CpG methylation within the 5' regulatory region of the BRCA1 gene is tumor specific and includes a putative CREB binding site.

Breast cancer is a genetic disease arising from a series of germ-line and/or somatic DNA changes in a variety of genes, including BRCA1 and BRCA2. DNA modifications have been shown to occur by a number of mechanisms that include DNA methylation. In some cases, the aberrant methylation of CpGs within 5' regulatory regions has led to suppression of gene activity. In this report we describe a variation in the pattern of DNA methylation within the regulatory region of the BRCA1 gene. We found no evidence of methylation at CpGs within the BRCA1 promoter in a variety of normal human tissues. However, screening of a series of randomly sampled breast carcinomas revealed the presence of CpG methylation adjacent to the BRCA1 transcription start site. One such methylated CpG occurs at a putative CREB (cAMP-responsive element binding) transcription factor binding site in the BRCA1 promoter. Gelshift assays with methylated and unmethylated BRCA1/CREB binding site oligonucleotides demonstrate that this site is sensitive to site-specific CpG methylation. These data suggest that aberrant DNA methylation at regulatory sequences in the BRCA1 locus may play a role in the transcriptional inactivation of the BRCA1 gene within subclones of breast tumors. This study represents the first evidence suggesting a role for DNA methylation in the transcriptional inactivation of the BRCA1 in human breast cancer.

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.

Genotype/phenotype correlations in X-linked dominant Charcot-Marie-Tooth disease.

We have studied the relationship between genotype, clinical phenotype, and pathology in 13 families with dominant X-linked Charcot-Marie-Tooth (CMT) neuropathy. Connexin32 (Cx32) gene mutations were spread throughout the coding region and included eight missense mutations, one 8-bp deletion/4-bp insertion frame shifting mutation, two nonsense mutations, and one deletion of the entire coding sequence. One hundred sixteen affected CMTX patients (53 males and 63 females) and 63 unaffected, at-risk individuals were compared by neurological and electrophysiological examinations and analyzed by gender; nerve biopsies were available from seven index cases. It was found that mutations within all regions of the Cx32 gene coding sequence caused an identical clinical phenotype. Male CMTX patients were affected more severely and showed an age-dependent progression of clinical signs and of the pathology; there was, however, variability in the severity of disease expression, irrespective of age, among males within families of defined genotype. All but 10% of female CMTX patients had only mild signs. Motor nerve conduction velocities were moderately slowed (median nerve MNCV: males 34.5 +/- 6.2 m/sec; females 45.8 +/- 7.3 m/sec), and motor and sensory nerve amplitudes were reduced (median nerve CMAP: males 3.7 +/- 3.7 mV; females 7.8 +/- 3.4 mV), with electromyographic evidence of chronic denervation. Differences were significant between gender and between affected and unaffected individuals. In agreement with the electrophysiological observations, pathological studies showed evidence of paranodal demyelination and of a length-related axonal degeneration in motor and sensory nerve fibers. Correlations between genotype and clinical phenotype suggested that missense mutations located within the second transmembrane domain and/or cytoplasmic loop might be associated with milder clinical phenotype, and therefore might be less disruptive of connexin32 gap junction function. Missense, chain-terminating, or deletion mutations in all other locations of the connexin32 protein caused severe forms of CMTX and disease onset in the first decade. Observed variability of disease severity among males within kinships suggests the influence of other modifying factors.

Variable expressivity of familial medullary thyroid carcinoma (FMTC) due to a RET V804M (GTG-->ATG) mutation.

