Interchromosomal duplications of the adrenoleukodystrophy locus: a phenomenon of pericentromeric plasticity

A 9.7 kb segment encompassing exons 7-10 of the adrenoleukodystrophy (ALD) locus of the X chromosome has duplicated to specific locations near the pericentromeric regions of human chromosomes 2p11,10p11, 16p11 and 22q11. Comparative sequence analysis reveals 92-96% nucleotide identity, indicating that the autosomal ALD paralogs arose relatively recently during the course of higher primate evolution (5-10 million years ago). Analysis of sequences flanking the duplication region identifies the presence of an unusual GCTTTTTGC repeat which may be a sequence-specific integration site for the process of pericentromeric- directed transposition. The breakpoint sequence and phylogenetic analysis predict a two-step transposition model, in which a duplication from Xq28 to pericentromeric 2p11 occurred once, followed by a rapid distribution of a larger duplicon cassette among the pericentromeric regions. In addition to facilitating more effective mutation detection among ALD patients, these findings provide further insight into the molecular basis underlying a pericentromeric-directed mechanism for non- homologous interchromosomal exchange.      

Human mini-chromosomes in mouse embryonal stem cells

We have introduced human mini-chromosomes of 4 Mb and approximately 15 Mb in size into mouse embryonal stem cells. Although these human mini- chromosomes are stable in hamster and chicken cells, they re-arrange or segregate aberrantly in the embryonal stem cells and are rapidly lost in the absence of selection. However, one of the mini-chromosomes re- arranged, acquired mouse centromeric sequences and was then stably maintained for at least 60 population doublings in culture. This mini- chromosome, which is 4 Mb in size, is a candidate for a mouse germ line chromosome vector.      

A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

Background  

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation.     

Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.      

Treatment characteristics of psychiatric programs that correlate with patient community adjustment

This large Veterans Administration cooperative study sought to identify the ward milieu characteristics of effective psychiatric programs. It was developed as a multivariable, correlational study that involved systematic observations of program characteristics and outcome effectiveness of wards as they operated in their usual manner. Seventy-nine wards in 18 hospitals provided 11,283 patients eligible for follow-up. Eleven treatment characteristics were found to be correlated to patients' community adjustment 3 months after discharge. These characteristics were classified into five general categories: Patient-staff interaction, patient activities, medication practices, ward physical environment, and nursing staff rotation. The major conclusions are: wards do differ in their effectiveness as measured by ratings of patient posthospital adjustment; and treatment characteristics make a difference in program effectiveness as measured by patients' posthospital adjustment.     

Dynamic and static phantoms for evaluation of digital subtraction angiographic systems

Two types of phantoms were developed with which to evaluate the overall performance of digital subtraction angiography (DSA) systems. A dynamic phantom, called a "fish bone" phantom, consists of polyethylene tubes that simulate blood vessels with various lesions, such as stenoses, ulcers, and aneurysms. With this phantom, washout curves were obtained representing the relationship between iodine content and time. It will be useful for qualitative assessment of DSA images, evaluation of different image-processing schemes, and studies of blood flow analysis. A static phantom, called a "C-D" phantom, can be used for measurement of quantitative contrast-detail (C-D) diagrams and for daily monitoring of DSA systems. This was constructed of tubes of seven different diameters (2.15-0.28 mm) and 14 different concentrations of contrast medium (100%-1.1% Renografin-76 [meglumine and sodium diatrizoate]). The C-D diagrams were determined from an observer performance study using C-D phantom images obtained at four different DSA settings.     

Severity of demyelination in vivo correlates with serum myelination inhibition activity in guinea pigs having a new form of experimental allergic encephalomyelitis

Guinea pigs received a suboptimal transfer of lymphocytes sensitized to myelin basic protein (BP) and were then immunized with guinea pig BP, BP plus chicken brain or chicken myelin, or chicken brain alone. Sera from these animals were tested for the presence of myelinotoxic antibodies, as detected by the myelination inhibition assay. Myelination inhibition activity correlated with the histologic severity of demyelination.     

Needle biopsy of renal allografts: comparison of two techniques

Two techniques for renal allograft biopsy were retrospectively evaluated to compare relative safety and efficacy. After ultrasound (US) localization of the kidney and biopsy with a hand-held 14-gauge cutting needle, an adequate specimen was obtained in 74 of 77 cases (96%). Major complications occurred in six of these 77 cases (8%). One hundred four biopsies were performed by using a smaller 18-gauge cutting needle with a spring-loaded biopsy "gun" and real-time US guidance. With this newer technique, specimens adequate for diagnosis were obtained in 99 biopsies (95%). There was a single major complication with this technique (1%). The 18-gauge needle with real-time US guidance yields comparably adequate specimens with a lower frequency of complications.     

Common Sheath Reimplantation Yields Excellent Results in the Treatment of Vesicoureteral Reflux in Duplicated Collecting Systems

Purpose

We evaluated our 10-year experience with the surgical treatment of vesicoureteral reflux in uncomplicated duplicated collecting systems.

Materials and Methods

Between 1984 and 1994, 54 refluxing renal units (8 bilateral) in 37 female and 9 male patients required surgery. Patient age ranged from 7 months to 17 years (average 4.9 at surgery). Postoperative followup (average 14.2 months) included voiding cystourethrography and renal sonography or excretory urography.

Results

Common sheath ureteral reimplantation via an intravesical approach was performed in 48 of the 54 refluxing renal units. Of the remaining 6 renal units detrussorrhaphy was performed in 4, and ureteroureterostomy combined with ureteral reimplantation and partial lower pole nephrectomy were done in 1 each. Two treated renal units had persistent postoperative vesicoureteral reflux, which resolved after subureteral polytetrafluoroethylene (Teflon) injection. No renal unit had postoperative hydronephrosis. Contralateral reflux was identified in 1 patient who underwent unilateral reimplantation. Our overall success rate was 96 percent for the surgical correction of vesicoureteral reflux in uncomplicated duplicated collecting systems. Common sheath reimplantation had a 98 percent success rate.

Conclusions

Although a duplicated collecting system increases the risk for surgical treatment, the presence of a duplication anomaly does not adversely affect surgical outcome. Modifications of procedures commonly performed in the surgical treatment of single system reflux to accommodate common sheath reimplantation have excellent surgical results with minimal morbidity.