Two cases of misinterpretation of molecular results in incontinentia pigmenti,and a PCR-based method to discriminate NEMO/IKKgamma dene deletion

Familial incontinentia pigmenti (IP) is a rare X-linked dominant disorder that affects ectodermal tissues. Over 90% of IP carrier females have a recurrent genomic deletion of exons 4-10 of the NEMO (IKBKG-IKKgamma) gene, which encodes a regulatory component of the IkB kinase complex, required to activate the NF-kB pathway. In IP, mutations in NEMOlead to the complete loss of NF-kB activation creating a susceptibility to cellular apoptosis in response to TNF-alpha. This condition is lethal for males during embryogenesis while females, who are mosaic as a result of X-inactivation, can survive. Recently, a second nonfunctional copy of the gene, DeltaNEMO, was identified, opposite in direction to NEMO in a 35.5-kb duplicated sequence tract. PCR-based detection of the NEMO deletion is diagnostic for IP disease. However, we present instances in which ex 4-10 DeltaNEMO pseudogene deletion occurs in unaffected parents of two females with clinically characteristic IP. These were missed by the currently standard PCR-based method, but can be easily discriminated by a new PCR-based test reported here that permits unambiguous molecular diagnosis and proper familial genetic counseling for IP.     

A rapid method for the inactivation of virus infectivity prior to assay for interferons

Virus infectivity in samples of culture medium or suspensions of animal tissue which are required for interferon assay can be rapidly and conveniently inactivated by overnight incubation with beta-propiolactone (BPL). As BPL hydrolyses spontaneously samples can be assayed with no further treatment. BPL does not affect the interfering activity of alpha, beta or gamma mouse interferons.     

The late component of L-type calcium current during guinea-pig cardiac action potentials and its contribution to contraction

 L-Type Ca²⁺ current (I _Ca,L) elicited during the action potential (AP) of guinea-pig ventricular myocytes exhibits an early and a late component. The whole-cell patch-clamp technique was used to characterize the process regulating the late I _Ca,L component and to assess its contribution to excitation-contraction coupling. A stepwise decrease in repolarization rate of AP-like voltage-clamp pulses led to an exponential increase in Ca²⁺ charge carried by I _Ca,L. This saturation behaviour was significantly reduced or absent when Ba²⁺ or monovalent cations were used as charge carriers, which suggests that the late component of I _Ca,L is controlled mainly by Ca²⁺-dependent processes. Simultaneously recording I _Ca,L and zero-load shortening or the internal Ca²⁺ concentration (fura-2) revealed that Ca²⁺ carried by the late component of I _Ca,L markedly contributes to the Ca²⁺ content of the sarcoplasmic reticulum (SR). Reducing the charge transfer by late I _Ca,L during a series of AP-like conditioning clamp pulses by 48% reduced the shortening amplitude during a subsequent test stimulation by 56%. This relationship was absent during long rectangular depolarizing conditioning clamps, during which Na⁺/Ca²⁺ exchange increased its influence on SR Ca²⁺ loading. The late component of I _Ca,L developed only a minor direct influence on the simultaneous cell shortening. Thus, the main contribution of the late I _Ca,L component is to supply Ca²⁺ for SR loading. Received: 5 November 1997 / Received after revision: 12 June 1998 / Accepted: 15 June 1998     

Magnesium restores high K-induced inactivation of the fast Na channel in guinea pig ventricular muscle

The effect of extracellular magnesium concentration (Mg_o) on the upstroke of the action potential was studied in guinea pig ventricular muscle under various K⁺ concentrations (2.7–19mM). Increased Mg_o shifted the steady state inactivation curve of the fast Na channel in the depolarizing direction and this effect was concentration-dependent (0–20mM). Such an effect could explain the Mg-induced increase in maximum rate of rise of the action potential which Späh and Fleckenstein (1979) proposed to be due to a Mg channel.     