BACKGROUND: Multiple endocrine neoplasia type 2 (MEN 2) and familial medullary thyroid carcinoma (FMTC) are autosomal dominantly inherited cancer syndromes that predispose to C-cell hyperplasia and MTC. MEN 2A and FMTC are caused by mutations in the RET proto-oncogene. METHODS: We used a multiplex polymerase chain reaction-based assay to screen exons 10, 11, 13, and 14 of RET for mutations in 2 families with FMTC. We correlated mutation status with calcitonin and pathologic studies to determine genotype-phenotype correlations. RESULTS: We identified a mutation in codon 804 in exon 14 (GTG-->ATG; V804M) in both families. An 86-year-old person who was a gene carrier and other individuals over age 70 who were suspected by pedigree analysis to be gene carriers had no overt clinical evidence of MTC. Four of 21 patients who underwent a thyroidectomy also had papillary thyroid cancer. One individual in each family had metastatic MTC at age 30 and 32 years, and all 26 people having thyroidectomies had either MTC or C-cell hyperplasia, leading us to continue to recommend prophylactic thyroidectomy for all identified patients who were gene carriers. CONCLUSIONS: Because of active MTC in younger members of these families, including metastases, we have continued to advocate thyroid surgery in mutation-positive individuals. While DNA diagnosis of gene carriers and subsequent genetic counseling was relatively straightforward, the acceptance of surgical recommendations was more difficult for some individuals. These families demonstrate that the search for RET mutations should include exons 13, 14, 15, and 16 in patients whose studies in exons 10 and 11 are negative.

CMT1X phenotypes represent loss of GJB1 gene function.

OBJECTIVE: To investigate possible genotype-phenotype correlations and to evaluate the natural history of patients with Charcot-Marie-Tooth disease type 1X (CMT1X). BACKGROUND: CMT1X is caused by over 260 distinct mutations in the gap junction beta 1 (GJB1) gene, located on the X chromosome, which encodes the gap junction protein connexin 32 (Cx32). The natural history of CMT1X is poorly understood, and it remains unknown whether particular mutations cause more severe neuropathies through abnormal gain-of-function mechanisms. METHODS: We evaluated 73 male patients with CMT1X, who each have 1 of 28 different GJB1 mutations predicted to affect nearly all domains of Cx32. Disability was evaluated quantitatively by the CMT Neuropathy Score (CMTNS) as well as by the CMT Symptom Score (CMTSS) and the CMT Examination Score (CMTES), which are both based on the CMTNS. Patients were also evaluated by neurophysiology. RESULTS: In all patients, disability increased with age, and the degree of disability was comparable with that observed in patients with a documented GJB1 deletion. Disability correlated with a loss of motor units as assessed by motor unit number estimates. CONCLUSIONS: Taken together, these data suggest that most GJB1 mutations cause neuropathy by a loss of normal connexin 32 function. Therefore, treatment of male patients with Charcot-Marie-Tooth disease type 1X may prove amenable to gene replacement strategies.

Identification and characterization of sporadic and inherited mutations in exon 31 of the neurofibromatosis (NF1) gene.

Neurofibromatosis type 1 (NF1) is one of the most common genetic disorders in humans, and presents with a variety of clinical symptoms, which are highly variable in expression. The mutation rate for NF1 is high, with as many as half of all cases resulting from new mutations. Although the NF1 gene has been cloned and its cDNA sequence determined, the specific role of the NF1 gene product in contributing to the NF1 mutations is one of the first steps in correlating genotype with clinical symptoms of the disease. In this paper we describe two independent mutations in exon 31 of the NF1 gene identified following polymerase chain reaction (PCR) amplification, heteroduplexing, and single strand conformational polymorphism (SSCP) analysis. One is a novel insertion that segregates with the disease phenotype in that particular family (5852insTT), while the other is a further example of the sporadic, recurrent C-->T mutation previously described in the literature (C5842T). The relationship between these mutations and clinical features of NF1 presented by the patients will be discussed.

Allele-specific non-CpG methylation of the Nf1 gene during early mouse development.

Recent reports of cytosine methylation occurring at CpA and CpT dinucleotides in murine ES cells as well as in Drosophila have renewed interest in methylation at sites other than CpGs. Our examination of the murine neurofibromatosis type 1 gene by sodium bisulfite genomic sequencing has revealed non-CpG methylation primarily in the oocyte and the maternally derived allele of the 2-cell embryo, with markedly lower levels found in sperm. Non-CpG methylation was not found in later stages of embryo development or in adult tissue. Our results suggest that maternal-specific de novo non-CpG methylation has occurred sometime between ovulation and formation of the 2-cell embryo, while during the same period the paternally derived allele has undergone site-specific active demethylation. Our data demonstrate both stage and parent-of-origin specific changes in methylation patterns within the neurofibromatosis type 1 coding region-involving cytosines located at both CpG and non-CpG dinucleotides.

A double mutation in exon 6 of the beta-hexosaminidase alpha subunit in a patient with the B1 variant of Tay-Sachs disease.

