R. J. Bartlett

Duchenne muscular dystrophy High frequency of deletions

DNA probes are available for Duchenne muscular dystrophy (DMD) carrier detection and prenatal diagnosis. With probes for about 25% of the proximal portion of the gene, we found the proximal probes detected deletions in 23% of nonselected DMD boys, while a single distal probe detected 17% more as deletions. The combined percentage was 39% for all probes tested. Prenatal diagnosis and carrier detection are more accurate if deletions are mapped rather than by use of restriction fragment length polymorphism analysis. The effort involved in screening all affected boys for deletions is considerably less, and provides an accurate genetic marker for subsequent prenatal diagnosis in the family and prospective counseling for female relatives. It seems likely that, once the entire gene (cDNA) is available for screening, most DMD boys will show deletions.

Genetic linkage studies in Alzheimer's disease families

Alzheimers disease is a devastating neurological disorder and the leading cause of dementia among the elderly. Recent studies have localized the gene for familial Alzheimers disease to chromosome 21 in a series of early onset AD families (mean age of onset less than 60). Familial late onset AD (mean age of onset greater than 60) is a more common clinical form of the disorder. Thirteen families with multiply affected Alzheimers disease family members were identified and sampled. Ten of these families were of the late onset Alzheimers disease type. Simulation studies were used to evaluate the usefulness of these pedigrees in linkage studies in familial Alzheimers disease. Linkage studies undertaken to test the localization of both early onset and late onset Alzheimers disease families to chromosome 21 failed to establish linkage and excluded linkage from a large portion of the region where the early onset Alzheimers disease gene was localized. These findings suggest that more than one etiology may exist for familial Alzheimers disease and indicate the need for continued screening of the genome in familial Alzheimers disease families.

In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide

In the canine model of Duchenne muscular dystrophy in golden retrievers (GRMD), a point mutation within the splice acceptor site of intron 6 leads to deletion of exon 7 from the dystrophin mRNA, and the consequent frameshift causes early termination of translation. We have designed a DNA and RNA chimeric oligonucleotide to induce host cell mismatch repair mechanisms and correct the chromosomal mutation to wild type. Direct skeletal muscle injection of the chimeric oligonucleotide into the cranial tibialis compartment of a six-week-old affected male dog, and subsequent analysis of biopsy and necropsy samples, demonstrated in vivo repair of the GRMD mutation that was sustained for 48 weeks. Reverse transcription–polymerase chain reaction (RT-PCR) analysis of exons 5–10 demonstrated increasing levels of exon 7 inclusion with time. An isolated exon 7-specific dystrophin antibody confirmed synthesis of normal-sized dystrophin product and positive localization to the sarcolemma. Chromosomal repair in muscle tissue was confirmed by restriction fragment length polymorphism (RFLP)–PCR and sequencing the PCR product. This work provides evidence for the long-term repair of a specific dystrophin point mutation in muscle of a live animal using a chimeric oligonucleotide.

Tight linkage of apolipoprotein C2 to myotonic dystrophy on chromosome 19

The cDNA and genomic probes for apolipoprotein C2 detect two restriction fragment length polymorphisms on chromosome 19. The combined estimated percentage of heterozygosity, assuming equilibrium, is approximately 75%, ie, apolipoprotein C2 is informative in 75% of matings. We have analyzed over 350 individuals in large multigenerational families for linkage of apolipoprotein C2 to myotonic muscular dystrophy. The maximum lod score was 16.29 with the maximum recombination fraction (𝛉) of 0.02, with 95% confidence limits for 𝛉 of 0.001 to 0.065. Thus, apolipoprotein C2 is useful in carrier detection and prenatal diagnosis with an accuracy of about 98%.

Notexin-induced muscle injury in the dog

Notexin, a myotoxic phospholipase, was used to induce focal necrosis in the sartorius muscles of normal mixed-breed adult dogs and in 12-week-old beagles. Notexin injury caused pathologic changes similar to those of Duchenne muscular dystrophy (DMD) and its canine homologue, golden retriever muscular dystrophy (GRMD). All three conditions are characterized by increased serum creatine kinase (CK) levels, sarcolemmal defects, delta lesions, hyaline degeneration of myofibers, calcium-positive myofibers, and minimal effects on neurovascular structures. Four and 24 h after exposure to notexin, serum CK levels were elevated, and many myofibers were necrotic. In addition, by 24 h the necrotic areas were heavily invaded by mononuclear cells, and calcium-positive myofibers were prominent. Capillaries appeared intact even in areas of marked myonecrosis. Massive cellular infiltrate and myotube formation was evident at 3 days post injury. By 7 days, most affected fascicles were occupied by small immature myofibers. Regeneration was largely complete at 21 days. Our results suggest that notexin-induced muscle injury in dogs will be useful in the evaluation of potential therapies for DMD such as myoblast transplantation.

