Anticipation in a unique family with Charcot-Marie-Tooth syndrome and deafness: delineation of the clinical features and review of the literature.

Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous group of polyneuropathies characterized by degeneration of peripheral nerves, resulting in distal muscle atrophy, sensory loss, and deformities of hands and feet. We have studied 34 individuals in a large 84-member four-generation central Illinois family with autosomal dominant Charcot-Marie-Tooth and deafness. Nerve conduction velocities are consistent with type 1 CMT. Audiological evaluation revealed both auditory neuropathy and cochlear involvement in affected individuals. There is increasing clinical severity and younger age of onset of CMT and deafness with each progressive generation, suggestive of anticipation (P < 0.05). The proband, a female diagnosed at birth with hypotonia, bilateral vocal cord palsy, swallowing incoordination, and hearing impairment, died at age 18 months. Another individual died at the age of 3 months from hypotonia later attributed to CMT. Genetic analysis indicated that affected individuals in this family do not have the common 1.4 Mb duplication associated with type 1A CMT; however, all affected individuals have a unique G to C transversion at position 248 in coding exon 3 of the peripheral myelin PMP22 gene located on chromosome 17p11.2-p12. This mutation is predicted to cause an Ala67Pro substitution in the second transmembrane domain of PMP22, consistent with the molecular cause of the CMT phenotype. However, it does not explain the cochlear component of the deafness, the clinical observation of anticipation, and other features in this family.

Clinical delineation and localization to chromosome 9p13.3-p12 of a unique dominant disorder in four families: hereditary inclusion body myopathy, Paget disease of bone, and frontotemporal dementia.

Autosomal dominant myopathy, Paget disease of bone, and dementia constitute a unique disorder (MIM 605382). Here we describe the clinical, biochemical, radiological, and pathological characteristics of 49 affected (23 male, 26 female) individuals from four unrelated United States families. Among these affected individuals 90% have myopathy, 43% have Paget disease of bone, and 37% have premature frontotemporal dementia. EMG shows myopathic changes and muscle biopsy reveals nonspecific myopathic changes or blue-rimmed vacuoles. After candidate loci were excluded, a genome-wide screen in the large Illinois family showed linkage to chromosome 9 (maximum LOD score 3.64 with marker D9S301). Linkage analysis with a high density of chromosome 9 markers generated a maximum two-point LOD score of 9.29 for D9S1791, with a maximum multipoint LOD score of 12.24 between D9S304 and D9S1788. Subsequent evaluation of three additional families demonstrating similar clinical characteristics confirmed this locus, refined the critical region, and further delineated clinical features of this unique disorder. Hence, autosomal dominant inclusion body myopathy (HIBM), Paget disease of bone (PDB), and frontotemporal dementia (FTD) localizes to a 1.08-6.46 cM critical interval on 9p13.3-12 in the region of autosomal recessive IBM2.

Clinical and molecular studies in a unique family with autosomal dominant limb-girdle muscular dystrophy and Paget disease of bone.

PURPOSE: To characterize the clinical features and perform linkage analysis of candidate loci in a large Illinois family with autosomal dominant limb-girdle muscular dystrophy (LGMD) and Paget disease of bone (PDB). METHODS: The family includes 11 affected individuals (8 M, 3 F). Clinical, biochemical and radiologic evaluations were performed to delineate clinical features of the disorder. Linkage analysis with polymorphic markers was performed for previously identified LGMD, PDB and cardiomyopathy loci. RESULTS: Onset of PDB is early, at a mean age of 35 y, with classic distribution involving the spine, pelvis, and skull. Muscle weakness and atrophy is progressive with mildly elevated to normal creatine phosphokinase levels. Muscle biopsy in the oldest male revealed vacuolated fibers, however, in others revealed nonspecific myopathy. Affected individuals die from progressive muscle weakness, and respiratory and cardiac failure in their 40s-60s. Linkage analysis excluded autosomal dominant and recessive LGMD, PDB, and cardiomyopathy loci. CONCLUSION: Autosomal dominant LGMD associated with PDB is an unusual disorder. Linkage analysis indicates a unique locus in this family.

Chromosome 3p14 homozygous deletions and sequence analysis of FRA3B.

