High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Anti-Interleukin-6 Therapy Decreases Hip Synovitis and Bone Resorption and Increases Bone Formation Following Ischemic Osteonecrosis of the Femoral Head

Legg-Calvé-Perthes disease (LCPD) is a juvenile form of ischemic femoral head osteonecrosis, which produces chronic hip synovitis, permanent femoral head deformity, and premature osteoarthritis. Currently, there is no medical therapy for LCPD. Interleukin-6 (IL-6) is significantly elevated in the synovial fluid of patients with LCPD. We hypothesize that IL-6 elevation promotes chronic hip synovitis and impairs bone healing after ischemic osteonecrosis. We set out to test if anti-IL-6 therapy using tocilizumab can decrease hip synovitis and improve bone healing in the piglet model of LCPD. Fourteen piglets were surgically induced with ischemic osteonecrosis and assigned to two groups: the no treatment group (n = 7) and the tocilizumab group (15 to 20 mg/kg, biweekly intravenous injection, n = 7). All animals were euthanized 8 weeks after the induction of osteonecrosis. Hip synovium and femoral heads were assessed for hip synovitis and bone healing using histology, micro-CT, and histomorphometry. The mean hip synovitis score and the number of synovial macrophages and vessels were significantly lower in the tocilizumab group compared with the no treatment group (p < .0001, p = .01, and p < .01, respectively). Micro-CT analysis of the femoral heads showed a significantly higher bone volume in the tocilizumab group compared with the no treatment group (p = .02). The histologic assessment revealed a significantly lower number of osteoclasts per bone surface (p < .001) in the tocilizumab group compared with the no treatment group. Moreover, fluorochrome labeling showed a significantly higher percent of mineralizing bone surface (p < .01), bone formation rate per bone surface (p < .01), and mineral apposition rate (p = .04) in the tocilizumab group. Taken together, tocilizumab therapy decreased hip synovitis and osteoclastic bone resorption and increased new bone formation after ischemic osteonecrosis. This study provides preclinical evidence that tocilizumab decreases synovitis and improves bone healing in a large animal model of LCPD. © 2020 American Society for Bone and Mineral Research (ASBMR).     

Efficacy of "high-intensity" blue-light and "standard" daylight phototherapy for non-haemolytic hyperbilirubinaemia

We report our clinical experience with phototherapy in 3802 infants; 3629 were exposed to "standard" daylight phototherapy and 173 to "high-intensity" blue-light phototherapy. High-intensity blue-light phototherapy was twice as effective as standard daylight phototherapy in decreasing bilirubin concentrations. No failures occurred with high-intensity phototherapy compared with an overall failure rate of 1.84/1000 with daylight lamps; these cases were transferred to high-intensity phototherapy with prompt response. Rebound after cessation of phototherapy was greater in those exposed to high-intensity blue light with a significantly greater number requiring a second exposure. However, the incidence was still low. No third exposure was required in any infant. Nursing of infants under high-intensity blue light was more difficult and inconvenient as was clinical monitoring. The light also caused more stress on the nursing and medical personnel. However, the infants tolerated both types of phototherapy equally well. High-intensity blue-light phototherapy would seem to be the treatment of choice for infants with rapidly increasing or very high bilirubin levels, as well as in those not responding adequately to daylight phototherapy.     

Recent advances in recognition of low-level methylmercury poisoning

Clinically evident neurologic damage from methylmercury exposure was well described following poisoning episodes in Japan and Iraq several decades ago. Paresthesias have been considered to be an early effect; however, additional data raise questions about whether this is the most sensitive adverse effect among adults. Fetuses are considered the most sensitive subpopulation because of the vulnerability of the developing nervous system. Over the past 5 years questions have been raised about what is an appropriate level of exposure for sensitive groups. A recent evaluation by a committee for the US National Research Council found that 0.1 microg/kg body weight per day is a scientifically justified level of methylmercury exposure for maternal-fetal pairs. The conclusions of this report and other issues are discussed in the present review. Because of anthropogenic release of mercury into the environment, methylmercury exposure from fish consumption is a pathway that is of increasing concern.     

