Mutational Analysis of the Rhodopsin Gene in Sector Retinitis Pigmentosa

Background: To determine the role of rhodopsin (RHO) gene mutations in patients with sector retinitis pigmentosa (RP) from Northern Ireland.

Design: A case series of sector RP in a tertiary ocular genetics clinic.

Participants: Four patients with sector RP were recruited from the Royal Victoria Hospital (Belfast, Northern Ireland) and Altnagelvin Hospital (Londonderry, Northern Ireland) following informed consent.

Methods: The diagnosis of sector RP was based on clinical examination, International Society for Clinical Electrophysiology of Vision (ISCEV) standard electrophysiology, and visual field analysis. DNA was extracted from peripheral blood leucocytes and the coding regions and adjacent flanking intronic sequences of the RHO gene were polymerase chain reaction (PCR) amplified and cycle sequenced.

Main Outcome Measure: Rhodopsin mutational status.

Results: A heterozygous missense mutation in RHO (c.173C?>?T) resulting in a non-conservative substitution of threonine to methionine (p. Thr58Met) was identified in one patient and was absent from 360 control individuals. This non-conservative substitution (p.Thr58Met) replaces a highly evolutionary conserved polar hydrophilic threonine residue with a non-polar hydrophobic methionine residue at position 58 near the cytoplasmic border of helix A of RHO.

Conclusions: The study identified a RHO gene mutation (p.Thr58Met) not previously reported in RP in a patient with sector RP. These findings outline the phenotypic variability associated with RHO mutations. It has been proposed that the regional effects of RHO mutations are likely to result from interplay between mutant alleles and other genetic, epigenetic and environmental factors.     

构建SD大鼠鱼藤酮中毒模型及其线粒体DNA突变的初步探索

目的构建SD大鼠鱼藤酮慢性中毒模型,同时对SD大鼠组织标本(脑组织和肝脏)进行线粒体DNA突变的初步探索,为后续实验提供线索。方法将75只雄性SD鼠随机分为三组,实验组:0.5 mg/(kg·d)鱼藤酮皮下注射4~8周,共45只;空白组:不予任何特殊处理4~8周,共15只;对照组:相同体积的二甲基亚砜(DMSO)皮下注射4~8周,共15只。其中实验组随机分为1、2、3小组,每小组15只;空白组和对照组也分别分为1、2、3小组,每小组5只。每组内相应的1、2、3小组分别在实验的第4、6、8周处死老鼠并留取相应的组织标本(中脑组织和肝脏),存储于-80℃冰箱中待用。在实验组的每个小组内分别随机选取3对组织标本,共9对;在空白组和对照组的每个小组内分别随机选取1对组织标本,共6对。随后进行组织DNA提取,采用2对引物进行长PCR并将PCR产物进行凝胶电泳分析,同时设计并利用线粒体DNA引物,用一代测序的方法进行线粒体全基因组测序。实验中观察各组大鼠的一般情况并进行行为学测试。结果1)实验组大鼠在实验中表现出较为明显的中毒症状。在体重增长速度上与对照组和空白组存在明显差异(P0.05)。在行为学上,实验组大鼠亦表现出了较为明显的行为学异常(P0.05)。2)大鼠脑以及肝脏组织的长PCR凝胶电泳结果未发现明显的异常条带;所有测序突变(缺失、插入、点突变)均为大鼠固有突变。结论 0.5 mg/(kg·d)鱼藤酮皮下注射4~8周可导致SD大鼠鱼藤酮慢性中毒。SD幼鼠的选择、鱼藤酮剂量及作用时间、一代测序等多种因素可能是导致本次实验未检测到有意义的线粒体DNA突变的原因。因此,后续实验应做相应改进。     

Phenotypic diversity of cytomegalovirus DNA polymerase gene variants observed after antiviral therapy

