Differential Stability of miR-9-5p and miR-9-3p in the Brain Is Determined by Their Unique Cis- and Trans-Acting Elements.

microRNAs (miRs) are fundamental regulators of protein coding genes. In the CNS, miR-9 is highly enriched and critical for neuronal development and function. Mature miRs are derived from a duplex precursor, and the -5p strand ("guide") is preferentially incorporated into an RNA-induced silencing complex (RISC) to exert its regulatory functions, while the complementary -3p strand ("passenger") is thought to be rapidly degraded. By contrast, both strands of the miR-9 duplex have unique functions critical for neuronal physiology, yet their respective degradation rates and mechanisms governing degradation are not well understood. Therefore, we determined the degradation kinetics of miR-9-5p and miR-9-3p and investigated the cis and trans elements that affected their stability in the brain. Using a combination of homogeneous neuronal/astrocyte cell models and heterogeneous brain tissue lysate, we demonstrate the novel finding that miR-9-3p was more stable than the miR-9-5p guide strand in all models tested. Moreover, the degradation kinetics of both miR-9-5p and miR-9-3p were brain-region specific, suggesting that each brain region was differentially enriched for specific degradation factors. We also determined that the 3' nucleotides harbor important cis elements required to not only maintain stability, but also to recruit potential protein degradation factors. We used mass spectrometry to assess the miR-9 interacting proteins and found that the -5p and -3p strands were associated with functionally distinct proteins. Overall, these studies revealed unique miR-9-5p and miR-9-3p degradation kinetics in the brain and proposed critical nucleotide sequences and protein partners that could contribute to this differential stability.

Histopathological features of breast cancer in carriers of ATM gene variants.

AIMS: Germline variants in the ataxia telangiectasia mutated (ATM) gene have been implicated in increased breast cancer risk. The aim of this study was to determine whether the histopathology of breast cancers occurring in ATM variant carriers is distinctive or resembles the described BRCA1 mutation-associated phenotype. METHODS: The histopathological features of breast cancers occurring in ATM variant carriers from multiple-case breast cancer families were compared with matched controls. The test group included 21 cases of in situ and/or invasive cancer from carriers of either the IVS10-6T-->G, 2424V-->G or 1420L-->F ATM variants in the absence of BRCA1 or BRCA2 mutations. An additional four invasive cancers from carriers of a pathogenic BRCA1 mutation in the context of a familial ATM variant were also examined. RESULTS: The histopathology of breast cancers in ATM variant-only carriers was not significantly different from controls and known features of BRCA1 mutation-associated cancer were rarely seen. In contrast, these features were prominent in the small group of cases with a pathogenic BRCA1 mutation. CONCLUSIONS: Breast cancer occurring in carriers of ATM variants is not associated with distinctive histopathological features and does not resemble the tumour phenotype commonly observed in BRCA1 mutation carriers.

Large genomic rearrangements of both BRCA2 and BRCA1 are a feature of the inherited breast/ovarian cancer phenotype in selected families.

INTRODUCTION: A strong family history of breast and/or ovarian cancer can often be explained by small insertions, deletions, or substitutions in BRCA1 or BRCA2 and large genomic rearrangements in BRCA1. However, there is little evidence that genomic rearrangements are a major factor in BRCA2 associated breast cancer and the frequencies of rearrangements in BRCA1 in large clinic based populations are unknown. OBJECTIVE: To investigate the frequency of large genomic rearrangements in BRCA1 and BRCA2 in a large clinic based population at high risk of developing breast and/or ovarian cancer. METHODS: Multiplex ligation dependent probe amplification was used to comprehensively screen BRCA1 and/or BRCA2 in 312 index cases. RESULTS: Three novel deletions detected in BRCA2 were found exclusively in families with at least one case of male breast cancer. Novel rearrangements in BRCA1 were detected mostly in families with both breast and ovarian cancer. Families with these mutations were significantly younger at average age of cancer diagnosis. CONCLUSION: Screening for large genomic rearrangements in both BRCA1 and BRCA2 is strongly supported by this study, in particular in multiple case breast/ovarian families with a young age of onset (BRCA1) and families containing at least one case of male breast cancer (BRCA2).

Absence of a weight gain response to Vitamin B12 supplementation in weaned dairy heifers grazing pastures of marginal cobalt content.

