Effect of the myotonic dystrophy (DM) mutation on mRNA levels of the DM gene.

Myotonic dystrophy (DM) results from the amplification of an unstable CTG repeat in the 3' untranslated region of a transcript encoding a putative serine/threonine kinase. We have analysed the amplification of the repeat and the steady state levels of the DM kinase (DMK) mRNA in tissues and cell lines from normal and congenital DM individuals. Southern blot analysis of DNA samples from a severely affected neonate shows somatic heterogeneity of the repeat in all tissues studied. RNA analyses on these tissues show a marked increase in DMK steady state mRNA levels. We demonstrate that the mutant DMK allele is expressed regardless of the number of CTG repeats and that the increase in DMK mRNA levels is due to elevated mutant mRNA levels. We postulate that elevated DMK levels explains the dominant inheritance pattern of DM.

Molecular characterization of the murine argininosuccinate synthetase locus.

The cDNA and gene encoding murine argininosuccinate synthetase were cloned and characterized. The cDNA sequence predicts a peptide of 412 amino acids (aa) including the initiator methionine. There is 98% identity with the aa sequence of the human enzyme. The 3'-untranslated region of the cDNA includes two regions of sequence which are conserved between mouse, rat, human and cow. The murine gene contains 16 exons with the start codon occurring in exon 3. Although alternative splicing occurs in primates to include or exclude exon 2, exon 2 sequences were included in the murine mRNA in all tissues and developmental stages examined. The inclusion of exon 2 in murine mRNA, compared to the usual exclusion in human mRNA, may be explained by differences in the donor splice sequences for exon 2.

Interstitial deletion of chromosome 18[del(18)(q11.2q12.2 or q12.2q21.1].

A 27-month old boy with mild developmental delay, growth delay, strabismus, midface hypoplasia, relative telecanthus, downslanting palpebral fissures, epicanthal folds, dental hypoplasia, and cardiac defects was found to have an interstitial deletion of chromosome 18 involving band q12.1 or q12.3

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.

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.

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).

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.

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.

Assignment of the structural gene for argininosuccinate synthetase to proximal mouse chromosome 2.

In order to develop linkage markers for the murine argininosuccinate synthetase locus (Ass-1), we have searched for restriction fragment length polymorphisms in the mouse genome using cloned sequences from the mouse arginosuccinate synthetase structural gene. Five restriction fragment length polymorphisms were found among the recombinant inbred progenitor strains AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, and SWR/J. Of these, four polymorphisms were found to distinguish the SWR/J strain from the other six strains, which all had the same fragment. The fifth polymorphism revealed differences among the progenitor strains for recombinant inbred strain sets AKXL, BXD, and SWXL. The strain distribution pattern for this polymorphism indicated close linkage of Ass-1 to Hc (the fifth component of complement) on proximal mouse chromosome 2 with a recombination fraction of 0.016 and a 95% confidence interval of 0.003 to 0.054. These data place Ass-1 in a syntenic group with the genes Hc, Abl, Fpgs, and Ak-1 whose linkage has been conserved between human chromosome 9q and mouse chromosome 2.

Characterization and polymerase chain reaction (PCR) detection of an Alu deletion polymorphism in total linkage disequilibrium with myotonic dystrophy.

The mutation causing myotonic dystrophy has been identified as an unstable trinucleotide CTG repeat located in the 3' untranslated region of a gene putatively encoding a serine-threonine protein kinase. The mutation has been reported to be in total linkage disequilibrium with an insertion/deletion polymorphism located within the kinase gene. To determine the nature of this polymorphism, we have sequenced this genomic fragment and have found that the sequence of this region consists of five consecutive Alu repeats. Further analysis suggests that the smaller of two alleles is actually due to a proposed deletion event that resulted in the loss of an equivalent of three Alu repeats. We have developed a PCR-based assay to detect this polymorphism, the closest, distal marker to the DM mutation.

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.

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.

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.