BackgroundCytomegalovirus UL54 DNA polymerase mutations observed in clinical specimens are of diagnostic significance if confirmed to affect antiviral drug susceptibility.ObjectivesValidate an updated recombinant phenotyping method to determine the degree of drug resistance conferred by previously uncharacterized UL54 sequence variants, in comparison with known resistance-related mutations.Study designBacterial artificial chromosome clones of viral DNA were mutagenized by recombination, transfected to produce live virus and phenotyped by standardized reporter-based yield reduction assays.ResultsSixteen recombinant viruses were constructed, representing baseline sequences, known resistance-related mutations and amino acid changes of unproven significance from clinical specimens. Phenotypes of baseline strains and known mutants were comparable to results from prior methods and helped to resolve some published inconsistencies. Mutations F412L, F412S, L545W were newly confirmed to confer ganciclovir and cidofovir resistance, while Q578H conferred ganciclovir and foscarnet resistance with borderline cidofovir resistance. Some foscarnet-resistant mutants were appreciably growth-retarded.ConclusionsResults add to known exonuclease domain mutations that confer ganciclovir-cidofovir cross-resistance, polymerase domain mutations that confer foscarnet resistance with variably decreased ganciclovir/cidofovir susceptibility, and increase the list of sequence variants with no measurable impact on drug susceptibility. The phenotypic diversity of similar UL54 genotypic variants complicates the interpretation of genotypic resistance testing. Technical improvements are facilitating the phenotyping of remaining unknown sequence variants.     

New insights into physiopathology of immunodeficiency-associated vaccine-derived poliovirus infection; systematic review of over 5 decades of data

Widespread administration of oral poliovirus vaccine (OPV) has decreased global incidence of poliomyelitis by ≈99.9%. However, the emergence of vaccine-derived polioviruses (VDPVs) is threatening polio-eradication program. Primary immunodeficiency (PID) patients are at higher risks of vaccine-associated paralytic poliomyelitis (VAPP) and prolonged excretion of immunodeficiency-associated VDPV (iVDPV).We searched Embase, Medline, Science direct, Scopus, Web of Science, and CDC and WHO databases by 30 September 2016, for all reports of iVDPV cases. Patient-level data were extracted form eligible studies. Data on immunization coverage and income-level of countries were extracted from WHO/UNICEF and the WORLD BANK databases, respectively. We assessed bivariate associations between immunological, clinical, and virological parameters, and exploited multivariable modeling to identify independent determinants of poliovirus evolution and patients’ outcomes. Study protocol was registered with PROSPERO (CRD42016052931).4329 duplicate-removed titles were screened. A total of 107 iVDPV cases were identified from 68 eligible articles. The majority of cases were from higher income countries with high polio-immunization coverage. 74 (69.81%) patients developed VAPP. Combined immunodeficiency patients showed lower rates of VAPP (p < .001) and infection clearance (p = .02), compared to humoral immunodeficiency patients. The rate of poliovirus genomic evolution was higher at early stages of replication, decreasing over time until reaching a steady state. Independent of replication duration, higher extent (p = .04) and rates (p = .03) of genome divergence contributed to a less likelihood of virus clearance. PID type (p < .001), VAPP occurrence (p = .008), and income-level of country (p = .04) independently influenced patients’ survival.With the use of OPV, new iVDPVs will emerge independent of the rate of immunization coverage. Inherent features of PIDs contribute to the clinical course of iVDPV infection and virus evolution. This finding could shed further light on poliomyelitis pathogenesis and iVDPV evolution pattern. It also has implications for public health, the polio eradication effort and the development of effective antiviral interventions.     

Clinical research in small genomically stratified patient populations

The paradigm of early drug development in cancer is shifting from ‘histology-oriented’ to ‘molecularly oriented’ clinical trials. This change can be attributed to the vast amount of tumour biology knowledge generated by large international research initiatives such as The Cancer Genome Atlas (TCGA) and the use of next generation sequencing (NGS) techniques developed in recent years. However, targeting infrequent molecular alterations entails a series of special challenges. The optimal molecular profiling method, the lack of standardised biological thresholds, inter- and intra-tumor heterogeneity, availability of enough tumour material, correct clinical trials design, attrition rate, logistics or costs are only some of the issues that need to be taken into consideration in clinical research in small genomically stratified patient populations. This article examines the most relevant challenges inherent to clinical research in these populations. Moreover, perspectives from the Academia point of view are reviewed as well as initiatives to be taken in forthcoming years.     

