Analysis of trinucleotide repeats in myotonic dystrophy.

Myotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat in the 3-untranslated region of a gene encoding a serine-threonine protein kinase. There is no one method to detect the entire range of expansion sizes possible in affected patients, so current diagnostic approaches rely on analyzing samples by hybridization of both polymerase chain reaction (PCR)-amplified CTG repeats (CTG-PCR) and genomic DNA. In this unit, the the Basic Protocol 1 describes the analysis of PCR-amplified repeats transferred to a nylon membrane by Southern blotting and hybridized to an alkaline phosphatase-labeled probe. The first support protocol describes a vacuum blotting technique for rapid transfer of the PCR product to the nylon membrane and the second support protocol describes the use of a radiolabeled oligonucleotide probe for hybridization. Analysis of genomic DNA by similar hybridization techniques is outlined in the second basic protocol. Myotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat Myotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat.

Measuring women's preferences for breast cancer treatments and BRCA1/BRCA2 testing.

In establishing decision models in the treatment and prevention of breast cancer, it is important to evaluate patients' preferences for such interventions. The objectives of the present study were: (i) to characterize women's preferences for breast cancer treatments and BRCA1/BRCA2 testing, using the rating scale and standard gamble techniques; and (ii) to identify factors associated with these quality of life indices. Data were collected from women with breast cancer (n = 60), high-risk relatives of women with breast cancer (n = 58), and women in the general population (n = 51). Regardless of group membership, participants favoured treatment and prevention options that involved minimal physical invasiveness. Women with breast cancer rated lumpectomy and radiation treatment more highly than high-risk relatives and women in the general population. Preferences did not differ according to participants' intentions to undergo BRCA testing. Age was the only demographic variable associated with health state preferences. These findings hold implications for the application of patient preferences to clinical decision making.

DNA methylation analysis with respect to prenatal diagnosis of the Angelman and Prader-Willi syndromes and imprinting.

The Angelman (AS) and Prader-Willi syndromes (PWS) are clinically distinct neurobehavioural syndromes resulting from loss of maternal (AS) or paternal contributions (PWS) of imprinted genes within the chromosomal 15q11-q13 region. The molecular diagnosis of both syndromes can be made by a variety of techniques, including DNA methylation, DNA polymorphism and molecular cytogenetic analyses. DNA methylation analysis at three major loci (ZNF127, PW71 and 5' SNRPN) has been successfully used for the postnatal diagnosis of AS and PWS. Methylation analysis, in contrast to other techniques, can reliably be used to diagnose all three major molecular classes (deletion, uniparental disomy and imprinting mutation) of PWS, and three of the four major classes of AS. In this study we demonstrate that methylation analysis can also be successfully used in prenatal diagnosis, by examining specimens obtained from amniocentesis and chorionic villus sampling. Correct prenatal diagnoses were obtained in 24 out of 24 samples using the 5' SNRPN locus; 4 out of 15 using the ZNF127 locus; and 10 out of 18 using the PW71 locus. Therefore, our data indicate that although the DNA methylation imprints of ZNF127 and 5' SNRPN arise in the germline and are present in brain, only 5' SNRPN maintains the imprint in tissues suitable for the prenatal diagnosis of AS and PWS.

Psychological and social determinants of women's decisions to undergo genetic counseling and testing for breast cancer.

This study examined the demand for breast cancer genetic testing and counseling among Canadian women diagnosed with breast cancer under the age of 50, together with some of the factors predicting both their intentions to be tested and the degree to which they act on their intentions. Participants were 110 women under the age of 50 and comprised of two groups: 1) women diagnosed with breast cancer (BC, n = 60): and 2) an index group of unaffected women from the general population (GP, n = 50). All participants completed a survey that addressed family history of breast and other cancers, demographic variables, knowledge and attitudes about breast cancer, and genetic testing. Members of the BC group were offered genetic counseling and testing for BRCA1 and BRCA2 free of charge. Overall, 60% of participants indicated they would like the test, and 40% either did not want it or were uncertain. Seventy-two percent of women in the BC group wanted to be tested. Of these, only 49% had actually contacted the genetic counselor about testing at follow-up 3-15 months later. Intention to be tested was associated with presence of breast cancer, greater perceived benefits of testing, fewer perceived 'costs' of testing, and higher levels of concern about the risk of relatives developing breast cancer. Actual arranging to meet with the genetic counselor among women in the BC group was associated with fewer perceived costs of having the test. Results suggest a moderate level of interest in gene testing, though intention to be tested may not translate into actual uptake. Women who do choose to have the test may believe the potential 'costs' of using this new genetic technology to be relatively few. This has implications for genetic counselors in terms of providing balanced and complete information to women considering genetic testing for breast cancer susceptibility.