AIM: To obtain information on serum and liver vitamin B12 and urinary methylmalonic acid concentrations as diagnostic tests to predict a weight gain response to supplementation with vitamin B12 in young dairy cattle when grazing pasture of low cobalt content. Methodology. Forty dairy cattle (12 Friesian, 14 Friesian x Jersey and 14 Jersey) were allocated to two equal sized groups, treated and untreated, based on liveweight. At monthly intervals for 14 months, all animals were weighed, their serum and urine sampled, their liver biopsied and the pasture sampled from the paddocks they were grazing and going to graze. Serum and liver were assayed for vitamin B12 concentrations. For the first 5 months of the trial, urine was assayed for methylmalonic acid concentrations. Both washed and unwashed pasture samples were assayed for cobalt concentrations. RESULTS: No weight gain response occurred vitamin B12 supplementation in young growing cattle grazing pasture with a cobalt concentration of 0.04-0.06 mg/kg DM. For 5 months of the trial, liver vitamin B12 concentrations from untreated calves were in the range 75-220 nmol/kg and serum vitamin B12 concentrations were as low as 72 pmol/1. There was no associated growth response to supplementation. CONCLUSION: Further trials involving young cattle grazing pastures with cobalt concentrations less than 0.04 mg/kg DM are required to reliably determine liver and serum vitamin B12 concentrations at which growth responses to vitamin B12 or cobalt supplementation are likely under New Zealand pastoral grazing conditions.

Loss of nuclear BRCA1 expression in breast cancers is associated with a highly proliferative tumor phenotype.

Recent studies have suggested that BRCA1-associated hereditary breast cancer may be a more aggressive form of disease than sporadic breast cancer. BRCA1-associated breast cancer has been reported to be significantly associated with grade 3 disease. Because grade 3 disease indicates a poor prognosis, this implies that women with a germ-line mutation in BRCA1 who develop breast cancer may have a poorer prognosis than those with sporadic disease. However, little is known about the association of BRCA1 expression with biological markers of prognosis. The present study examined the expression of BRCA1 in a total of 40 archival breast tumor specimens from three patient cohorts (sporadic, familial, and early onset breast cancer) to determine localization of the protein. Furthermore, BRCA1 staining was compared with expression of markers of tumor biology. We found that BRCA1 is generally located in the nucleus and the cytoplasm of normal and malignant breast tissue. Nuclear staining for BRCA1 was observed in most sporadic tumors, but nuclear BRCA1 was reduced or absent in the majority of familial and early onset breast tumors. Although no correlation was found between nuclear BRCA1 expression and estrogen and progesterone status, a significant inverse correlation was found between nuclear BRCA1 and expression of the proliferation marker Ki-67 (P = 0.01). Our findings suggest that tumors associated with a germ-line mutation in one of the breast cancer genes may be highly proliferative and support the view that loss of BRCA1 expression in breast tumors may lead to a more aggressive tumor phenotype.

Association of hereditary angioedema and hereditary breast cancer.

A family is presented in whom hereditary angioedema (HAE) and hereditary breast cancer were coexistent, an association not previously reported. A potential for genetic and treatment-related interactions between the two conditions exists. The use of the hormonal agent danazol to suppress HAE is unlikely to adversely affect the development or outcome of breast cancer. Surgery, chemotherapy, and radiotherapy were received by affected family members, without triggering edema. Whether hormonal breast cancer treatment affects the suppression of HAE by danazol remains unknown.

Pediatric trauma.

In the United States, trauma continues to be the leading cause of death in children between the ages of 1 and 15 years of age. Children die from trauma at a rate five times greater than from leukemia which is the next leading cause of death in this age group. The acutely injured child is brought to community hospitals as well as university hospitals. The CRNA that is called to care for the injured child, either in the emergency room or the operating room, must be knowledgeable of the anatomic, physiological, and emotional differences between the adult and pediatric trauma patient and their response to treatment. Only with this knowledge will there be a decrease in the morbidity of the traumatized pediatric patient. The CRNA should be able to rapidly assess and gain control of the pediatric airway and assure adequate respiration. Initial assessment of the pediatric trauma patient also includes the restoration or maintenance of hemodynamic stability.

Unusual expression of mRNA typical of Philadelphia positive acute lymphoblastic leukemia detected in chronic myeloid leukemia.

