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PubMed2001

An effective strategy of using molecular testing to screen mentally retarded individuals for fragile X syndrome.

Fragile X syndrome (FXS) is the most common form of familial mental retardation (MR). It is caused by the expansion of the CGG repeat in the FMR1 gene on the X chromosome. To date, FXS is not treatable, but can be prevented by prenatal genetic examination. Identifying women who carry a full mutation or premutation FMR1 gene is thus very important, and can be done by tracing family members of FXS subjects. However, most of the FXS subjects in Taiwan as well as those in many other countries have not been identified. In this study the authors attempt to develop reliable and inexpensive tests suitable for a large-scale screen of subjects with MR for FXS. Together with their previous study, a total of 311 male and 160 female subjects with MR were screened with nonradioactive Southern blot assay using mixed deoxyribonucleic acid from three subjects of the same sex. From these subjects, nine male subjects and one female FXS subject were diagnosed. All male subjects were also screened with nonradioactive polymerase chain reaction (PCR). These nine male FXS subjects were also detected on the basis of PCR amplification failure. No false-negative results were discerned. The PCR procedure was simplified further by combining it with an analysis of a blood spot on filter paper, which is a much simpler and cheaper method for sample collection and DNA preparation. This method was then used to screen 104 boys with MR. Two of them were suspected, and later confirmed with Southern blot assay, as subjects with FXS. This study suggests that simple PCR combined with blood spot analysis could be a reliable, inexpensive test that is feasible for a large-scale screening of male subjects with MR for FXS. However, Southern blot assay with mixed deoxyribonucleic acid is appropriate for screening female subjects. Based on this strategy, most FXS subjects could be identified easily for further management.

Blotting, Southern↗
PubMed2007

Molecular testing for Fragile X Syndrome: lessons learned from 119,232 tests performed in a clinical laboratory.

PURPOSE: To examine the data from over 119,000 Fragile X Syndrome tests and 307 prenatal tests to detect unsuspected findings and obtain clinical data when indicated to optimize genetic counseling. METHODS: A proprietary database containing 119,232 consecutive postnatal and 307 prenatal FXS tests performed between November 2, 1992 and June 1, 2006 was queried. RESULTS: The distribution of normal FMR1 alleles was a bimodal distribution with a major peak at 30 repeats and a minor peak at 21 repeats. Of 59,707 tests performed for males, 1.4% had a fully expanded and methylated FMR1 allele. Of 59,525 tests performed for females, 0.61% had an affected FMR1 allele, and 1.7% had a premutation FMR1 allele for a total carrier frequency of 1.3%. When fetuses inherited an expanded maternal allele, the risk of expansion to a full affected allele was 0%, 5%, 30% and 100% for allele sizes of <50, 50-75, 76-100 and >100 repeats, respectively. CONCLUSIONS: These figures can be used for genetic counseling of patients presenting for carrier detection and prenatal diagnosis for Fragile X Syndrome.

Adult↗
PubMed2005

An optimized DHPLC protocol for molecular testing of the EXT1 and EXT2 genes in hereditary multiple osteochondromas.

Hereditary multiple osteochondromas (MO) is an autosomal dominant bone disorder characterized by the presence of bony outgrowths (osteochondromas or exostoses) on the long bones. MO is caused by mutations in the EXT1 or EXT2 genes, which encode glycosyltransferases implicated in heparan sulfate biosynthesis. Standard mutation analysis performed by sequencing analysis of all coding exons of the EXT1 and EXT2 genes reveals a mutation in approximately 80% of the MO patients. We have now optimized and validated a denaturing high-performance liquid chromatography (DHPLC)-based protocol for screening of all EXT1- and EXT2-coding exons in a set of 49 MO patients with an EXT1 or EXT2 mutation. Under the optimized DHPLC conditions, all mutations were detected. These include 20 previously described mutations and 29 new mutations - 20 new EXT1 and nine new EXT2 mutations. The protocol described here, therefore, provides a sensitive and cost-sparing alternative for direct sequencing analysis of the MO-causing genes.

Chromatography, High Pressure Liquid↗
PubMed2003

Comparison of molecular tests for detection and quantification of cell-associated cytomegalovirus DNA.

A cell-based standard was developed to compare the COBAS Amplicor CMV Monitor test, the Hybrid Capture System CMV DNA test, and the NucliSens CMV test. The standard was prepared by infecting human foreskin fibroblasts (HFFs) with cytomegalovirus (CMV) strain AD169 at low multiplicity of infection (0.03) and harvesting the cells at 6 h postinfection. Buffy coat cells were added to produce concentrations of from 0 to 10(5) HFFs per 10(6) total cells. Five laboratories performed the Amplicor PCR test and two laboratories performed the NucliSens and Hybrid Capture tests. The Amplicor PCR test was 1.5 to 2.0 log(10) more sensitive than the Hybrid Capture test. The specificities of the Amplicor PCR and Hybrid Capture tests were 100 and 93.8%, respectively. The linear range of the Amplicor PCR and Hybrid Capture tests were 2 to 4.48 log(10) and 3.48 to at least 5.0 log(10) CMV target cells, respectively. The standard deviations of the Amplicor PCR and Hybrid Capture tests varied throughout their linear range, and for both tests the variability was greater for lower concentrations of input CMV DNA. These data allow the direct comparison of viral load values between the Amplicor and Hybrid Capture tests. The analytical sensitivity of the NucliSens test could not be determined by using the 6-h standard, because the low multiplicity of infection and short culture time did not allow for adequate transcription of pp67 late mRNA measured in the test. Extending the incubation time of the standard to 24 h increased the analytical sensitivity of the NucliSens test to 3.0 log(10) target cells.

