral antigens, and virus-induced stimulation of cellular DNA synthesis. In general, subconfluent, actively growing cells yielded higher amounts of infectious virus than did confluent contact-inhibited cells. The higher yield of infectious virus was correlated with a greater percentage of cells producing viral antigens within the first 48 h after infection. In confluent cultures, 25 to 50% of the cells produced viral antigens within the first 48 h postinfection. This proportion did not change over a 10-fold range of multiplicity of infection, indicating that many of the cells in confluent cultures did not support productive infection. However, virtually all the cells in subconfluent cultures were susceptible. Also, in contrast to herpes simplex virus and pseudorabies virus, infectious CMV is not produced by cells treated with 5-fluorouracil and thymidine. Virus-induced stimulation of cellular DNA synthesis in cells infected at high multiplicities of infection could be detected only in confluent cultures, in which cellular DNA synthesis had been previously suppressed, but could not be detected in similarly treated cultures of subconfluent cells. The lack of detectable stimulation of cellular DNA synthesis in the latter was related to the fact that practically all the cells in the culture synthesized viral antigens within the first 48 h after infection, productive infection and detectable synthesis of cellular DNA being mutually exclusive.