The Philadelphia chromosome (Ph) is found in both chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). The Ph translocation, t(9;22)(q34;q11), can disrupt the BCR gene on chromosome 22 in one to two areas called the major (Mbcr1) and minor (mbcr1) breakpoint cluster regions. In CML the breakpoint has been mapped almost exclusively to Mbcr1, whereas in Ph positive ALL both Mbcr1 and the upstream mbcr1 breakpoints have been described. In this communication we describe an unusual patient with typical chronic phase Ph positive CML and evidence of the uncharacteristic mbcr1 breakpoint, predicting expression of the ALL-type p190 fusion protein. Fluorescence in situ hybridization demonstrated BCR gene rearrangement, the reverse transcription polymerase chain reaction detected the BCR-ABL fusion mRNA characteristic of the mbcr1 breakpoint, and failed to detect BCR-ABL mRNA characteristic of the Mbcr1 breakpoint. Southern blot analysis revealed no rearrangement in Mbcr1, and direct sequencing of the PCR product confirmed it to be the ALL-type mbcr1 fusion mRNA with the first exon of the BCR gene fused to ABL exon a2. This case differs from the previously reported cases of "p190" CML in that the patient presented without abnormal hematopoietic features other than those found in typical CML and provides further evidence that the p190 mRNA is not sufficient to cause an acute rather than chronic leukemia.

Benign marrow progenitors are enriched in the CD34+/HLA-DRlo population but not in the CD34+/CD38lo population in chronic myeloid leukemia: an analysis using interphase fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) was used to discriminate between benign and malignant cells in sorted populations of chronic myelogenous leukemia (CML) marrow. FISH has the advantage of allowing for a cell by cell analysis of the breakpoint cluster region (BCR) gene rearrangement immediately after flow sorting in nondividing G0/G1 cells that are potentially transcriptionally inactive. We initially selected CD34+ cells with very low expression of the activation antigen CD38 as a candidate phenotype for an immature and hypothetically more benign cell population, but found no enrichment for Ph negativity in that subtype. In five CML samples, 55% +/- 3.3% (mean +/- SE) of CD34+/CD38hi cells had the BCR gene rearrangement, similar to 57% +/- 3.7% seen in the CD34+/CD38lo population. In contrast, subsequent experiments (n = 4) determined that the CD34+/HLA-DRlo population in CML marrow does contain an increased proportion of benign cells: 15% +/- 1% of the CD34+/DRlo cells were BCR rearranged, compared with 52% +/- 5.8% of the CD34+/DRhi cells (P = .001). Our results indicate that benign progenitors in CML are enriched within the CD34+ cells with low DR antigen expression, but not low CD38 expression. One possible interpretation of these observations is that low CD38 antigen expression is not as useful as low HLA-DR expression for isolating immature cells.

Y chromosome loss in chronic myeloid leukemia detected in both normal and malignant cells by interphase fluorescence in situ hybridization.

Loss of the Y chromosome in bone marrow (BM) cells is a normal age-associated event. Y chromosome loss is also observed in the Philadelphia chromosome (Ph) positive BM cells of some patients with chronic myeloid leukemia (CML) in chronic phase, but at a younger age than in normal individuals. While the significance of loss of the sex chromosome in normal males is uncertain, -Y marrow cells are not believed to be of clonal origin. However, because CML is a clonal disease, CML sub-populations with Y loss may constitute a disease-related sub-clone. We used a PCR-amplified yeast artificial chromosome containing the BCR gene region for single color interphase analysis of BCR rearrangement by fluorescence in situ hybridization (FISH). Then, using two color FISH, with one fluorochrome detecting the BCR gene region and the other detecting Y chromosome repeat sequences, we surveyed peripheral and BM Y loss in both normal Ph- (BCR not disrupted) and CML Ph+ (BCR rearranged) interphase nuclei of two patients with Y loss in Ph positive cells observed by metaphase analysis. -Y was seen in a proportion of Ph+ cells in both cases, and the proportion matched that seen in Ph- cells, indicating that Y loss is probably sporadic in both normal and CML populations, and that the propensity for Y loss in normal BM cells may be a phenotype that can be retained by malignant cells in CML.

Growth and growth hormone therapy of children treated for leukaemia.

