Integrating Functional Analysis in the Next-Generation Sequencing Diagnostic Pipeline of RASopathies.

RASopathies are a group of heterogeneous conditions caused by germline mutations in RAS/MAPK signalling pathway genes. With next-generation sequencing (NGS), sequencing capacity is no longer a limitation to molecular diagnosis. Instead, the rising number of variants of unknown significance (VUSs) poses challenges to clinical interpretation and genetic counselling. We investigated the potential of an integrated pipeline combining NGS and the functional assessment of variants for the diagnosis of RASopathies. We included 63 Chinese patients with RASopathies that had previously tested negative for PTPN11 and HRAS mutations. In these patients, we performed a genetic analysis of genes associated with RASopathies using a multigene NGS panel and Sanger sequencing. For the VUSs, we evaluated evidence from genetic, bioinformatic and functional data. Twenty disease-causing mutations were identified in the 63 patients, providing a primary diagnostic yield of 31.7%. Four VUSs were identified in five patients. The functional assessment supported the pathogenicity of the RAF1 and RIT1 VUSs, while the significance of two VUSs in A2ML1 remained unclear. In summary, functional analysis improved the diagnostic yield from 31.7% to 36.5%. Although technically demanding and time-consuming, a functional genetic diagnostic analysis can ease the clinical translation of these findings to aid bedside interpretation.

The Effect of Individualized Versus Standardized Parenteral Nutrition on Body Weight in Very Preterm Infants.

BACKGROUND: This study was designed to evaluate whether standardizing total parenteral nutrition (TPN) is at least non-inferior to TPN with individualized composition in premature infants with a gestational age (GA) < 32 weeks. METHODS: In this retrospective cohort study, all preterm born in or transferred to Maxima Medical Center (MMC) within 24 hours after birth with a GA < 32 weeks were included. The individualized group (2011) was compared to the partially standardized group (2012) and completely standardized group (2014) consequently. The primary endpoint was difference in growth. Secondary endpoints included differences in electrolyte concentrations. RESULTS: A total of 299 preterm were included in this study. When comparing weight gain, the infants in the (partially) standardized group demonstrated significantly (P < 0.05) less weight loss during the first days of life and grew faster subsequently in the following days than the individualized TPN regimen. Furthermore, significant differences in abnormal serum sodium, chloride, calcium, creatinine, magnesium and triglycerides values were demonstrated. CONCLUSION: TPN with a (partially) standardized composition revealed to be at least non-inferior to TPN with an individualized composition.

William Boog Leishman: parasitologist and politician.

Famous for the discovery of the parasite, Leishmania, named after him, and the invention of Leishman's stain, William Boog Leishman should perhaps be better known for his work in military and public health, particularly the prevention of typhoid. Leishman was a Medical Officer in the British Army from 1887 until his death in 1926. His early research was on diseases affecting troops posted to stations within the British Empire. He saw cases of Leishmaniasis while stationed in India, and was able to identify the causative organism from his detailed records of his observations. Leishman's most important contribution to public health, however, was his work with typhoid, a major cause of morbidity and mortality in the army. Leishman planned experiments and the collection of data to demonstrate the efficacy of anti-typhoid inoculation and, using his considerable political skills, advocated the adoption of the vaccine. He planned for the inoculation of troops in an emergency so, when war broke out in 1914, the vaccine was available to save thousands of lives. Leishman's colleagues and mentors included Ronald Ross and Almroth Wright. Leishman was less outspoken than either Ross or Wright; this paper shows how the different contributions of the three men overlapped.

Versatile DNA damage detection by the global genome nucleotide excision repair protein XPC.

To investigate how the nucleotide excision repair initiator XPC locates DNA damage in mammalian cell nuclei we analyzed the dynamics of GFP-tagged XPC. Photobleaching experiments showed that XPC constantly associates with and dissociates from chromatin in the absence of DNA damage. DNA-damaging agents retard the mobility of XPC, and UV damage has the most pronounced effect on the mobility of XPC-GFP. XPC exhibited a surprising distinct dynamic behavior and subnuclear distribution compared with other NER factors. Moreover, we uncovered a novel regulatory mechanism for XPC. Under unchallenged conditions, XPC is continuously exported from and imported into the nucleus, which is impeded when NER lesions are present. XPC is omnipresent in the nucleus, allowing a quick response to genotoxic stress. To avoid excessive DNA probing by the low specificity of the protein, the steady-state level in the nucleus is controlled by nucleus-cytoplasm shuttling, allowing temporally higher concentrations of XPC in the nucleus under genotoxic stress conditions.

Malarial antigens on infected erythrocytes.

Absorption experiments with washed membranes derived from erythrocytes infected with schizonts of Plasmodium knowlesi (PE) showed that the antigen responsible for agglutination of PE in SICA tests is firmly bound to membranes. Further experiments demonstrated that the number of antibody binding sites at the surface of parasitized erythrocytes can be measured using iodinated globulins. The conditions under which this could be achieved have been defined and a preliminary estimate of the number of high affinity surface antigen sites (greater than or equal to 10(4) per cell) has been made. Suggestions are given for further work to obtain a more accurate value.

Reduced lectin binding on erythrocytes of monkeys infected with malaria.

The number of binding sites for Axinella lectin (specific for D-galactose) on monkey erythrocytes containing schizonts of Plasmodium knowlesi was found to be reduced by about a third, from approximately 11 X 10(6) sites on normal erythrocytes to approximately 8 X 10(6) sites on parasitized erythrocytes. In contrast, only minor differences were found in the amounts of iodinatable external proteins. The results indicate that there is a general reduction of accessible sugars to which lectins may bind on parasitized erythrocytes.