Some caveats in PCR-based prenatal diagnosis on direct amniotic fluid versus cultured amniocytes.

The polymerase chain reaction (PCR) offers new advances in prenatal genetic diagnosis particularly with limitations in amount of sample, turn-around time of results, and costs. However, maternal contamination is a concern in any fetal sampling, and even more so with PCR given its potential to detect at the level of a few cells. We report our experience with 53 matched pairs of direct and cultured amniocytes using three independent DNA markers amplified by PCR within the setting of a service molecular diagnostic laboratory. Despite 15/53 (30 per cent) of the amniotic fluids showing visible red blood cells prior to culturing, only 5/53 (9 per cent) showed trace PCR contamination. Of note, this was found on only one marker with a particularly robust PCR product of small size and at such a low level that it was unlikely to have resulted in ambiguous interpretation. One of the cultures also showed a similar type of contamination with this same marker. However, in addition, there were 2/53 (3.7 per cent) cultures which showed substantial maternal contamination detected by all three PCR markers, but not visualized on the originating direct samples. Our results suggest that the careful use of direct amniocytes for molecular genetic testing by PCR is reliable and reproducible in most cases.

Development and Evaluation of a Decision Aid About Prenatal Testing for Women of Advanced Maternal Age.

OBJECTIVE: To develop and evaluate a decision aid designed to prepare patients of advanced maternal age for counseling about prenatal diagnostic testing. SETTING: A regional genetics center. DESIGN: A before/after study. INTERVENTIONS: Participants used an audioguided workbook to learn about options and outcomes and to clarify personal risks, values, questions, and predispositions. SUBJECTS: 21 women of advanced maternal age and 17 spouses. MAIN OUTCOME MEASURES: Knowledge of prenatal testing alternatives, decisional conflict, level of anxiety, and acceptability of the decision aid. RESULTS: After using the decision aid, participants had significantly reduced decisional conflict (uncertainty) and a significant increase in knowledge. There was no effect on state or trait anxiety. More than three-quarters of participants were satisfied with the length, clarity, balance, and acceptability of the decision aid. CONCLUSIONS: The decision aid shows promise as a useful aid for preparing couples for counseling.

Physician knowledge and attitudes towards molecular genetic (DNA) testing of their patients.

OBJECTIVE: To better define the knowledge and attitudes of practicing physicians about genetics; specifically molecular genetics. Further, to examine differences between four practice specialties and to assess variables that affect both knowledge and attitudes. DESIGN: A mail-in survey was sent to a random sample of non-geneticist physicians, with a second copy sent to non-responders. Questions included sociodemographic variables, sources of current knowledge and education in genetics, clinical experience with genetic disease, self-confidence in providing genetic counseling, attitudes towards referring patients to a genetic center, awareness of molecular genetic testing and attitudes towards its use in clinical practice and population screening. SETTING: Responses were obtained from over 900 practicing physicians in the Canadian province of Ontario (population 10 million). Genetic services are provided through nine major and several outreach centers. Molecular diagnostic services are provided through six provincially funded laboratories. There are no direct costs to the patient for any genetic service. PARTICIPANTS: A random sample of family physicians, obstetricians, pediatricians and internists was surveyed from both private and hospital based practices. RESULTS AND CONCLUSION: Responses varied by specialty, years from graduation, gender, and type of practice. Pediatricians and obstetricians were more knowledgeable about genetics, had more interaction with genetic services and were more supportive of their utility. A major proportion of physicians continue to rely upon undergraduate and medical school courses for knowledge, and the specialties showed different preferences for seeking information. A majority of physicians considered their knowledge of genetics to be adequate, but a minority were confident to provide genetic counseling for simple genetic scenarios. Relatively few had actually made use of DNA diagnostic services and there was relatively poor knowledge of what services were available.

Clinical application of the molecular diagnosis of spinal muscular atrophy: deletions of neuronal apoptosis inhibitor protein and survival motor neuron genes.