The B1 variant form of Tay-Sachs disease is enzymologically unique in that the causative mutation(s) appear to affect the active site in the alpha subunit of beta-hexosaminidase A without altering its ability to associate with the beta subunit. Most previously reported B1 variant mutations were found in exon 5 within codon 178. The coding sequence of the alpha subunit gene of a patient with the B1 variant form was examined with a combination of reverse transcription of mRNA to cDNA, PCR, and dideoxy sequencing. A double mutation in exon 6 has been identified: a G574----C transversion causing a val192----leu change and a G598----A transition resulting in a val200----met alteration. The amplified cDNAs were otherwise normal throughout their sequence. The 574 and 598 alterations have been confirmed by amplification directly from genomic DNA from the patient and her mother. Transient-expression studies of the two exon 6 mutations (singly or together) in COS-1 cells show that the G574----C change is sufficient to cause the loss of enzyme activity. The biochemical phenotype of the 574 alteration in transfection studies is consistent with that expected for a B1 variant mutation. As such, this mutation differs from previously reported B1 variant mutations, all of which occur in exon 5.

Example of somatic mosaicism in a series of de novo neurofibromatosis type 1 cases due to a maternally derived deletion.

Neurofibromatosis type 1 (NF1), affecting primarily the growth of neural crest-derived tissues, is one of the most common autosomal dominant genetic disorders with an unusually high spontaneous mutation rate. In four cases of sporadic NF1, demonstrated by hemizygosity to have a deletion involving the NF1 gene, we were able to assign the deletion event to the maternally derived chromosome. One of these individuals was determined to be a somatic mosaic for NF1, as a trace of the maternally derived haplotype was detected at the NF1 locus. This indicated a postzygotic, as opposed to gametic, deletion event. It may be that somatic mosaicism is more common in NF1 than has hitherto been appreciated and may be responsible in part for the high mutation rate in this disorder. In addition, it is suggested that the mechanism(s) of gene deletion is subject to a parent of origin effect, being more frequent on the maternally derived chromosome. This is in contrast to the other types of mutations which, in sporadic NF1, have been found to occur preferentially on the paternally derived chromosome.

Clinical and pathological observations in men lacking the gap junction protein connexin 32.

The X-linked form of Charcot-Marie-Tooth disease has been associated with mutations in the connexin 32 (Cx 32) gene, which encodes a gap junction protein. The majority of identified mutations are missense, but a few nonsense mutations or frame-shifting microdeletions have been encountered. Functional assessments of the mutated gap junction protein have demonstrated altered or simple losses of function. Mutations segregate with a typical clinical phenotype, which is the result of an age-related, progressive neuropathy. The mechanisms that cause the nerve damage are unknown. This report describes the consequences of a unique deletion mutation that eliminates the entire coding sequence of Cx 32, resulting in the absence of the Cx 32 gap junction protein in affected, hemizygous men. The clinical expression of this unique mutation was studied by the clinical, electrophysiological, and pathological evaluation of this kinship of five generations. The resulting severe neuropathy combines features of demyelination, notably in paranodal distribution, and distal accentuated axonal degeneration. The predicted absence of Cx 32 gap junctions is shown to be associated with a severe dysfunction of the axon-Schwann cell unit. Observed changes resemble those of Cx 32-null mice. No central nervous system changes were demonstrated.

A nongenetic basis of cycle frequency in bipolar disorder: study of a monozygotic twin pair.

The authors describe a pair of monozygotic twins with bipolar disorder but with a different course of the illness including age of onset, sequence of episodes, and cycle length. Based on these findings, the clinical course of bipolar illness does not appear to be genetically determined.

Family cancer histories predictive of a high risk of hereditary non-polyposis colorectal cancer associate significantly with a genomic rearrangement in hMSH2 or hMLH1.