Loss of heterozygosity (LOH) involving 3p occurs in many carcinomas but is complicated by the identification of four distinct homozygous deletion regions. One putative target, 3p14.2, contains the common fragile site, FRA3B, a hereditary renal carcinoma-associated 3;8 translocation and the candidate tumor suppressor gene, FHIT. Using a approximately 300 kb comsid/lambda contig, we identified homozygous deletions in cervix, breast, lung and colorectal carcinoma cell lines. The smallest deletion (CC19) was shown not to involve FHIT coding exons and no DNA sequence alterations were present in the transcript. We also detected discontinuous deletions as well as deletions in non-tumor DNAs, suggesting that FHIT is not a selective target. Further, we demonstrate that some reported FHIT aberrations represent normal splicing variation. DNA sequence analysis of 110 kb demonstrated that the region is high in A-T content, LINEs and MER repeats, whereas Alu elements are reduced. We note an intriguing similarity in repeat sequence composition between FRA3B and a 152 kb segment from the Fragile-X region. We also identified similarity between a FRA3B segment and a small polydispersed circular DNA. In contrast to the selective loss of a tumor suppressor gene, we propose an alternative hypothesis, that some putative targets including FRA3B may undergo loss as a consequence of genomic instability. This instability is not due to DNA mismatch repair deficiency, but may correlate in part with p53 inactivation.

Distinct 3p21.3 deletions in lung cancer and identification of a new human semaphorin.

Loss of chromosome 3p is a critical event in the pathogenesis of lung cancer. Overlapping homozygous 3p21.3 deletions in lung cancer cell lines involving GNAI2 were characterized and found to involve a region of genomic instability. A new widely expressed Semaphorin, H.SemaIV, was isolated from the GNAI2 deletion region. Reduced H.SemaIV expression allowed identification of additional cell lines with submicroscopic or larger deletions of the locus which occurred in a heterogeneous manner. We also demonstrate the presence of a distinct 3p21.3 homozygous deletion region, adjacent to the DNA mismatch repair gene, hMLH1, and identified deletions in direct tumors. This appears to represent one of the first demonstrations of homozygous deletions affecting 3p in direct lung tumors.

A second-generation YAC contig map of human chromosome 3.

A map of human chromosome 3 which integrates both physical and genetic data has been developed from the fusion of two large collections of markers and corresponding yeast artificial chromosome (YAC) clones. The map contains 972 megabase-sized YACs identified with 593 primary markers, of which 162 are highly polymorphic sequence-tagged sites (STSs) and form a closely spaced genetic linkage map; the remaining markers are hybridization-based. Chromosome 3 is now represented by 24 large YAC contigs whose order and orientation is largely known. The map generated by fusion of these hybridization- and STS-based datasets covers about 80% (over 160 megabases) of the chromosome and will provide the foundation necessary for rapid development of a detailed genetic understanding for this large autosome.

Integrated YAC contig containing the 3p14.2 hereditary renal carcinoma 3;8 translocation breakpoint and the fragile site FRA3B.

An extended YAC contig has been developed for the 3p14 region containing the hereditary renal carcinoma 3;8 translocation breakpoint and the 3p14.2 fragile site FRA3B. This region of chromosome 3 has been implicated by chromosomal translocation, deletion, and loss of heterozygosity in the pathogenesis of several malignant diseases. The contig allows accurate positioning of candidate genes, polymorphic markers, and other 3p rearrangements within this region. The contig, spanning approximately 6 Mb of DNA, contains 51 YACs identified by 27 markers, including a subset of CA repeats located in the 3p14.1-14.2 interval. The order of CA microsatellites, derived from marker content of the YACs, is in agreement with the order previously determined by genetic linkage studies. We find that the protein-tyrosine phosphatase gamma gene, PTPRG, is located minimally 1 Mb proximal to the t(3;8) breakpoint. The more proximal 3p homozygous deletion in the small-cell lung cancer cell line, U2020, is more than 5 Mb from the site of the 3;8 translocation. This integrated physical and genetic map provides a framework for further investigations of malignant diseases associated with proximal 3p loss. In addition, the positioning of separate 3p14.2 aphidicolin-induced breakpoints suggests that FRA3B may represent a region rather than a single site.

Terrestrial soft-bodied protists and other microorganisms in triassic amber.

Protozoa, cyanobacteria, sheathed algae, sheathed fungi, germinating pollen or spores, and fungal spores have been found in amber 220 to 230 million years old. Many of these microorganisms can be assigned to present-day groups. This discovery of terrestrial, soft-bodied protists that can be referred to modern groups indicates that morphological evolution is very gradual in many protists and that both structural and probably functional stasis extend back at least to the Upper Triassic period.

Identification of the bacteriophage Mu kil gene.

The kil gene encoded in bacteriophage Mu DNA was previously shown to reside between the end of the B gene at 4.3 kb and the EcoRI site at 5.1 kb from the left end. To precisely map the kil gene within this region, two series of BAL-31 deletion derivatives were created: one removed Mu DNA rightward from the Hpal site (4.2 kb) and the other removed Mu DNA leftward from the EcoRI site. The deleted Mu DNA was subcloned into the expression vector pUC19 under lac promoter control and tested for the expression of the killing function following IPTG induction. Using DNA sequencing analysis, the Mu DNA in Kil+ and Kil- clones was precisely determined, and the kil gene was mapped to the first open reading frame beyond the B gene. The expression of the kil gene was sufficient to induce dramatic morphological changes: cells became enlarged and predominantly spherical, reminiscent of the phenotype of certain cell mutants.