The molecular genetic diagnosis of spinal muscular atrophy (SMA) has recently been complicated by the identification of two candidate genes, which are often deleted in affected individuals but are also occasionally deleted in apparently unaffected carriers. We present a compilation of genotypes, from our laboratory and recent reports, for the survival motor neuron (SMN) and neuronal apoptosis inhibitor protein (NAIP) genes. Bayesian analyses were used to generate probabilities for SMA when deletions are present or absent in SMN. We found that when the SMN(T) exon 7 is deleted, the probability of SMA can reach greater than 98% in some populations, and when SMN(T) is present, the probability of SMA is approximately 17 times less than the prior population risk. Deletion of NAIP exon 5, as well as SMN(T) exon 7, is associated with a 5-fold increased risk of type I SMA. Case studies are used to illustrate differing disease risks for pre- and postnatal testing, depending on the presence of information about clinical status or molecular results. These analyses demonstrate that deletion screening of candidate genes can be a powerful tool in the diagnosis of SMA.

Molecular diagnosis of non-deletion SMA patients using quantitative PCR of SMN exon 7.

The telomeric survival motor neuron (SMN(T)) gene is a valuable molecular diagnostic tool for childhood-onset spinal muscular atrophy (SMA) as homozygous deletions of SMN(T) exon 7 (delta7SMN(T)) are present in approximately 94% of patients. In this report, we provide the first comprehensive study of 32 unrelated non-deletion SMA patients. Quantitative polymerase chain reaction (PCR) studies established that 90% had two intact copies of SMN(T) exon 7 suggesting that these patients do not have 5q SMA. Once 5q SMA is confirmed, the SMN(T) gene can be screened for subtle mutations. Using single strand conformation analysis, we identified two missense mutations (P245L and Y272C) in exon 6 of the SMN(T) gene of two SMA patients shown to have a single copy of SMN(T) exon 7. Y272 is most likely critical for SMN(T) function as it is a target for recurring mutations and is associated with type I SMA. These results emphasize the need for dosage analysis in the differential diagnosis of 5q SMA in nondeletion patients, consistent with extensive clinical heterogeneity and some genetic heterogeneity in this disease. Homozygosity or heterozygosity for a delta7SMN(T) allele confirms the diagnosis of 5q SMA with greater precision than clinical examination alone.

Analysis of parent of origin specific DNA methylation at SNRPN and PW71 in tissues: implication for prenatal diagnosis.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct developmental disorders caused by absence of paternal or maternal contributions of the chromosome region 15q11-q13, resulting from deletions, uniparental disomy (UPD), or rare imprinting mutations. Molecular cytogenetic diagnosis is currently performed using a combination of fluorescence in situ hybridisation (FISH), DNA polymorphism analysis, and DNA methylation analysis. Only methylation analysis will detect all three categories of PWS abnormalities, but its reliability in tissues other than peripheral blood has not been examined extensively. Therefore, we examined the methylation status at the CpG island of the small nuclear ribonucleoprotein associated polypeptide N (SNRPN) gene and at the PW71 locus using normal and abnormal lymphoblast (LB) cell lines (n = 48), amniotic fluid (AF) cell cultures (n = 25), cultured chorionic villus samples (CVS, n = 17), and fetal tissues (n = 18) by Southern blot analysis with methylation sensitive enzymes. Of these samples, 20 LB cell lines, three AF cultures, one CVS, and 15 fetal tissues had been previously diagnosed as having deletions or UPD by other molecular methods. Methylation status at SNRPN showed consistent results when compared with FISH or DNA polymorphism analysis using all cell types tested. However, the methylation pattern for PW71 was inconsistent when compared with other tests and should therefore not be used on tissues other than peripheral blood. We conclude that SNRPN, but not PW71, methylation analysis may be useful for diagnosis of PWS/AS on LB cell lines, cultured amniotic fluid, or chorionic villus samples and will allow, for the first time, prenatal diagnosis for families known to carry imprinting centre defects.

Direct detection of expanded trinucleotide repeats using PCR and DNA hybridization techniques.

