Cryptic duplication of 12q24.33 --> qter in a child with Angelman syndrome-simultaneous occurrence of two unrelated cytogenetic events.

Simultaneous occurrence of two unrelated cytogenetic events is rare. We present a case of Angelman Syndrome (AS) deletion and 12q duplication in a child with a history of developmental delay, microcephaly, cerebral palsy, and seizures. Traditional cytogenetic studies showed a normal 46,XY karyotype. Fluorescence in situ hybridization (FISH) using probe D15S10 (AS region/15q11.2) revealed a deletion. In addition, we serendipitously detected 12q24.3 duplication by FISH with 12q subtelomere probe. He inherited this duplication from the mother who presented with a balanced translocation karyotype 46,XX,add(12)(q24.3).ish t(12;13)(q24.3;p11.2)(12qtel-;12qtel+,D13Z1/D21Z1+,RB1+). Array comparative genomic hybridization (array-CGH) revealed a duplication of three bacterial artificial chromosome (BAC) clones (RP11-46H11, RP11-386I8, and RP11-309H3) covering about 423 Kb of DNA sequence. The published 12q terminal duplication cases had a detectable segment by classical banded cytogenetics techniques. To our knowledge, this is the smallest 12q cryptic rearrangement characterized by array-CGH and confirmed by BAC-clone FISH analysis. Based on these findings, we attempted to separate the clinical features associated with AS deletion and those features that are probably due to partial 12q duplication. We then reviewed the genes mapped in the duplicated region using the human genome database to understand the clinical significance. A subsequent pregnancy in the mother revealed an apparently balanced t(12;13) karyotype. We compare our case with the published cases, and discuss the implications of our findings and its relevance in addressing genetic counseling issues.

Surgical outcomes after breast cancer surgery: measuring acute lymphedema.

BACKGROUND: Studies of lymphedema have used inconsistent measures and criteria. The purpose of this pilot study was to measure the onset and incidence of acute lymphedema in breast cancer survivors using strict criteria for limb evaluation. MATERIALS AND METHODS: Eligible women were those undergoing breast cancer surgery that included axillary staging and/or radiation therapy of the breast. Arm volume, strength, and flexibility were measured preoperatively and quarterly. Lymphedema was defined as a greater than 10% increase in limb volume. Additional strength and flexibility assessments were done at these times. RESULTS: In 30 evaluable patients, half underwent modified radical mastectomy and half lumpectomy, with half of the lumpectomy patients undergoing axillary node staging. Of the 30 patients 27% were Stage 0; the rest were Stage I (27%), IIA (13%), IIB (23%), and IIIA (7%). One subject was IIIB postoperatively. There were 2 women with a 10% or greater change in limb volume; the change was detected in one woman at 3 months (5% incidence) and in the second woman at 6 months (11% incidence). Both had undergone mastectomy and axillary dissection and one of these two women had symptoms of tingling and numbness in the affected arm that began at 3 months. Overall, 35% of the sample experienced symptoms by 3 months, which included numbness, aching, and tingling of the entire upper extremity, but without volume changes. The relationship between undergoing modified radical mastectomy and experiencing symptoms in the affected limb at 3 months was significant (P = 0.05). CONCLUSIONS: In this interim report strict methods of measurement and limb volume comparisons detected acute lymphedema at 3 months in 5% of the sample, and at 6 months in 11% of the sample. Furthermore, symptoms were detected in 35% without volume changes at 3 months postoperatively, which may warn of lymphedema occurrence within the next 3 months. This may assist clinical evaluation of symptoms in the postoperative period and support early referral to lymphedema experts.

Condensation of rat telomere-specific nucleosomal arrays containing unusually short DNA repeats and histone H1.

Vertebrate telomeres contain arrays of nucleosomes with unusually short and regular repeat lengths (Makarov, V. L., Lejnine, S., Bedoyan, J., and Langmore, J. P.(1993) Cell 73, 775-787; Lejnine, S., Makarov, V., and Langmore, J. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2393-2397). In order to better define the specific structural features of telomere chromatin, we examined the condensation and H1 content of telomere nucleoproteins from rat liver. Velocity sedimentation analysis shows that telomeric nucleosome arrays condense with increasing ionic strength and molecular weight in a manner comparable with that of bulk chromatin despite the very short repeat length. However, these condensed structures do not exhibit the approximately 100-base pair deoxyribonuclease II repeat characteristic of condensed bulk chromatin. Frictional coefficient calculations suggest that telomere-specific higher order structure is more compact than bulk chromatin. Nucleoprotein gel electrophoresis shows that telomeric dinucleosomes from soluble chromatin contain H1. Finally, direct isolation and analysis of telomere nucleoproteins from formaldehyde-cross-linked nuclei indicate the presence of core histone proteins and H1. These results are consistent with the view that a major fraction of the long telomeres of rat are organized as specialized nucleosome arrays with features similar but not identical to those of bulk chromatin.