Identification of the bacteriophage D108 kil gene and of the second region of sequence nonhomology with bacteriophage Mu.

To identify the second region of sequence nonhomology between the genomes of the transposable bacteriophages Mu and D108 originally observed by electron-microscopic analysis of DNA heteroduplexes and to localize functions ascribed to the 'accessory' or 'semi-essential' early regions of the phages between genes B and C, a 0.9-kb fragment of each genome located immediately beyond the B gene was cloned and sequenced. Three open reading frames (ORFs) were identified in each. The region of nonhomology is located within the 3' portion of the third ORF. D108 is shown to possess a Kil function similar to that previously shown for Mu, and that function is encoded by the first ORF.

Transposition and replication of maxi-Mu derivatives of bacteriophage Mu.

The insertion of DNA fragments within the lac sequence of a MudI(Ap,lac) prophage resulted in the formation of a set of maxi-Mu genomes which were 39.8, 59, 85.6, and 88.2 kb long, respectively. The comparison of these maxi-Mu's with the 37.2-kb-long parental MudI(Ap,lac) indicated that the transposition frequency decreased as the length of the prophage increased. No replication of the two longest maxi-Mu's could be detected. The 59- and the 39.8-kb-long chimeric genomes were noted to replicate at approximately 1-2 and 30%, respectively, of the rate found with the MudI(Ap,lac) prophage. The length dependence of the transposition and replication could be explained by the impairment of an early step of the transposition/replication mechanism.

Multiple factors and processes involved in host cell killing by bacteriophage Mu: characterization and mapping.

The regions of bacteriophage Mu involved in host cell killing were determined by infection of a lambda-immune host with 12 lambda pMu-transducing phages carrying different amounts of Mu DNA beginning at the left end. Infecting lambda pMu phages containing 5.0 (+/- 0.2) kb or less of the left end of Mu DNA did not kill the lambda-immune host, whereas lambda pMu containing 5.1 kb did kill, thus locating the right end of the kil gene between approximately 5.0 and 5.1 kb. For the Kil+ phages the extent of killing increased as the multiplicity of infection (m.o.i.) increased. In addition, killing was also affected by the presence of at least two other regions of Mu DNA: one, located between 5.1 and 5.8 kb, decreased the extent of killing; the other, located between 6.3 and 7.9 kb, greatly increased host cell killing. Killing was also assayed after lambda pMu infection of a lambda-immune host carrying a mini-Mu deleted for most of the B gene and the middle region of Mu DNA. Complementation of mini-Mu replication by infecting B+ lambda pMu phages resulted in killing of the lambda-immune, mini-Mu-containing host, regardless of the presence or absence of the Mu kil gene. The extent of host cell killing increased as the m.o.i. of the infecting lambda pMu increased, and was further enhanced by both the presence of the kil gene and the region located between 6.3 and 7.9 kb. These distinct processes of kil-mediated killing in the absence of replication and non-kil-mediated killing in the presence of replication were also observed after induction of replication-deficient and kil mutant prophages, respectively.

Synchronization of bacteriophage Mu DNA replicative transposition: analysis of the first round after induction.

The lytic cycle of bacteriophage Mu includes a large number of coupled DNA replication and integration events, each of which is equivalent in several respects to the process of transposition of genetic elements. To aid us in studying the process of Mu DNA replicative transposition, we developed a technique for synchronizing the first round of replication following induction of a lysogen. Synchronization was achieved by inducing a lysogen in the absence of DNA replication for a time sufficient to develop the potential for Mu DNA replication in all cells in the population; upon release of the inhibition of replication, a synchronized round of Mu DNA replication was observed. Development of the potential for Mu DNA replication in the entire population took approximately 12 min. Protein synthesis was required for development of the potential, but the requirement for protein synthesis was satisfied by approximately 9 min suggesting that other, as yet unspecified, reactions occupied the last 3 min. Replication proceeded predominantly from the left end of the prophage, though a significant amount of initiation from the right end was observed. The usefulness of the technique for studying the mechanism of replicative transposition and the end products of a single round of replication are discussed.

A family planning risk scoring system for health care providers.

The potential usefulness of a Family Planning Risk Scoring Sheet was studied in 1720 consecutive women who completed a family planning visit and were prescribed a specific contraceptive method. The results demonstrated that many women had relative or absolute contraindications to their prescribed method that were detected later by the Family Planning Risk Scoring Sheet. There were 29 women in the oral contraceptive and intrauterine device groups who had absolute contraindications detected (2.8 and 2.4%, respectively). The nurse practitioners tended to have fewer unrecognized problems in their groups than did the physicians. The usefulness of the Family Planning Risk Scoring Sheet was demonstrated and its routine use in a busy family planning unit is recommended.