Feeding suppression induced by intra-ventricle III infusion of 1,5-anhydroglucitol

1,5-Anhydroglucitol (1-DG) has been known as an antimetabolic glucose analogue. Using gas chromatography, 1-DG was found to be physiologically present in rat serum. In order to investigate its direct and long-term effects on feeding, 1-DG was infused during the light period into the rat third ventricle in doses of 3.0, 6.0 and 12.0 mumol/rat. Its effects were then compared to those of similarly applied 2-deoxy-D-glucose (2-DG). Following initial hyperphagia, both of these glucose-analogues produced suppressive effects on feeding during the subsequent day throughout the light and dark periods. On the third day after 2-DG injection reduction of feeding did not recover completely to the pretreatment baseline levels, but it did recover after 1-DG. Both 1-DG and 2-DG caused linear dose-related hypophagia, with the slope for 1-DG being about half of that for 2-DG. It is suggested that the delayed hypophagia which followed the initial hyperphagia produced by deoxyglucose was a result of sustained inactivation of the Na-pump due to intracellular ATP deficiency caused by accumulation of deoxy-glucose-6-phosphate.     

Grandparental genotyping enhances exome variant interpretation

Trio exome sequencing is a powerful tool in the molecular investigation of monogenic disorders and provides an incremental diagnostic yield over proband‐only sequencing, mainly due to the rapid identification of de novo disease‐causing variants. However, heterozygous variants inherited from unaffected parents may be inadvertently dismissed, although multiple explanations are available for such scenarios including mosaicism in the parent, incomplete penetrance, imprinting, or skewed X‐inactivation. We report three probands, in which a pathogenic or likely pathogenic variant was identified upon exome sequencing, yet was inherited from an unaffected parent. Segregation of the variants (in NOTCH1, PHF6, and SOX10) in the grandparent generation revealed that the variant was de novo in each case. Additionally, one proband had skewed X‐inactivation. We discuss the possible genetic mechanism in each case, and urge caution in data interpretation of exome sequencing data. We illustrate the utility of expanding segregation studies to the grandparent generation and demonstrate the impact on exome interpretation strategies, by showing that objective genotype data can overcome subjective parental report of lack of symptoms.     

Reversible and irreversible inhibition of rat brain muscarinic receptors is related to different substitutions on bisquaternary pyridinium oximes

The role of the functional substituents on the pyridinium ring of bisquaternary pyridinium compounds, mostly oximes, in exerting reversible and irreversible inhibition of binding of [³H]-N-methyl-4-piperidyl benzilate ([³H]-4NMPB) to rat brain stem muscarinic receptors was studied. The drugs tested, i.e. HGG-42, HGG-12, HGG-52, HI-6, obidoxim, SAD-128 and TMB-4, could reversibly inhibit binding of [³H]-4NMPB, with the highest potency (K_I=1.7–6 M) exhibited by analogs possessing hydrophobic substituents at position 3 or 4 of the pyridinium ring. Bisquaternary drugs possessing an oxime moiety at position 2, but not at position 4 of the pyridinium ring, could also induce about 30% reduction of maximal binding capacity (B_max) (loss of muscarinic receptors) in addition to their reversible effect. Thus the structural correlates of the reversible and the irreversible effects of these drugs are different.     

2015—2020年四川省手足口病流行病学特征分析

目的 分析2015—2020年四川省手足口病流行病学及病原学特征,为手足口病的防控提供科学依据。 方法 利用描述流行病学方法对2015—2020年四川省手足口病监测资料进行统计分析。 结果 2015—2020年四川省累计报告手足口病520 147例,重症1 759例,死亡38例,2015—2020年四川省手足口病发病率呈波动状,年均发病率为104.82/10万,2015—2017和2019年呈现明显的双峰(4—7月和10—12月),2018、2020年呈单峰,发病高峰分别在7—11月和10—12月;年龄以 5 岁及以下儿童为主,男女性别比为1.38∶1;发病率居前五位的地区是成都市(221.25/10万)、眉山市(145.51/10万)、德阳市(115.52/10万)、雅安市(108.02/10万)和遂宁市(100.56/10);共报告实验室确诊病例44 410例,其中其他肠道病毒、CoxA16、EV71分别占65.53%、22.35%、12.12% 。 结论 2015—2020年四川省手足口病发病水平呈上升趋势,防控压力逐渐加大。四川省手足口病发病以5岁以下儿童为主,成都及周边,川东北发病率较高,优势病原分型构成发生变化,其他肠道病毒比重逐渐提高,病原学监测工作需要调整。     

