CottonGVD: A Comprehensive Genomic Variation Database for Cultivated Cottons

Cultivated cottons are the most important economic crop, which produce natural fiber for the textile industry. In recent years, the genetic basis of several essential traits for cultivated cottons has been gradually elucidated by decoding their genomic variations. Although an abundance of resequencing data is available in public, there is still a lack of a comprehensive tool to exhibit the results of genomic variations and genome-wide association study (GWAS). To assist cotton researchers in utilizing these data efficiently and conveniently, we constructed the cotton genomic variation database (CottonGVD; http://120.78.174.209/ or http://db.cngb.org/cottonGVD). This database contains the published genomic information of three cultivated cotton species, the corresponding population variations (SNP and InDel markers), and the visualized results of GWAS for major traits. Various built-in genomic tools help users retrieve, browse, and query the variations conveniently. The database also provides interactive maps (e.g., Manhattan map, scatter plot, heatmap, and linkage disequilibrium block) to exhibit GWAS and expression GWAS results. Cotton researchers could easily focus on phenotype-associated loci visualization, and they are interested in and screen for candidate genes. Moreover, CottonGVD will continue to update by adding more data and functions.

Personalized reference intervals – statistical approaches and considerations

For many measurands, physicians depend on population-based reference intervals (popRI), when assessing laboratory test results. The availability of personalized reference intervals (prRI) may provide a means to improve the interpretation of laboratory test results for an individual. prRI can be calculated using estimates of biological and analytical variation and previous test results obtained in a steady-state situation. In this study, we aim to outline statistical approaches and considerations required when establishing and implementing prRI in clinical practice. Data quality assessment, including analysis for outliers and trends, is required prior to using previous test results to estimate the homeostatic set point. To calculate the prRI limits, two different statistical models based on ‘prediction intervals’ can be applied. The first model utilizes estimates of ‘within-person biological variation’ which are based on an individual’s own data. This model requires a minimum of five previous test results to generate the prRI. The second model is based on estimates of ‘within-subject biological variation’, which represents an average estimate for a population and can be found, for most measurands, in the EFLM Biological Variation Database. This model can be applied also when there are lower numbers of previous test results available. The prRI offers physicians the opportunity to improve interpretation of individuals’ test results, though studies are required to demonstrate if using prRI leads to better clinical outcomes. We recommend that both popRIs and prRIs are included in laboratory reports to aid in evaluating laboratory test results in the follow-up of patients.

In Silico Analysis of Pathogenic CRB1 Single Nucleotide Variants and Their Amenability to Base Editing as a Potential Lead for Therapeutic Intervention

Mutations in the Crumbs homolog 1 (CRB1) gene cause both autosomal recessive retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). Since three separate CRB1 isoforms are expressed at meaningful levels in the human retina, base editing shows promise as a therapeutic approach. This retrospective analysis aims to summarise the reported pathogenic CRB1 variants and investigate their amenability to treatment with currently available DNA base editors. Pathogenic single nucleotide variants (SNVs) were extracted from the Leiden open-source variation database (LOVD) and ClinVar database and coded by mutational consequence. They were then analyzed for their amenability to currently available DNA base editors and available PAM sites from a selection of different Cas proteins. Of a total of 1115 unique CRB1 variants, 69% were classified as pathogenic SNVs. Of these, 62% were amenable to currently available DNA BEs. Adenine base editors (ABEs) alone have the potential of targeting 34% of pathogenic SNVs; 19% were amenable to a CBE while GBEs could target an additional 9%. Of the pathogenic SNVs targetable with a DNA BE, 87% had a PAM site for a Cas protein. Of the 33 most frequently reported pathogenic SNVs, 70% were targetable with a base editor. The most common pathogenic variant was c.2843G>A, p.Cys948Arg, which is targetable with an ABE. Since 62% of pathogenic CRB1 SNVs are amenable to correction with a base editor and 87% of these mutations had a suitable PAM site, gene editing represents a promising therapeutic avenue for CRB1-associated retinal degenerations.