Recently, unstable trinucleotide repeats have been shown to be the etiologic factor in seven neuropsychiatric diseases, and they may play a similar role in other genetic disorders which exhibit genetic anticipation. We have tested one polymerase chain reaction (PCR)-based and two hybridization-based methods for direct detection of unstable DNA expansion in genomic DNA. This technique employs a single primer (asymmetric) PCR using total genomic DNA as a template to efficiently screen for the presence of large trinucleotide repeat expansions. High-stringency Southern blot hybridization with a PCR-generated trinucleotide repeat probe allowed detection of the DNA fragment containing the expansion. Analysis of myotonic dystrophy patients containing different degrees of (CTG)n expansion demonstrated the identification of the site of trinucleotide instability in some affected individuals without any prior information regarding genetic map location. The same probe was used for fluorescent in situ hybridization and several regions of (CTG)n/(CAG)n repeats in the human genome were detected, including the myotonic dystrophy locus on chromosome 19q. Although limited at present to large trinucleotide repeat expansions, these strategies can be applied to directly clone genes involved in disorders caused by large expansions of unstable DNA.

A recombination event occurring within two complex 5q13.1 microsatellite repeat polymorphisms suggests a telomeric mapping of spinal muscular atrophy.

The gene for the childhood spinal muscular atrophies (SMAs) has been mapped to 5q13.1. The interval containing the SMA gene has been defined by linkage analysis as 5qcen-D5S629-SMA-D5S557-5qter. We have identified a recombination event within this interval on a type-I SMA chromosome. The recombination maps to a region of multilocus microsatellite repeat (MSR) markers, and occurs between different subloci of two such markers, CMS-1 and 7613. While the possibility of a novel mutation caused by the recombination cannot be discounted, we believe when viewed in the context of a similar recombination in a Dutch SMA family, a centromeric boundary at the recombination site for the critical SMA interval is likely. This new proximal boundary would reduce the minimal region harboring the SMA locus from approximately 1.1 Mb to approximately 600 kb.

Delivery of molecular genetic services within a health care system: time analysis of the clinical workload. The Molecular Genetic Study Group.

The most recent discoveries in molecular genetics today are rapidly incorporated into clinical practice and have resulted in an unprecedented expansion of medical options. Despite this, the impact of molecular genetics on health care services has yet to be evaluated. In order to begin this assessment, clinical genetic workload was prospectively collected from cases where molecular genetic testing was considered. Participation involved all 16 urban and outreach genetic centers regionalized to service the entire population of 10 million within the Canadian province of Ontario. Molecular genetic testing has been clinically available for > 5 years, as part of a publicly supported genetic network in which there are no direct costs to residents. Cross-sectional data were collected on 1,101 clients from 544 families involving 1,742 clinical actions relating to diseases in which molecular (DNA) tests were considered. Median times per clinical genetic action were as follows: formal counseling (60 min), case review (15 min), phone call (10 min), letter (15 min), specimen arrangement (15 min), and interpretation of molecular test results (10 min). Times varied significantly with the inheritance pattern of the disease, topics involved, and location. For any given genetic case, multiple clinical actions resulted in substantial time spent by the genetic professional. Clerical and administrative times were not captured. Workload unit measurements similar to those currently employed in hospital laboratories may be helpful for predicting the clinical resources and personnel that will be required as the use of molecular genetics by other medical specialties increases.

Convergent myotonic dystrophy (DM) haplotypes: potential inconsistencies in human disease gene localization.

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease which has been shown to be caused by an unstable trinucleotide repeat located on chromosome 19q. We have conducted extensive haplotype analysis on 105 DM chromosomes using twelve 19q13.2 loci identifying 18 RFLPs, spanning a physical distance of 1.3 Mb containing the DM gene. Three major haplotypes (H1, H2 and H3) comprising 46.7% of the DM chromosomes in our population, were observed. With the exception of H1 and H2 derivatives (H4, H5 and H6), the remainder of the DM chromosomes analyzed were found to have unique haplotypes. Haplotypes H2 and H3 observed exclusively on DM chromosomes of French-Canadian origin contain identical 500-kb core regions. The low frequency of this core haplotype in normal chromosomes (0.8%) is consistent with a mapping of the DM gene within this region. However, the DM mutation is found 160 kb distal to the point of divergence between the two haplotypes. In contrast, the 450-kb region shared by haplotypes H1 and H2 contains the DM mutation. Further analysis of the DM region using a polymorphic microsatellite (GJ-VSSM2; D19S207) located 15 kb distal to the DM mutation revealed strong allelic association of one of the (CA)n repeat alleles to DM; allele 5 was observed on 88.2% of DM chromosomes and 6% of normal chromosomes. The fact that the (CA)n allele 5 was found on all 56 DM chromosomes containing the three major haplotypes indicates that DM chromosomes in our population, including the two French-Canadian haplotypes which have a common region outside the DM gene, are probably derived from the same mutational event.(ABSTRACT TRUNCATED AT 250 WORDS)

Two 5q13 simple tandem repeat loci are in linkage disequilibrium with type 1 spinal muscular atrophy.