Hereditary non-polyposis colorectal cancer (HNPCC) results from inactivating germline mutations in a set of DNA-mismatch-repair genes, of which the most clinically relevant are hMSH2 and hMLH1. Computer-assisted pedigree risk assessment tools are available to assist in the calculation of an individual's likelihood of bearing such a deleterious mutation. One such tool, cancergene version 3.4 (http://www3.utsouthwestern.edu/cancergene) was used to assess the risk of a deleterious mutation in the genes hMSH2 and/or hMLH1 in a series of probands selected from a panel of 67 South-western Ontario kindred previously identified as likely candidates for HNPCC by established clinical criteria. A DNA sample isolated from peripheral blood leukocytes obtained from each of these probands was examined for genomic rearrangement using the multiplex ligation-dependent probe amplification (MLPA) method. Of the individuals calculated to have a risk of >50% of a hMSH2 or hMLH1 gene mutation by the CancerGene risk assessment tool, 69% (9/13) were shown to have a genomic rearrangement resulting in the deletion of one or more exons of one of these two genes. Family cancer histories predictive of a high risk of HNPCC significantly associate with a genomic rearrangement in hMSH2 or hMLH1.

Effect of radiosensitizing agents on electron transport systems.

Experiments have been carried out to study the interaxtion between chemical radiosensitizing agents and model electron transport systems. Using an NAD(P)H:O2 oxidoreductase enzyme as such a model, it was demonstrated that radiosensitizers can act as intermediates in the transfer of electrons from NADH to O2, even in the presence of classical inhibitors of electron transport, with anefficiency related to both their redox potentials and their radiosensitizing abilities. This work which was further confirmed in mammalian mitochondria and microsomes as well as in a cultured cell system indicated that these sensitizers can accept electrons from a variety of organelle systems. This action was shown to be related to the concentration of reduced pyridine nucleotides present both in vivo and in vitro. Of the electron-affinic agents tested, those whose redox potential was more negative than -0.39 V may possibly serve as better radiotherqpeutic mediators.

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.

Pathological findings in the x-linked form of Charcot-Marie-Tooth disease: a morphometric and ultrastructural analysis.

Mutations in the connexin 32 gene (Cx 32) are associated with the x-linked form of Charcot-Marie-Tooth disease (CMTX) and segregate with a CMT 1 phenotype. The gap junction protein Cx 32 is expressed in myelinating Schwann cells and has been localized to regions of non-compacted cytoplasm in paranodes and in Schmidt-Lanterman incisures. Mutant Cx 32 myelin proteins are predicted to impair Schwann cell functions. We have studied the resulting pathology in motor and sensory nerves from the probands of 13 CMTX kindreds with precisely defined genotype. This report provides a detailed descriptive and morphometric analysis of 14 CMTX nerve biopsy samples, taken at various stages in the development of the neuropathy and studied by light and electron microscopic examination. Findings indicated unusually prominent changes in paranodal myelin with resulting widened nodes of Ranvier, but with segmental demyelination being less common. In parallel early axonal cytoskeletal abnormalities were noted, which were followed later by axonal atrophy, degeneration and loss of myelinated nerve fibers, occurring in a length-dependent fashion. Regenerative sprouting was also unusually prominent. Ultrastructural abnormalities included a frequent dilatation of the adaxonal spaces, prominence of the adaxonal Schwann cell cytoplasm and widening of the Schmidt-Lanterman incisures. We conclude that mutations in Cx 32 gap junction protein lead to a compromise of Schwann cell functions and to impaired Schwann cell-axon interactions with subsequent pathology in both myelin and axons.

Genotype/phenotype correlation in affected individuals of a family with a deletion of the entire coding sequence of the connexin 32 gene.

X-linked Charcot-Marie-Tooth disease (CMTX) is a peripheral nerve disorder that has been linked to mutations in the connexin 32 gene (Cx32). These mutations have been shown to be genetically heterogeneous, though recurrences of specific mutations in apparently unrelated families have been seen. The majority of mutations have been shown to be missense, resulting in non-conservative amino acid changes. A few mutations resulting in a premature termination of protein translation, including both nonsense mutations as well as frameshifting microdeletions, have been documented. We would like to report a deletion mutation that appears to eliminate the entire coding sequence of the Cx32 gene, but which has been shown to segregate with a clinical phenotype not unlike that seen in individuals with a less severe alteration of the Cx32 gene. The causes at a cellular level of the CMTX phenotype are still not fully clear, though there has been speculation that these may involve a dominant negative effect where the mutant connexin 32 suppresses the function of other connexins. Studies of kindreds such as this, where in CMTX-affected males the Cx32 gene product is totally absent, will help us to better understand the molecular mechanisms underlying the clinical phenotype associated with this disorder.