A total of 37 children (24 male, 13 female) who had been treated for leukaemia with chemotherapy and 24 Gy cranial irradiation, and who were disease free for at least 18 months, were commenced on somatrem at a mean of 7.6 years (range, 4.8-12.1 years) after leukaemia diagnosis because of growth rate below the 25th centile for bone age. Peak GH response to provocation (exercise, arginine, insulin hypoglycaemia) was less than 20 milliunits/litre in 27 children (deficient group) and 20 milliunits/litre or more in 10 children (non-deficient group). The mean height SD decrease from diagnosis of leukaemia to commencement of somatrem was 1.98, 86% of the children decreasing by more than 1 SD. Those who were tall for age at leukaemia diagnosis and females were more severely affected. Mean (+/- SD) height velocity increased on somatrem from 2.7 +/- 1.1 to 6.6 +/- 2.2 cm/year during the first 6 months (n = 25), and to 6.0 +/- 1.7 cm/year during the first 12 months (n = 19). No difference in growth response was seen between the sexes or between the deficient and non-deficient groups. Catch-up growth occurred for the first 6 months only. It is concluded that children with a low growth rate after treatment of leukaemia should be considered for GH therapy irrespective of the results of GH provocative tests.

Growth failure and growth-hormone deficiency after treatment for acute lymphoblastic leukaemia.

In a study of 77 children who had been treated for acute lymphoblastic leukaemia (ALL) with an LSA2L2 (Memorial Sloan-Kettering) chemotherapy protocol plus radiotherapy (24 Gy) as cranial prophylaxis, growth was examined 3.0-9.5 years after diagnosis. The children's growth slowed and they crossed height percentiles towards the end of or after treatment. The Z-score, which reflects the deviation of height measurements from the population mean, was used to assess height change. The mean Z-score was 0.16 at diagnosis, -0.30 2 years later, -0.71 4 years later, and -1.37 6 years later. Height for age had fallen by more than 1 standard deviation of the population mean in 32% of survivors 4 years after diagnosis and in 71% 6 years after diagnosis. Younger children and those tall for age at diagnosis were more severely affected. Growth-hormone (GH) response to standard provocation tests was measured in 46 patients; 30 had partial or complete GH deficiency. Mean pulsatile GH secretion was low in the 34 patients tested. Cranial irradiation is probably the most important causative factor in the development of GH deficiency in survivors of ALL.

Unusual skin rash following withdrawal of oral 6-mercaptopurine in children with leukemia.

Sixteen episodes of a distinctive, papular rash occurred in eight patients following withdrawal of 6-mercaptopurine (6MP) and methotrexate (MTX) used as maintenance therapy for acute lymphoblastic leukemia (ALL). The rash also developed in one of the eight patients when only 6MP was discontinued. The eruption occurred mainly on the face, and in this site resembled the perioral dermatitis seen following withdrawal of topical fluorinated steroids. The rash generally began within 3 weeks of stopping 6MP and lasted 3 to 4 weeks. It failed to improve with the use of topical corticosteroid. We conclude that this rash is caused by the withdrawal of oral 6MP.

Glutathione peroxidase activity in bovine serum and erythrocytes in relation to selenium concentrations of blood, serum and liver.

The concentration of selenium (Se) in liver, serum and whole blood, and the activity of glutathione peroxidase (GSH-px) in serum and erythrocytes were monitored for seven months after the administration of Se to Se-deficient calves. There was a rapid increase in Se concentration in liver and serum, followed by an exponential decline with half-lives of 22.1 +/- 0.2 days and 28.3 +/- 2.0 days respectively. Whole blood Se concentration also increased rapidly but declined more slowly than liver or serum Se concentraations. The rise in Se concentration and GSH-px activity in erythrocytes was delayed and both levels remained elevated several months after liver and serum Se concentrations had become marginal or deficient. GSH-px activity in serum increased more rapidly after dosing and declined more rapidly than GSH-px activity in erythrocytes. The results suggest that liver and serum Se concentration and serum GSH-px activity respond to changes in dietary Se intake more rapidly than either whole blood Se or erythrocyte GSH-px activity. Two-monthly administrations of Se, at the current recommended therapeutic dose (0.1 mg Se/kg as sodium selenate) appears to be necessary to maintain adequate Se levels in calves on Se-deficient pasture.

A renal lesion in ankylosing spondylitis.

2 patients with ankylosing spondylitis (A Sp) were found to have renal lesions similar to those seen in IgA nephropathy. In 1 patient the changes were extremely severe and progressive and in the other they were mild. Vascular changes were also noted in 1 patient. The findings suggest an immune complex mediated glomerulonephritis and support an earlier report that there may be a specific renal lesion in patients with A Sp. The significance of IgA deposition in the mesangium, and of an increase in the serum levels of IgA in some patients with A Sp is unclear.