The poliovirus sensitivity (PVS) gene is on chromosome 19q12→q13.2

Sensitivity to nonmodified poliovirus infection is an autosomal dominant trait, specific to primates. The gene for poliovirus sensitivity (PVS) is encoded on human chromosome 19. In order to sublocalize the PVS gene, we infected rodent-human hybrid cell lines that divide human chromosome 19 into four regions with poliovirus 1 and/or 3. When infected, these hybrid cell lines showed the typical cytopathic effect of poliovirus infection only if they contained 19q12----q13.2 as the smallest region of overlap. Appropriate negative and positive controls were used. PVS may be of relevance to myotonic dystrophy (DM) and the inherited motor neuron diseases: to DM because it localizes to the same region of chromosome 19 and to the inherited motor neuron diseases because it encodes a cell-surface receptor expressed on motor neurons.

Golden Retriever Muscular Dystrophy: Monitoring for Success

There are many phenotypic features that can be monitored to, in the end, assess the success of any form of treatment for muscular dystrophy (Table 1). Of course, in this setting, we are speaking principally of myoblast transplantation.

Tight linkage of creatine kinase (CKMM) to myotonic dystrophy on chromosome 19

The myotonic dystrophy (DM) gene is localized to the proximal long arm of chromosome 19. There have been reports of tight linkage to a number of chromosome 19 markers, including APOC2 and creatine kinase muscle type (CKMM), but they did not establish orientation of the 2 markers to DM. We screened several large multi-generational DM families for linkage to a series of chromosome 19 markers including CKMM. CKMM is tightly linked to DM in these data with z(𝛉) = 28.41; 𝛉 = 0.01. Analysis of cross-over data indicates CKMM is on the same side and closer to DM than APOC2. Thus, CKMM is a useful probe for carrier detection studies in presymptomatic individuals as well as for prenatal diagnosis.

Long-lasting gene repair

on an elegantly simple idea: Treat disease by correcting the underlying mutation in situ. In this way, the mutant gene—with all of its existing enhancers, promoter(s), and cellspecific controls and context—can function as a template to produce the normal gene product after the mutation is reverted. Thus far, it has proved difficult to demonstrate the permanence of such genetic changes. But in this issue, Alexeev and Yoon present convincing evidence that gene repair can induce prolonged and reproducible genetic changes at the genomic, mRNA, protein, and phenotypic levels in cultured melanocytes. After continuous passage for over three months in culture, the resulting cells maintain not only the reverted wild-type allele, but also the wild-type phenotypes. The potential promise of gene therapies for the treatment of human genetic diseases has produced a variety of conceptual and experimental approaches. All have one goal in common—the permanent expression of the normal version of the missing gene product in the appropriate tissue. The most commonly used gene therapy approach relies on complementing the missing gene product via introduction of a functional cDNA engineered into one of a series of delivery vectors. Both nonviral and viral (e.g., retroviruses and adeno-associated viruses) vectors have been employed to deliver therapeutic genes, with the aim of gene expression through integration of an engineered transgene into host chromosomes. However, all of the various strategies that seek to introduce a cDNA transgene delivered to the target tissue have shown only modest success. This is due to limitations of delivery to the target tissue, cellular shutdown of the transgene, and/or immunological responses against either the transduced cell (due to viral or transgene antigenic recognition) or the vector on re-administration. Moreover, although a variety of recombinational strategies are also under study,

RFLPs at the D19S19 locus of human chromosome 19 linked to myotonic dystrophy (DM).

SOURCE/DESCRIPTION: Probe LDR152 a 1.68 Kb single copy Hind III fragment isolated from a chromosome 19 bivariate flow sorted enriched Charon 21A library and subcloned into SP64. CHROMOSOMAL LOCALISATION: 1 9cent-1 9q 1 •3 by somatic cell hybrid and linkage data. POLYMORPHISMS: Recognises two, two allele polymorphisms when U3ed to probe human genomic DNA digested with P3t I and Msp I. Pst I Polymorphisms Size Frequency Upper Al 19 Kb 0.11 Lower A2 11 Kb 0.86 M3p I Polymorphisms Upper Al 2.1Kb 0. 16 Lower A2 1.3 Kb 0.84 NOT POLYMORPHIC FOR: Taq I, Stu I, Bgl I, Sac I, EcoRV, Bst XI, Bst NI, Xmn I, Hind 3, Bam HI when tested against 14 individuals. MENDELIAN INHERITANCE: Co-dominant segregation in seven large DM pedigrees. Closely linked to DM. Recombination frequency is 0.0 (95