Twins with hereditary sensory and autonomic neuropathy type IV with preserved periodontal sensation

Turkish twin brothers affected with hereditary sensory and autonomic neuropathy type IV (HSAN IV) are reported. Their clinical findings were generally typical for HSAN IV. Interestingly they both had preserved periodontal sensation. Mutation analysis of the NTRK1 gene showed a homozygous c.2001C>T substitution in exon 15 in both twins. This base substitution is predicted to change a polar, positively charged amino acid arginine to the highly active amino acid cystein at position 654 (p.Arg654Cys). The parents were heterozygous for the mutation. This mutation has been reported previously in one Japanese and one Arab patients. The preserved periodontal sensation has not previously been reported in patients affected with HSAN IV. This preserved sensation in our patients might have been through Ruffini endings, the periodontal mechanoreceptors which have been reported to be present in TrkA knockout mice. Here we report the first twins affected with HSAN IV and the observation that periodontal sensation is not affected by mutation in NTRK1.     

芯片检测结合测序技术在遗传性耳聋产前基因筛查与诊断中的应用

目的 探讨遗传性耳聋基因检测芯片在中国孕妇人群常见遗传性耳聋基因突变位点检测中的作用,并评估其在遗传性耳聋产前诊断中的应用.方法 对3056例孕妇采集外周血并抽提DNA,采用遗传性耳聋基因芯片检测GJB2、SLC26A4、线粒体12S rRNA、GJB3等4个中国人群常见遗传性耳聋基因共9个突变热点.根据检测结果对有耳聋生育风险的夫妇提供遗传咨询与生育指导.结果 3056例孕妇中,共检测到156例携带至少一种基因突变,占总抽查人数的5.11％.其中7例为线粒体12S rRNA突变,预测后代亦为此突变携带者,需终生避免使用氨基糖苷类抗生素.149例为另外三种隐性耳聋基因突变,对其中的124例生育配偶进行了相关基因的全序列测序分析,共有20对夫妇具有遗传性耳聋生育风险.5对夫妇要求进行耳聋产前诊断,4例产前诊断结果均为相应致病基因单杂合突变或野生型,出生后听力随访均未发现异常;Ⅰ例为p.V37I/c.235delC复合杂合突变,有产生轻、中度耳聋的风险.结论 利用遗传性耳聋基因芯片进行临床耳聋基因突变产前筛查具有高度可靠性和可操作性,结合产前诊断技术能有效预防先天性耳聋患儿的出生.     

Emergence of KRAS-mutation in liver metastases after an anti-EGFR treatment in patient with colorectal cancer: Are we aware of the therapeutic impact of intratumor heterogeneity?

Tumors represent a dynamic system where the genomic plasticity permits to adapt to the perturbation induced by environmental pressures, supporting the importance of longitudinal tumor sampling strategies to deciphering the temporal acquisition of driver event that could impact treatment outcome. We describe the case of a metastatic colorectal cancer (mCRC) patient, RAS wild-type, who responded to anti-EGFR therapy and underwent liver surgery, revealing a KRAS mutations in the metastatic lesion, not detectable prior to initiation of therapy in the colonic biopsy. After liver surgery, the patient received chemotherapy alone, then underwent left colectomy and the final pathological report confirmed the KRAS wild-type status. We can speculate the existence of two distinct populations of KRAS wild-type and mutant CRC cells sharing the same genetic origin. The anti-EGFR treatment represented a selective pressure which allowed the selection of KRAS mutant subclones. The prognostic and /or predictive role of intratumor heterogeneity has not been assessed prospectively. Our case report is of clinical relevance because patients with mCRC who respond to anti-EGFR antibodies often develop resistance within several months of initiating therapy, thus outlining the importance to better ascertain the molecular landscape of tumors to design better therapeutic strategies.