Computer-assisted prenatal aneuploidy screening for chromosome 13, 18, 21, X and Y based on multiplex ligation-dependent probe amplification (MLPA)

In routine prenatal diagnostics we used a commercial multiplex ligation-dependent probe amplification (MLPA) kit for aneuploidy screening for chromosomes 13, 18, 21, X and Y. We present the results of 1593 consecutive prenatal samples analysed and diagnosed prior to knowledge of the G-banding analysis during 8-month routine use of computer-assisted MLPA aneuploidy screening. In total, 27 aneuploidies were detected. There were no false positive results while two false negative results could be explained by a placental mosaicism and a partial monosomy, respectively. In total, 3.2% of the samples were inconclusive. We conclude that automatic computer assisted MLPA is a rapid, simple and reliable method for detection of aneuploidies in prenatal diagnostics.     

Chromosomal radiosensitivity of breast cancer with a CHEK2 mutation

Recently, multiple studies have shown that a sequence variant in CHEK2 (CHEK2 1100delC) plays a role in the susceptibility to breast cancer. This mutation should confer about a twofold increased breast cancer risk in women and a 10-fold increased risk in men. Because the CHEK2 gene plays a critical role in DNA damage repair and the CHEK2 1100delC variant confers susceptibility to breast cancer, we investigated if patients carrying the CHEK2 1100delC mutation are characterized by an enhanced chromosomal radiosensitivity. To this end, familial breast cancer patients, sporadic breast cancer patients, and healthy women, considered in our previously studied to determine their chromosomal radiosensitivity with the G2 and G0-MN assay, were all tested in present study for the presence of the CHEK2 1100delC variant. The 1100delC variant was detected in none of the 100 healthy individuals, in 1 of 100 (1%) unselected breast cancer patients and in 3 of 78 (3.8%) breast cancer patients with a family history of breast cancer. The breast cancer patients with the CHEK2 1100delC genotype had a mean radiation-induced yield of chromatid breaks that was not significantly different from that of the healthy control group. Although the mean yield of micronuclei (MN) was significantly higher compared to the healthy control group, this higher mean MN yield was due to a single patient who had a very high number of MN compared to the parallel control. Our data suggest that breast cancer patients with a CHEK2 1100delC mutation are in general not characterized by a distinct enhanced chromosomal radiosensitivity. These conclusions are, however, very preliminary, because of the small numbers of CHEK2 1100delC breast cancer patients studied.     

Matrix metalloproteinases (MMP), EMMPRIN (extracellular matrix metalloproteinase inducer) and mitogen-activated protein kinases (MAPK): Co-expression in metastatic serous ovarian carcinoma

Activation or suppression of intracellular signaling via the mitogen-activated protein kinase (MAPK) family has been linked to expression of matrix metalloproteinases (MMP) in experimental models, but this association has not been demonstrated in clinical material. The objective of this study was to investigate the possible association between expression and activity of MMP, expression of the MMP inducer EMMPRIN, and the expression (level) and phosphorylation status (activity) of the extracellular-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK) and high osmolarity glycerol response kinase (p38) in effusions from patients diagnosed with serous ovarian carcinoma. MAPK level and activity were studied in 55 effusions using immunoblotting. MMP-1, MMP-2, MMP-9 and EMMPRIN expression was studied using immunocytochemistry (ICC) and mRNA in situ hybridization (ISH). The gelatinolytic activity of MMP-2 and MMP-9 was measured by zymography. ERK and phospho-ERK (p-ERK) were detected in 54/55 (98%) and 50/55 (91%) specimens, respectively. JNK and p-JNK were detected in 53/55 (96%) and 38/55 (69%) specimens, respectively. p38 was expressed in 54/55 (98%) specimens, and its phosphorylated form was found in 51/55 (92%). MMP-2 mRNA expression (P=0.048), protein expression (P=0.046) and gelatinolytic activity (P=0.039) correlated with ERK phosphorylative activity. MMP-2 activity also correlated with p38 activity (P=0.017). MMP-9 protein expression correlated with phosphorylation of p38 (P=0.046), but enzyme activity showed inverse relationship with both p-ERK (P=0.05) and p-p38 (P=0.033) expression. EMMPRIN expression correlated with MMP-1 (P<0.001), MMP-2 (P=0.042) and MMP-9 (P=0.029) expression, as well as with ERK activity (P=0.001). Our results present the first evidence of a possible link between MAPK signaling and MMP expression and activity in vivo. These data may expand our understanding regarding the mechanisms by which MMP synthesis is regulated in effusions and possibly affect treatment strategies for this form of malignancy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mutation analysis of the BRCA1 and BRCA2 genes results in the identification of novel and recurrent mutations in 6/16 flemish families with breast and/or ovarian cancer but not in 12 sporadic patients with early-onset disease. Mutations in brief no. 224. Online