The gene for the common recessive neuromuscular disorder spinal muscular atrophy (SMA) has been previously mapped to chromosome 5q. We report here linkage disequilibrium analyses of two polymorphic simple tandem repeat (STR) sequences which map into the critical region of 5q13 containing the SMA gene. The polymorphisms presented are constituents of CATT-1, a complex STR which is present in as many as four or more copies per chromosome 5. The PCR can amplify as many as eight CATT-1 products of different sizes from genomic DNA samples due to differing numbers of CA dinucleotides at each STR location (sublocus). Oligonucleotide primers for two of these subloci have been developed for specific PCR assays; a variety of allele sizes can be generated with each assay and, in some cases, no amplification products are detected due to null alleles. The genotyping of 149 SMA Type 1 chromosomes and 142 normal chromosomes from Canadian and American kindreds reveals the presence of significant linkage disequilibrium between the null allele of the sublocus referred to as CATT-40G1 and mutation(s) causing SMA Type 1 (Werdnig-Hoffmann disease). Allele 2 of the second sublocus, CATT-192F7, is also in linkage disequilibrium with SMA Type 1 although the degree of this association is less than that found for CATT-40G1. The proximal and distal STRs from the critical region, D5S435 and D5S351, showed no linkage disequilibrium with SMA. The data presented here will serve as a framework for future linkage disequilibrium analyses, expediting the final stage of the search for the SMA gene.

Cystic fibrosis carrier screening in a high-risk population. Participation based on a traditional recruitment process.

OBJECTIVE: Recent advances in molecular genetic (DNA) technology have permitted identification of previously undetectable cystic fibrosis (CF) carriers. Although research has been initiated in the general population, to our knowledge no published studies have looked at the utilization of DNA-based carrier screening in the high-risk CF population (family history of CF). DESIGN: Cross-sectional, diagnostic open trial. SETTING: Carrier testing was offered to a high-risk CF population via adult patients with CF or parents of pediatric patients with CF attending two regional CF clinics over a 3-year period. PARTICIPANTS: Consecutive sample of virtually all patients with CF (n = 118) from a population of 1 million. MAIN RESULTS: Despite free services, written follow-up, and counseling for 99% of patients attending the CF clinic, there was less than 10% participation from high-risk family members (168 blood relatives and 26 spouses of identified carriers or patients with CF; 38 and 156 persons from the adult and pediatric clinic families, respectively). Nevertheless, we identified 91 CF carriers among the 168 high-risk relatives. This is comparable to the number of carriers detected in general population carrier screening that has tested substantially more individuals (> 3000 per study). CONCLUSIONS: Our results suggest that research concerning CF carrier screening not only focus on data about fundamental program resources and numbers of carriers detected but also investigate how information about the availability of carrier screening is disseminated, the motivation behind testing, and the perceived relevance of test results by those tested in the high-risk population. These issues are increasingly relevant as screening becomes feasible using DNA testing for far more prevalent disorders (such as breast cancer and diabetes).

A multicopy dinucleotide marker that maps close to the spinal muscular atrophy gene.

Spinal muscular atrophy (SMA) is a common autosomal recessive disorder resulting in loss of motor neurons. The interval containing the SMA gene has been defined by linkage analysis as 5qcen-D5S435-SMA-D5S557-5qter. We have isolated a new dinucleotide repeat marker, CATT1, that lies between these two closest markers. The marker CATT1 has 16 alleles and is highly polymorphic. The marker can have 1 to 4 (or more) copies per chromosome, giving rise to individuals with up to 8 (or more) alleles. All of the subloci map between the markers D5S557 and D5S435 and lie in close proximity to one another. The marker CATT1 is linked to the SMA gene with a lod score of Zmax = 34.42 at theta = 0 and crosses all available recombinants. Certain alleles occurred more frequently in either the SMA or normal populations, indicating significant allelic association between CATT1 and the SMA locus. Haplotype analysis combining U.S. and Canadian SMA families reveals that one haplotype group (VII) occurs significantly more frequently in the SMA population than in the normal. This confirms the allelic association of CATT1 with the SMA locus.