2018—2020年徐州市食源性疾病监测分析

目的 分析徐州市2018—2020年食源性疾病哨点医院主动监测结果,了解该地区食源性疾病流行特征。方法 收集2018—2020年徐州市食源性疾病哨点医院监测的病例信息,并对部分病例的粪便样本进行病原学检测。结果 3年共监测食源性疾病病例7 548例,其中25～45岁年龄组占比最高(26.81%);6—9月为发病高峰;肉与肉制品(20.72%)为主要的可疑暴露食品;可疑食物进食场所主要为家庭(80.49%);农民(26.75%)和散居儿童(24.95%)病例构成比较高。共采集1 835份腹泻病例粪便样本,其中诺如病毒检出率最高为(4.69%)。结论 徐州市食源性疾病高发期为6—9月,具有明显的季节性,好发于家庭,肉与肉制品为主要暴露食品,感染患者集中在>25～45岁年龄组,诺如病毒感染率较高。     

全国细菌耐药监测网2014—2019年尿标本细菌耐药监测报告

目的探讨全国尿标本分离细菌菌种分布及耐药变迁。方法按照全国细菌耐药监测网(CARSS)技术方案,应用WHONET5.6软件对2014—2019年所有CARSS成员单位上报的尿标本分离细菌及药敏结果数据进行分析。结果男性患者尿标本分离细菌居前5位者分别为大肠埃希菌(33.1%~34.6%)、粪肠球菌(9.2%~10.2%)、肺炎克雷伯菌(9.0%~9.4%)、屎肠球菌(7.8%~10.2%)和铜绿假单胞菌(5.6%~6.9%),女性患者尿标本分离细菌居前5位者分别为大肠埃希菌(57.0%~57.4%)、肺炎克雷伯菌(7.5%~8.3%)、屎肠球菌(6.8%~8.7%)、粪肠球菌(5.5%~6.0%)和奇异变形杆菌(3.3%~3.5%)。男性和女性患者尿标本分离粪肠球菌对氨苄西林和呋喃妥因耐药率分别<12%和7%,对万古霉素耐药率<3%;屎肠球菌对氨苄西林、左氧氟沙星耐药率均为90%左右,对万古霉素耐药率<4%。大肠埃希菌对头孢曲松耐药率>47%,对头孢哌酮/舒巴坦、哌拉西林/他唑巴坦、呋喃妥因耐药率≤8%,对于β-内酰胺类耐药率男性比女性高,其中头孢曲松的耐药率高12个百分点左右。男性患者分离肺炎克雷伯菌对头孢曲松耐药率为58%左右,女性患者耐药率为45%左右。男性和女性患者尿标本分离铜绿假单胞菌对头孢哌酮/舒巴坦和哌拉西林/他唑巴坦的耐药率均<14%,对碳青霉烯类耐药率为15%左右。鲍曼不动杆菌对头孢哌酮/舒巴坦和米诺环素耐药率分别<27%和22%,对碳青霉烯类耐药率,男性为31.7%~47.7%,女性为26.5%~41.2%。结论尿标本分离细菌在不同性别构成上有所不同,且部分肠杆菌目细菌耐药率不同性别间也有一定差异,不同年度间部分细菌的耐药率也有一定变化。尿标本分离细菌的耐药监测,可为尿路感染抗菌药物合理应用提供参考数据。