双能量X线骨质密度测量仪监测小儿下肢骨延长骨矿物质的变化

目的　在儿童骨延长的患儿中 ,为了能够有效地控制骨延长的速率 ,达到骨延长的目的 ,采用双能量X线骨质密度测量仪 (dualenergyX Rayabsorptiometry ,DEXA)监测延长断端骨矿含量 (bonemineralcontent,BMC)的变化。方法　 30例患儿中有 5 0处下肢作了骨延长术 ,平均年龄10 .9岁 (5～ 17岁 ) ,引起短肢的病因不同。术后 7～ 10d开始行骨延长 ,每次延长 0 .2 5mm ,每天 4次。牵引延长期间每周扫描一次 ,拆除外固定器后每 2周扫描一次到术后 2年。DEXA扫描的分辨率是 1mm× 1mm ,扫描速度 30mm/s。比较不同延长时期中骨矿含量的变化。分析不同病因和不同外固定器之间骨矿含量变化的差别。结果　不同固定器之间骨矿含量的差别无著性意义。根据骨延长区BMC增加速率 ,将患儿分为快速组、一般组和慢速组。快速组每日BMC增加速率为 0 .3%～ 0 .6 % ,新骨生长快速 ;一般组每日BMC增加 0 .1%～ 0 .3% ,新骨中速生长 ;慢速组每日增加 <0 .1% ,新骨生成缓慢。骨矿化速率与原发病因相关。结论　DEXA能动态监测骨延长中新生骨的骨矿含量的变化 ,根据骨矿含量变化的程度 ,能够调整骨延长的速率 ,从而达到预期骨延长的目的。     

Comparing sulindac with indomethacin for closure of ductus arteriosus in preterm infants

Objectives: A prospective study comparing the efficiacy and side-effects of oral sulindac with intravenous indomethacin in clinically stable preterm infants (<1750 g) requiring non-invasive closure of haemodynamically significant patent ductus arteriosus.
Methodology: As maturity and birthweight are the two major determinants of ductal closure, infants were matched as closely as possible for these parameters. An eligible patient was first assigned to the sulindac group and a subsequent patient with similar gestational age (± 1 week) and birthweight (±100 g) to the previously recruited infant would automatically receive indomethacin. A total of eight infants were enrolled in each group.
Results: The ductus arteriosus was successfully closed in all eight infants receiving indomethacin, and in seven of eight infants receiving sulindac. No significant differences were found with regards to the ductal size between the two groups at diagnosis or on each of the consecutive days of treatment ( P >0.25). More renal adverse effects were encountered in the indomethacin group. Significant differences in changes from baseline value for urine output, plasma sodium, urea and creatinine concentrations were noted at 24, 48 and 72 h after commencement of treatment between the two groups ( P <0.05). All the parameters returned to normal or pre-treatment levels 48 h after stopping therapy. Unexpectedly, severe gastrointestinal complications were encountered in the sulindac group.
Conclusions: Sulindac is capable of promoting ductal constriction in clinically stable preterm infants without compromising the renal function. The spectrum of gastrointestinal complications observed in sulindac treated infants were similar to those described for indomethacin. The use of sulindac for ductal closure in the preterm infant should remain experimental.     

Insights from CREDENCE trial indicate an acute drop in estimated glomerular filtration rate during treatment with canagliflozin with implications for clinical practice

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MAD2 associates with the cyclosome/anaphase-promoting complex and inhibits its activity

Cell cycle progression is monitored by checkpoint mechanisms that ensure faithful duplication and accurate segregation of the genome. Defects in spindle assembly or spindle-kinetochore attachment activate the mitotic checkpoint. Once activated, this checkpoint arrests cells prior to the metaphase-anaphase transition with unsegregated chromosomes, stable cyclin B, and elevated M phase promoting factor activity. However, the mechanisms underlying this process remain obscure. Here we report that upon activation of the mitotic checkpoint, MAD2, an essential component of the mitotic checkpoint, associates with the cyclin B-ubiquitin ligase, known as the cyclosome or anaphase-promoting complex. Moreover, purified MAD2 causes a metaphase arrest in cycling Xenopus laevis egg extracts and prevents cyclin B proteolysis by blocking its ubiquitination, indicating that MAD2 functions as an inhibitor of the cyclosome. Thus, MAD2 links the mitotic checkpoint pathway to the cyclin B destruction machinery which is critical in controlling the metaphase-anaphase transition.