Cytomegalovirus↗
PubMed2002

NF1 mutations and molecular testing.

Neurofibromatosis 1 is a progressive autosomal dominant condition caused by mutations in the NF1 gene on chromosome 17. The condition shows clinical variable expressivity, with varying features even between family members who share the same mutation. Furthermore, it is impossible to precisely predict the severity and course of the condition, a source of frustration for families and physicians. Neurofibromatosis 1 is also heterogeneous at the mutation level, with more than 300 independent mutations having been reported in this gene. The mutation data have accumulated slowly owing to the variability of the mutation types and the size and complexity of the gene. This is also reflected in the lack of a simple, inexpensive, highly accurate DNA-based test for neurofibromatosis 1 at present. This article reviews current NF1 mutation spectrum and testing, discussing and illustrating mutation mechanisms and pathogenetic effects, as well as factors affecting DNA testing and interpretation/diagnosis.

Child↗
PubMed2005

Molecular testing (strand displacement assay) for identification of urethral gonorrhoea in men: can it replace culture as the gold standard?

The objective was to evaluate the performance of Becton Dickinson's BD Probe Tec(TM) (BDPT) strand displacement amplification (SDA) test for the detection of Neisseria gonorrhoeae on urethral specimens from men with urethritis compared with conventional culture and to show that SDA improves the diagnostic yield of gonorrhoea infections (GC). Anonymized retrospective testing of stored urethral swab samples from men attending genitourinary services in East London was performed using SDA. The prevalence of GC culture positive infections in this sample was 20/152 (13%). The sensitivity, specificity, positive predictive value and negative predictive value for the BDPT-SDA system compared with culture were 100%, 95%, 77% and 100%, respectively. In this study population, the BDPT-SDA assay was a highly sensitive and specific test for the diagnosis of N. gonorrhoeae from urethral swabs in men. Therefore, SDA can be used to complement culture in the diagnosis of N. gonorrhoeae infection. No ethics committee approval was obtained as all samples were anonymized.

Bacterial Typing Techniques↗
PubMedDate not supplied

[Use of molecular tests of human papilloma virus (HPV) as screening test for cervix cancer: a review].

Infection from some types of human papillomavirus has been recognised as carcinogenic for the uterine cervix. With reliable techniques (PCR and Hybrid Capture II) "high risk" HPV types are found in a very high proportion of women with invasive cancer and high-grade pre-invasive lesions. On the other hand prevalence is low in cytologically normal women, except in young women, who seem to have a high frequency of transient infections in the years following the beginning of sexual activity. Some studies found a role of the presence and persistence of "high-risk" HPV types in the progression of low-grade pre-invasive lesions vs. high-grade rather then vs. spontaneous regression. For these reasons HPV testing has been suggested as a possible tool for primary screening. A few studies suggest that it could allow increasing sensitivity, although problems of extrapolability of results exist. It must, however, be considered that traditional cytological screening is already very protective and that simply adding a further test would lead to an unfavourable cost-benefit ratio. An appealing possibility is applying HPV testing with long intervals between screening rounds. This would reduce the burden for women and plausibly allow reaching higher coverage at each round. A key element is the duration of infection before progression to pre-invasive lesion. A long duration would allow selecting women at low risk of developing a lesion for years (those HPV-negative), who could have long-interval test, and others (those HPV-positive) at high risk, to be followed more strictly. Published data from longitudinal studies are limited and don't allow an adequately precise estimate. Therefore the use of HPV testing for primary screening is very promising but to the moment must be considered as a research object.

DNA Probes, HPV↗
PubMed2000

[Development of molecular tests for rapid detection of enteropathogens].

Amplification of specific DNA sequences by polymerase chain reaction (PCR), enables rapid, sensitive and direct, specific identification of pathogens at very low concentrations in clinical samples. Studies in recent years have reported identification of several enteropathogens directly from stool samples by PCR. The amplification process includes the use of primers complementary to the DNA sequences specific to the pathogen, thus relying on the pathogen's genotype, rather than its phenotype on which identification by the methods of classical microbiology were based. We have developed PCR protocols for the differential identification of enteropathogens resembling the normal flora (enterotoxigenic E. coli (ETEC), E. coli O-157), Shigella spp, and the detection of enteropathogens that can not be grown on classic growth media (Norwalk virus). The amplification process is inhibited by several substrates present in fecal material (phenol, hemoglobin), limiting DNA extraction by phenol. The protocols we have developed for direct detection of Shigella spp and ETEC in stools circumvent inhibition of PCR by the use of a 4-hour pre-enrichment step in brain-heart infusion broth. Rapid and accurate identification of enteropathogens is important for prompt and focused intervention to stop the chain of transmission in outbreaks of gastroenteritis in military and civilian populations.

Dysentery, Bacillary↗