Jack Jung

Homonucleotide tracts, short repeats and CpG/CpNpG motifs are frequent sites for heterogeneous mutations in the neurofibromatosis type 1 (NF1) tumour-suppressor gene.

Neurofibromatosis type 1 (NF1) is among the most common human genetic disorders, having a constellation of cutaneous and skeletal manifestations, intellectual impairment, and an increased risk for a variety of malignancies. The NF1 gene has a high spontaneous mutation rate and is also associated with a variety of sporadic cancers in the general population. While a number of laboratories are involved in a coordinated effort to identify NF1 mutations, an important gap in our knowledge is an understanding of the mechanisms responsible for NF1 mutagenesis. In this present paper we describe our analysis of the sequence environment in the NF1 gene at those sites where small deletions, insertions and nucleotide substitution mutations have been reported. Our objective was to determine whether specific nucleotide sequences commonly occur at these mutation sites within the NF1 gene. We assessed how frequently independent NF1 mutations occur at the site of short direct repeats, single nucleotide repeats (homonucleotides) and at CpG and CpNpG motifs. We have established that homonucleotide and short direct repeats are commonly involved in the majority of small deletions and insertions analysed. Substitution mutations are frequently associated with homonucleotide repeats and methylatable CpG dinucleotides and CpNpG trinucleotides. We suggest that NF1 mutations are acquired and retained by cells through an intricate balancing of repair and replication mechanisms. Such mutations may provide a proliferative advantage for that cell and its progeny.

A large novel deletion in the APC promoter region causes gene silencing and leads to classical familial adenomatous polyposis in a Manitoba Mennonite kindred

Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome caused by the inheritance of germline mutations in the APC tumour suppressor gene. The vast majority of these are nonsense and frameshift mutations resulting in a truncated protein product and abnormal function. While APC promoter hypermethylation has been previously documented, promoter-specific deletion mutations have not been reported. In a large Canadian Mennonite polyposis kindred, we identified a large novel germline deletion in the APC promoter region by linkage analysis and MLPA. By RT-PCR and sequence analysis, this mutation was found to result in transcriptional silencing of the APC allele. A few genetic disorders have been characterized as over-represented in the Manitoba Mennonite population, however, the incidence of cancer has not been recognized as increased in this population as compared to other Manitoba ethnic groups. This study strengthens the likelihood that this novel APC promoter mutation is linked to this unique population as a founder mutation.

A genetic study of neurofibromatosis type 1 (NF1) in south-western Ontario. II. A PCR based approach to molecular and prenatal diagnosis using linkage.

Neurofibromatosis type 1 (NF1) is a common, autosomal dominant genetic disorder with a variety of highly variable symptoms including cutaneous manifestations (such as café au lait spots), Lisch nodules, plexiform neurofibromas, skeletal abnormalities, an increased risk for malignancy, and the development of learning disabilities. The wide clinical variability of expression of the disease phenotype and high (spontaneous) mutation rate of the NF1 gene indicate that careful clinical examination of patients and family members is necessary to provide an accurate diagnosis of the disease. Since very few NF1 mutations have been identified, and with the apparent lack of a predominant mutation in this large, highly mutable gene, molecular diagnosis of NF1 will continue to be based on haplotypes using linkage analysis. Here we report our experiences while providing a molecular diagnostic service for NF1 in the ethnically diverse region of south-western Ontario. Molecular diagnoses with at least one informative probe/enzyme combination are reported for 19 families including two families requesting prenatal diagnosis for NF1. We have augmented the classical Southern based approach to linkage analysis with the use of PCR based assays for molecular linkage. Furthermore, criteria have been established in our laboratory for executing molecular linkage based on heterozygosity values, recombination fractions, and the use of intragenic probes/markers.

A genetic study of neurofibromatosis 1 in south-western Ontario. I. Population, familial segregation of phenotype, and molecular linkage.

This report is concerned with neurofibromatosis type 1 (NF-1, 17q11.2) in south-western Ontario, an ethnically diverse population derived from multiple immigrations. The population incidence, prevalence, and mutation rates for this disease are similar in most racial groups of this population and are also comparable to earlier reports. NF-1 is one of the most common single gene disorders in this population. The occurrence of the disease is not affected by the birth order or sex of the transmitting parent. The severe manifestation of this disease is statistically related to paternal transmission. Five polymorphic DNA probes (pA1041, pHHH202, pTH1719, NF1, pEW206, pEW207) were evaluated in relation to segregation of NF-1 using appropriate restriction enzymes. The observed heterozygosity was found to be relatively high, ranging from 25% to 55% for all the probes on 17q and flanking the NF-1 gene. We recommend the use of pHHH202/pTH1719 and pEW206 in any linkage analysis for detection of the presence of the NF-1 mutation. For informative families the degree of certainty is as high as 99.5%. Some future modifications may include the use of NF-1 exon specific probes and primers that remain to be evaluated for heterogeneity and heterozygosity among populations.

A method for transforming lymphocytes from very small blood volumes suitable for paediatric samples

SummaryPermanent lymphoblastoid cell lines are important in the molecular analysis and characterization of human genetic disorders, when immortalized cells must be banked for future diagnostic or research purposes. However, routine methods for transformation using Epstein-Barr virus (EBV) require blood volumes that may be difficult to collect from clinically compromised neonates and small children. Here we report a modified transformation procedure utilizing blood samples of small volume (less than 1.0ml), which we have found to be particularly useful for the immortalization of lymphocytes destined for future molecular genetic studies.

Strategies and applications of DNA level diagnosis in genetic diseases: past experiences and future directions.

The development of technologies towards the detection of mutations represents one of the most important areas of molecular biology. It has played a pivotal role in the tremendous success of the elucidation of complex biological problems, including genetic diseases. Today, these proven and emerging technologies have become the basis of successful biological investigations. More importantly, they are expected to play a central role in medicine, particularly the diagnosis and prognosis of genetic diseases including genetic predispositions, the assessment of treatments including transplants and decisions on reproductive choices. In addition, these technologies hold the key to future breakthroughs. This review provides an up-to-date examination of the principles of genetic diseases, the theories behind current methods of genetic diagnosis and detection of mutations including strategies for modification and the development of future technologies as they impact on the practice of medicine and on society as a whole.