In vivo distribution of immune protein scolexin in bacteria-injected Manduca sexta larvae.

The Manduca sexta larva-specific immune protein, scolexin, was isolated and (14)CH(3)-labelled by reductive alkylation. The influence of the bacterium Streptococcus faecalis on the hemocoelic distribution of the labelled scolexin was then analyzed. During bacterial challenge, most of the scolexin signal was detected in association with the hemocyte aggregations and nodules which formed; in this respect the protein sometimes appeared to be associated with hemocytes which had phagocytized bacteria, while at other times it was most concentrated in the nodule-associated, and free, coagulum. Areas of high scolexin activity were sometimes detected at various sites on the surface of the fat body. The scolexin did not appear to bind directly to bacterial cells. Up to 24 hr following the injection of S. faecalis, the larvae were still carrying out the formation of nodules; unlike the nodules of the 3 and 6 hr intervals, the nodules observed at 21-24 hr were covered with an apparently humorally derived, coagular capsule.

Nucleosomal organization of telomere-specific chromatin in rat.

Rat liver interphase chromosomes have telomeres 20-100 kb in length. Micrococcal nuclease digestion of nuclei cleaves telomeres with a uniform 157 bp periodicity, producing soluble particles that sediment in sucrose gradients exactly like oligonucleosomes. The monomeric telomere particles comigrate with nucleosome core particles on nucleoprotein and DNA gels but do not bind H1. DNAase I cleaves telomere nucleoprotein into a series of bands spaced by about 10.4 bp and with the same intensity distribution as bands from bulk nucleosomes. Removal of H1 from chromatin alters the sedimentation properties of telomeres in parallel with bulk chromatin. Thus, telomeres of mammals are constructed of closely spaced nucleosomes, in contrast with the telomeres of lower eukaryotes, which show no evidence of nucleosomal structure.

Transcription, nucleosome stability, and DNA repair in a yeast minichromosome.

The template strand of the URA3 gene in the minichromosome YRpTRURAP is repaired of UV-induced cyclobutyl pyrimidine dimers (PD) much more efficiently than the nontemplate strand in growth-arrested Saccharomyces cerevisiae cells (Smerdon, M. J., and Thoma, F. (1990) Cell 61, 675). However, other regions of the plasmid are also efficiently repaired. We have examined the transcription and chromatin structure of these regions in growth-arrested cells to allow a more detailed comparison of transcription, nucleosome stability, and excision repair efficiency. Northern analysis, using strand-specific probes, indicates that four different transcripts are made from YRpTRURAP in addition to the URA3 mRNA in both growing and growth-arrested cells. The templates for these transcripts encompass all of the efficiently repaired regions outside of the URA3 gene. Nucleosome mapping indicates that the structure of the minichromosome in growth-arrested cells is indistinguishable from that of growing cells except for two nucleosomes in the region 500-800 base pairs 5' of the URA3 gene which become much less stable in growth-arrested cells. Comparison of the distribution of YRpTRURAP topoisomers in the two states, however, indicates that these nucleosomes are not lost from the majority of plasmid molecules. One of the four transcripts initiates in this region and increases by more than 5-fold in growth-arrested cells. Another transcript extends into a "slowly repaired" region which contains a very stable nucleosome. By determining the stability and relative amounts (at equilibrium) of the RNAs made from YRpTRURAP, transcription rates were determined and compared with the average PD repair rate for the different template regions. The results indicate that: 1) the rate of excision repair increases once a low, basal rate of transcription is achieved; 2) beyond this rate of transcription there is no simple correlation between the rates of transcription and PD repair; 3) nucleosome stability may "override" the coupling between transcription and repair if the transcription rate is low; and 4) at higher transcription rates, repair may be insensitive to nucleosome stability.

DNA repair in a small yeast plasmid folded into chromatin.

The question of whether excision repair of yeast plasmids accurately reflects the repair of yeast genomic chromatin has yielded conflicting answers. These conflicts could have arisen from differences in the conformation of plasmid molecules used during these studies. We have examined excision repair of UV photoproducts in a small (2619 bp) autonomously replicating plasmid (YRp-TRURAP), known to be folded into chromatin with positioned nucleosomes in vivo, in the yeast Saccharomyces cerevisiae. A quantitative assay was used to measure the yield of cyclobutane pyrimidine dimers (PD) in plasmid DNA by measuring the fraction of Form I molecules resistant to T4 endonuclease V. After a UV dose of 100 J/m2, which yields 1.2 PD/plasmid in irradiated cells, radiation insensitive (wt) cells repair approximately 70% of the PD in TRURAP chromatin in 2 hr (a rate comparable to that of genomic chromatin). On the other hand, no measurable repair occurs in TRURAP chromatin in radiation sensitive cells (rad1) during the same time period. Thus, this small plasmid contains sufficient chromatin structure in vivo to reflect the incompetent repair of genomic chromatin seen in a rad mutant, while maintaining the competent repair level in wt cells.