Preliminary experience with a family planning risk-scoring system.

PIP: Utilizing a risk scoring system for selection of the proper contraceptive method for women seeking family planning care is discussed and recommended as being attractive and practical. Because there are risks associated with specific contraceptive methods, such as thrombosis with oral contraception and pelvic infections with intrauterine contraception, it is necessary to preselect the safest method for individual women. Testing 495 women upon their first visit to the clinic, the authors compared each woman's desired method with the methods selected for by the risk-scoring system. Based on a point score of 1-10 with 10 indicating high risk, it is shown that 42% of the women desiring oral contraception had some risk to be considered while 5.1% had a high risk score. For women desiring the intrauterine device, 28.6% had some risk factor while 4.2% had a high risk score. They conclude that 3 advantages are: 1) the system was easy to use and pointed out problem areas before a method was selected, 2) the system provided a means of quickly monitoring the patient's care by a large number of persons, and 3) the scoring sheet served as a teaching device.^ieng

Cellular location of Mu DNA replicas.

To ascertain the form and cellular location of the copies of bacteriophage Mu DNA synthesized during lytic development, DNA from an Escherichia coli lysogen was isolated at intervals after induction of the Mu prophage. Host chromosomes were isolated as intact, folded nucleoids, which could be digested with ribonuclease or heated in the presence of sodium dodecyl sulfate to yield intact, unfolded nucleoid DNA. Almost all of the Mu DNA in induced cells was associated with the nucleoids until shortly before cell lysis, even after unfolding of the nucleoid structure. We suggest that the replicas of Mu DNA are integrated into the host chromosomes, possibly by concerted replication-integration events, and are accumulated there until packaged shortly before cell lysis. Nucleoids also were isolated from induced lambda lysogens and from cells containing plasmid DNA. Most of the plasmid DNA sedimented independently of the unfolded nucleoid DNA, whereas 50% or more of the lambda DNA from induced lysogens cosedimented with unfolded nucleoid DNA. Possible explanations for the association of extrachromosomal DNA with nucleoid DNA are discussed.

Fate of plasmids containing Mu DNA: chromosome association and mobilization.

The fluorescent dye, diamidinophenylindole-dihydrochloride (DAPI) can be added to CsCl gradients to enhance the density resolution of DNA species, independent of their topological configurations. When Proteus mirabilis and Escherichia coli strains carrying an RP4::Mucts plasmid were examined with the use of such a technique, it was found that after thermal induction of the prophage essentially al of the plasmid DNA became associated with the chromosome. This quantitative association is detergent-RNase- and pronase-resistant and dependent on the expression of Mu genes. The association is temporally, and probably functionally, correlated with the onset of Mu DNA replication. Genetic studies with F'::mini Mu plasmids indicate that some of the association results in stable Hfr formation, and does not require the product of Mu gene B.

Early events in the replication of Mu prophage DNA.

To determine whether the early replication of Mu prophage DNA proceeds beyond the termini of the prophage into hose DNA, the amounts of both Mu DNA and the prophage-adjacent host DNA sequences were measured using a DNA-DNA annealing assay after induction of the Mu vegetative cycle. Whereas Mu-specific DNA synthesis began 6 to 8 min after induction, no amplification of the adjacent DNA sequences was observed. These data suggest that early Mu-induced DNA synthesis is constrained within the boundaries of the Mu prophage. Since prophage Mu DNA does not undergo a prophage lambda-like excision from its original site after induction (E. Ljungquist and A. I. Bukhari, Proc. Natl. Acad. Sci. U.S.A. 74:3143--3147, 1977), we propose the existence of a control mechanism which excludes prophage-adjacent sequences from the initial mu prophage replication. The frequencies of the Mu prophage-adjacent DNA sequences, relative to other Escherichia coli genes, were not observed to change after the onset of Mu-specific DNA replication. This suggests that these regions remain associated with the host chromosome and continue to be replicated by the chromosomal replication fork. Therefore, we conclude that both the Mu prophage and adjacent host sequences are maintained in the host chromosome, rather than on an extrachromosomal form containing Mu and host DNA.

Re-examination of F plasmid replication in a dnaC mutant of Escherichia coli.

The replication of an F' plasmid in a dnaC mutant, thermolabile for initiation of chromosomal replication, has been re-examined using a novel DNA-DNA annealing assay. Plasmid replication ceases rapidly at non-permissive conditions, consistent with a direct role for the dnaC product in the replication of F.