Search

By use of cDNA probes, molecular deletions were identified in 66.6% of 42 patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). Owing to this high deletion rate, a new strategy for detecting DMD/BMD carriers is feasible in which the polymerase chain reaction is used as an initial screen for detecting the deletions occurring in specific deletion-prone exons. Because the deletions do not occur randomly, specific cDNA probes are utilized first with Southern blot analysis. Identification of a deletion permits direct analysis for DMD carrier status and removes the inherent limitations of the conventional restriction fragment length polymorphism technique. Carrier status is determined by scanning the autoradiographs with a densitometric spectrophotometer or by detection of a junction fragment.

Hereditary non-polyposis colorectal cancer is characterized by mutations in one of the DNA mismatch repair genes, primarily MLH1, MSH2, or MSH6. We report here the identification of a genomic deletion of approximately 11.4 kb encompassing the first two exons of the MSH2 gene in two generations of an Ohio family. By Southern blot analysis, using a cDNA probe spanning the first seven exons of MSH2, an alteration in each of three different enzyme digests (including a unique 13-kb band on HindIII digests) was observed, which suggested the presence of a large alteration in the 5' region of this gene. Mouse-human cell hybrids from a mutation carrier were then generated which contained a single copy each of human chromosome 2 on which the MSH2 gene resides. Southern blots on DNA from the cell hybrids demonstrated the same, unique 13-kb band from one MSH2 allele, as seen in the diploid DNA. DNA from this same monosomal cell hybrid failed to amplify in polymerase chain reactions (PCRs) using primers to exons 1 and 2, demonstrating the deletion of these sequences in one MSH2 allele, and the breakpoints involving Alu repeats were identified by PCR amplification and sequence analysis.

Gastrinomas and other gastrointestinal neuroendocrine tumors may occur sporadically or as part of the inherited syndrome multiple endocrine neoplasia type 1 (MEN1). Mutations in the recently identified MEN1 gene have been described in sporadic gastrinomas and insulinomas. This study describes techniques used to identify mutations in the MEN1 gene in DNA extracted from paraffin-preserved tissue. Two novel mutations are identified in the MEN1 gene from nine archived paraffin-embedded neuroendocrine tumors, demonstrating that retrospective genetic analysis can be used to identify mutations in the MEN1 gene from preserved tissue. Conditions are provided by which paraffin-embedded tissue can be used as a source of genetic material for sequence information of sufficient quality for mutational studies of the MEN1 gene. It should also be possible to apply this retrospective genetic analysis of paraffin-embedded tissue to other disease models.

Hereditary hemochromatosis, a common autosomal recessive trait caused by mutations in the HLA-H gene, is often diagnosed by the pathologist at the time of histologic examination. Unfortunately, histologic parameters alone do not differentiate between hereditary hemochromatosis and other causes of iron overload. We performed a retrospective study to determine the frequency of familial hemochromatosis in patients diagnosed with he mochromatosis by abnormal liver histology.DNA was isolated from paraffin-embedded tissue sections from 15 patients and used in a polymerase chain reaction-based assay in which we tested for the C282Y and H63D mutations. We found that in this group of patients, 5 (33%) were homozygous for the common C282Y genetic mutation, 3 (20%) were heterozygous, and 7 (47%) were normal.Our study shows that the molecular assay is the gold standard for the diagnosis of hereditary hemochromatosis. The case study also illustrates that a definitive diagnosis of familial hemochromatosis has significant counseling implications allowing for accurate family studies.

Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by defects in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central portion of the gene. The nondeletion/duplication cases are most likely the result of smaller mutations that cannot be identified by current diagnostic screening strategies. We screened approximately 80% of the dystrophin coding sequence for small mutations in 158 patients without deletions or duplications and identified 29 mutations. The study indicates that many of the DMD and the majority of the BMD small mutations lie in noncoding regions of the gene. All of the mutations identified were unique to single patients, and most of the mutations resulted in protein truncation. We did not find a clustering of small mutations similar to the deletion distribution but found > 40% of the small mutations 3' of exon 55. The extent of protein truncation caused by the 3' mutations did not determine the phenotype, since even the exon 76 nonsense mutation resulted in the severe DMD phenotype. Our study confirms that the dystrophin gene is subject to a high rate of mutation in CpG sequences. As a consequence of not finding any hotspots or prevalent small mutations, we conclude that it is presently not possible to perform direct carrier and prenatal diagnostics for many families without deletions or duplications.

Duchenne muscular dystrophy (DMD) patients with mutations that disrupt the translational reading frame produce little or no dystrophin. Two exceptions are the deletion of exons 3-7 and the occurrence of rare dystrophin-positive fibers (revertant fibers) in muscle of DMD patients. Antibodies directed against the amino-terminus and the 5' end of exon 8 did not detect dystrophin in muscle from patients who have a deletion of exons 3-7. However, in all cases, dystrophin was detected with an antibody directed against the 3' end of exon 8. The most likely method of dystrophin production in these cases is initiation at a new start codon in exon 8. We also studied two patients who have revertant fibers: one had an inherited duplication of exons 5-7, which, on immunostaining, showed two types of revertant fibers; and the second patient had a 2-bp nonsense mutation in exon 51, which creates a cryptic splice site. An in-frame mRNA that uses this splice site in exon 51 was detected. Immunostaining demonstrated the presence of the 3' end of exon 51, which is in agreement with the use of this mRNA in revertant fibers. The most likely method of dystrophin production in these fibers is a second mutation that restores the reading frame.

Molecular biology techniques have changed the way in which we now consider a patient with Duchenne muscular dystrophy or Becker muscular dystrophy. Using cDNA probes, it has been shown that approximately 65% of the patients with Duchenne muscular dystrophy or Becker muscular dystrophy have gene deletions. The identification of a deletion allows the disease to be confirmed by noninvasive DNA testing. Furthermore, the identification of the gene defect can help distinguish Becker muscular dystrophy from other clinically similar neuromuscular disorders. Most importantly, the elucidation of the gene defects has resulted in the application of direct carrier and prenatal diagnostics.

Proximal spinal muscular atrophy (SMA) is a common motor neuron disease in humans and in its most severe form causes death by the age of 2 years. It is caused by defects in the telomeric survival motor neuron gene ( SMN1 ), but patients retain at least one copy of a highly homologous gene, centromeric SMN ( SMN2 ). Mice possess only one survival motor neuron gene ( Smn ) whose loss is embryonic lethal. Therefore, to obtain a mouse model of SMA we created transgenic mice that express human SMN2 and mated these onto the null Smn (-/-)background. We show that Smn (-/-); SMN2 mice carrying one or two copies of the transgene have normal numbers of motor neurons at birth, but vastly reduced numbers by postnatal day 5, and subsequently die. This closely resembles a severe type I SMA phenotype in humans and is the first report of an animal model of the disease. Eight copies of the transgene rescues this phenotype in the mice indicating that phenotypic severity can be modulated by SMN2 copy number. These results show that SMA is caused by insufficient SMN production by the SMN2 gene and that increased expression of the SMN2 gene may provide a strategy for treating SMA patients.

Detection of Duchenne muscular dystrophy carriers by genetic analysis is illustrated by four case studies. The technique is most useful in obligate families, in excluding carrier status in isolated cases, and in families in which the affected child demonstrates a molecular deletion. A major limitation of this technique is that the accuracy of carrier status in isolated (i.e., no family history) cases is limited by the probability that the affected child may represent a new mutation. To improve the carrier risk estimate generated by the DNA data, particularly in isolated cases, we suggest that measuring creatine kinase activities in the serum and performing the genetic analysis on the nonaffected males may be helpful.