Since the identification of the BRCA1 and BRCA2 genes (MIM#s 113705 and 600185), more than hundred different mutations throughout both genes have been reported. Recurrent mutations are rare and mainly due to founder effects. We analyzed 12 sporadic female patients with breast cancer before age 35, as well as 16 unrelated families, presenting with either (i) at least 3 first degree relatives with breast and/or ovarian cancer diagnosed at any age, or (ii) at least 2 first and/or second degree relatives with breast and/or ovarian cancer before age 45 years. We performed a protein truncation test for BRCA1 exon 11 and BRCA2 exons 10 and 11 and heteroduplex analysis for all the remaining exons of BRCA1 and 2. Presence of genomic deletions encompassing exons 13 or 22 of BRCA1, known to be Dutch founder mutations, was investigated by PCR. In 6/16 (37.5%) unrelated families the causal mutation in either the BRCA1 or BRCA2 gene was identified. Four different mutations were found in the BRCA1 gene: IVS5+3A>G (intron 5), 1191delC (exon 11), R1443X (exon 13), IVS22+5G>A (intron 22) and two in the BRCA2 gene: 6503delTT (exon 11), 6831delTG (exon 11). 1191delC (BRCA1) and 6831delTG (BRCA2) are novel mutations. IVS5+3A>G in exon 5 of BRCA1 published by Peelen et al. (1997) as a novel Belgian mutation, was identified in one additional family, not fulfilling our inclusion criteria. In the group of 12 sporadic female patients no mutations were found.     

Role of group A streptococcal IgG-binding proteins in triggering experimental glomerulonephritis in the rabbit

Our previous studies have indicated that the IgG-binding M-family proteins (IgGBP) of group A streptococci may be involved in eliciting experimental acute poststreptococcal glomerulonephritis (APSGN) in the rabbit. These surface proteins were also found to trigger production of anti-IgG, which might conceivably act to enhance renal deposition of immune complexes (IC). In the present study, a clinical isolate of serotype M22 (strain AL168), an isogenic double mutant deficient for both the IgGBPs Mrp and Emm, as well as mutants deficient in only one of the proteins were tested for capacity to induce glomerulonephritis. Streptococci to be used for injecting rabbits were heat-killed. Surface-bound IgG was removed by 1 M KSCN and cells were then repeatedly washed in PBS before use. Rabbits were injected intravenously with 109 cells three times a week for 8 weeks and, following one month of rest, for another 6 weeks. Deposits of IgG and C3 as well as induced chemokines TNF-alpha, IL-1beta and IL-6 were traced in cryostat sections using specific antibodies and appropriate peroxidase-labelled anti-antibodies. In four rabbits immunized with the double mutant strain, no deposits were found, and as examined by TEM, only subtle and transient renal changes were observed. In contrast, the original strain AL168 induced pronounced inflammatory and degenerative glomerular changes in all four rabbits injected, and deposits of TNF-alpha, IL-1beta and IL-6 were found in mesangial and endothelial cells. Similar deposits and glomerular changes were seen in all eight rabbits injected with the mrp-emm+ mutant and in four out of seven animals receiving the mrp+emm- mutant. There was a highly significant correlation between high levels of circulating anti-IgG and development of APSGN. These results confirm an important role of streptococcal IgGBP in triggering experimental APSGN as earlier proposed by our group.     

The use of blood components in surgical transfusion therapy

Blood components can be prepared either by separation of ordinary whole blood units or, selectively, by apheresis techniques. In recent years, new methods for improvement of quality and length of storage have been developed. The additive solution approach is now being applied increasingly. Its advantages and the difference between some available systems are described. Blood component therapy must be integrated into the patients' water/electrolyte balance and nutrition schedule. An outline is given of the role of blood components in the treatment of shock. The question of excessive bleeding, the possibilities of making the diagnosis of its cause(s) in the individual case, and the use of blood components is described. Coronary pulmonary bypass is used as an example of a complicated situation that can be handled effectively by a limited number of diagnostic and therapeutic tools.

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Resumen Los componentes sanguíneos pueden ser preparados bien por separación de unidades ordinarias de sangre total o, selectivamente, por técnicas de aferesis. Estas últimas tienen la ventaja de permitir la selección de donantes particularmente apropiados en relación a compatibilidad inmunológica o a la ausencia de agentes infecciosos transmisibles, así como a la posibilidad de que un mismo donante pueda ser usado en forma repetida dentro de un período de tiempo corto. Nuevos métodos de mejoramiento y prolongación del período de almacenamiento han sido desarrollados. El enfoque de las soluciones aditivas es empleado con creciente frecuencia; se describen sus ventajas y las diferencias con otros métodos disponibles en la actualidad.La terapia con componentes sanguíneos debe ser parte integral del manejo del equilibrio de agua y electrolitos y del programa de soporte nutricional. Se provee una guía sobre el uso de componentes sanguíneos en el tratamiento del shock.Se describe el problema del sangrado excesivo, la posibilidad de establecer el diagnóstico de su causa en cada caso individual, y el uso de los componentes sanguíneos. El bypass coronario es presentado como ejemplo de una situación compleja que puede ser manejada en forma efectiva por medio de un número limitado de métodos de diagnóstico y tratamiento.

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Résumé Les constituants du sang peuvent être isolés soit par séparation des unités de sang total ordinaire, soit sélectivement par techniques d'apharèse. Au cours des récentes années des méthodes pour améliorer la qualité et la durée du stockage ont été mises au point. Pour ce faire, l'emploi d'additifs est largement répandu. Les avantages et les différences entre quelques méthodes disponibles sont décrits par les auteurs. Le traitement par constituants du sang doit être associé à l'équilibre hydroélectrolytique et nutritif. Le rôle des différentes parties constituantes du sang dans le traitement du choc est souligné. La question de l'hémorragie excessive, les possibilités de faire le diagnostic de ses causes dans les cas individuels et l'emploi des parties constituantes adéquates du sang sont décrits. A titre d'exemple le By pass pulmo-coronarien, situation particulièrement délicate, peut être contrôlé efficacement par un nombre limité de méthodes diagnostiques et thérapeutiques.

     

Sarcopenia Definitions as Predictors of Fracture Risk Independent of FRAX®, Falls,and BMD in the Osteoporotic Fractures in Men (MrOS) Study: A Meta-Analysis

Dual-energy X-ray absorptiometry (DXA)-derived appendicular lean mass/height² (ALM/ht²) is the most commonly used estimate of muscle mass in the assessment of sarcopenia, but its predictive value for fracture is substantially attenuated by femoral neck (fn) bone mineral density (BMD). We investigated predictive value of 11 sarcopenia definitions for incident fracture, independent of fnBMD, fracture risk assessment tool (FRAX^®) probability, and prior falls, using an extension of Poisson regression in US, Sweden, and Hong Kong Osteoporois Fractures in Men Study (MrOS) cohorts. Definitions tested were those of Baumgartner and Delmonico (ALM/ht² only), Morley, the International Working Group on Sarcopenia, European Working Group on Sarcopenia in Older People (EWGSOP1 and 2), Asian Working Group on Sarcopenia, Foundation for the National Institutes of Health (FNIH) 1 and 2 (using ALM/body mass index [BMI], incorporating muscle strength and/or physical performance measures plus ALM/ht²), and Sarcopenia Definitions and Outcomes Consortium (gait speed and grip strength). Associations were adjusted for age and time since baseline and reported as hazard ratio (HR) for first incident fracture, here major osteoporotic fracture (MOF; clinical vertebral, hip, distal forearm, proximal humerus). Further analyses adjusted additionally for FRAX-MOF probability (n = 7531; calculated ± fnBMD), prior falls (y/n), or fnBMD T-score. Results were synthesized by meta-analysis. In 5660 men in USA, 2764 Sweden and 1987 Hong Kong (mean ages 73.5, 75.4, and 72.4 years, respectively), sarcopenia prevalence ranged from 0.5% to 35%. Sarcopenia status, by all definitions except those of FNIH, was associated with incident MOF (HR = 1.39 to 2.07). Associations were robust to adjustment for prior falls or FRAX probability (without fnBMD); adjustment for fnBMD T-score attenuated associations. EWGSOP2 severe sarcopenia (incorporating chair stand time, gait speed, and grip strength plus ALM) was most predictive, albeit at low prevalence, and appeared only modestly influenced by inclusion of fnBMD. In conclusion, the predictive value for fracture of sarcopenia definitions based on ALM is reduced by adjustment for fnBMD but strengthened by additional inclusion of physical performance measures. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Molecular Human Reproduction: Evidence for {gamma}-aminobutyric acid specific binding sites on human spermatozoa

The possible presence of -aminobutyric acid (GABA) specificbinding sites on human spermatozoa was investigated. Swim-uppreparations of human spermatozoa were incubated with radiolabelledGABA in the presence of unlabelled GABA, alternatively displacersof GABA_A/B receptors and GABA transport proteins. The resultsindicate that GABA specific binding sites are present on thesurface of human spermatozoa, and that these binding sites possiblyindicate the presence of GABA transport proteins. Furthermore,GABA at different concentrations was added to swim-up preparationsof human spermatozoa. Possible effects of GABA on sperm motility,hyperactivation and acrosome reaction were explored. No significantdifferences were observed between treated groups and controlsconcerning motility parameters and hyperactivation. Incubationwith GABA did not cause any increase in spontaneous acrosomereaction. However, spermatozoa treated with the calcium ionophoreA-23187 showed a small but significantly increased ability toundergo the acrosome reaction following preincubation in 10–⁴M GABA (P < 0.05).