Total lab automation: sample stability of clinical chemistry parameters in an automated storage and retrieval module

Objectives Automated storage and retrieval modules (SRM), as part of total lab automation (TLA) systems, offer tremendous practical and economic benefits. In contrast to manual storage systems, SRMs indicate continuous motion of samples and may leave samples prone to temperature fluctuations. This study investigates analyte stability in serum and heparin plasma within an automated storage module. Methods The stability of 28 common biochemistry analytes was investigated using 57 freshly obtained routine serum samples and 42 lithium-heparin plasma samples. Following baseline measurement, samples were stored at 2–8 °C in the automated SRM of the Accelerator a3600 TLA and reanalyzed at fixed time points (2, 4, 8, 12, 24, 48 and 72 h) on the Abbott Architect c16000 chemistry analyzer. The concentration at each time point was expressed as %-difference to the baseline value and mean results were compared to the criteria for desirable bias derived from the biological variation database. Results Nine of the analytes exceeded the bias criterion within 72 h after initial measurement in either serum samples, plasma samples or both. Lithium-heparin plasma samples showed increasing values for phosphor, potassium and lactate dehydrogenase (LDH), which were only considered stable for respectively 24, 12 and 4 h, glucose was considered stable for 8 h. Electrolyte concentrations and LDH activity significantly increased in serum samples beyond 48 h. Bicarbonate should not be performed as add-on test at all. Conclusions The presented data indicate that the conditions within an SRM have no clinical impact on sample stability and allow stable measurement of routine analytes within 72 h, comparable to manual storage facilities.

International initiative for a curated SDHB variant database improving the diagnosis of hereditary paraganglioma and pheochromocytoma

BackgroundSDHB is one of the major genes predisposing to paraganglioma/pheochromocytoma (PPGL). Identifying pathogenic SDHB variants in patients with PPGL is essential to the management of patients and relatives due to the increased risk of recurrences, metastases and the emergence of non-PPGL tumours. In this context, the ‘NGS and PPGL (NGSnPPGL) Study Group’ initiated an international effort to collect, annotate and classify SDHB variants and to provide an accurate, expert-curated and freely available SDHB variant database.MethodsA total of 223 distinct SDHB variants from 737 patients were collected worldwide. Using multiple criteria, each variant was first classified according to a 5-tier grouping based on American College of Medical Genetics and NGSnPPGL standardised recommendations and was then manually reviewed by a panel of experts in the field.ResultsThis multistep process resulted in 23 benign/likely benign, 149 pathogenic/likely pathogenic variants and 51 variants of unknown significance (VUS). Expert curation reduced by half the number of variants initially classified as VUS. Variant classifications are publicly accessible via the Leiden Open Variation Database system (https://databases.lovd.nl/shared/genes/SDHB).ConclusionThis international initiative by a panel of experts allowed us to establish a consensus classification for 223 SDHB variants that should be used as a routine tool by geneticists in charge of PPGL laboratory diagnosis. This accurate classification of SDHB genetic variants will help to clarify the diagnosis of hereditary PPGL and to improve the clinical care of patients and relatives with PPGL.

The effect of drone transport on the stability of biochemical, coagulation and hematological parameters in healthy individuals

Objectives Transport of blood tubes is mainly by car or pneumatic transport. The transportation of blood tubes by drones is a novel approach for rapid transportation of blood tubes over long distances. However, limited data on the stability of biochemical, coagulation and hematological parameters is available after transport of blood tubes by drone. Methods To investigate the effect of drone transport on the stability of blood parameters, four test flights were performed. Blood was drawn from 20 healthy individuals and 39 of the most frequently measured blood parameters were compared between 4 groups; immediate measurement (control), late measurement, transport by car and transport by drone. Total Allowable Error (TAE) of the EFLM Biological Variation Database was used to determine the clinical relevance of significant differences. Results The majority of blood parameters were not affected by drone transport. Eight of the measured parameters showed significant differences between all the groups; glucose, phosphate, potassium, chloride, hemoglobin, platelet count, APTT and Lactate dehydrogenase (LD). A clinically relevant increase for LD after transport and a decrease for glucose values in time and after transport compared with the control group was shown. Conclusions Transportation of blood tubes from healthy individuals by drones has a limited clinically relevant effect. From the 39 investigated blood parameters only LD and glucose showed a clinically relevant effect.

PRPH2 mutation update: In silico assessment of 245 reported and 7 novel variants in patients with retinal disease

Mutations in PRPH2, encoding peripherin-2, are associated with the development of a wide variety of inherited retinal diseases (IRDs). To determine the causality of the many PRPH2 variants that have been discovered over the last decades, we surveyed all published PRPH2 variants up to July 2020, describing 720 index patients that in total carried 245 unique variants. In addition, we identified seven novel PRPH2 variants in eight additional index patients. The pathogenicity of all variants was determined using the ACMG guidelines. With this, 107 variants were classified as pathogenic, 92 as likely pathogenic, one as benign, and two as likely benign. The remaining 50 variants were classified as variants of uncertain significance. Interestingly, of the in total 252 PRPH2 variants, more than half (n=137) were missense variants. All variants were uploaded into the Leiden Open source Variation Database. Our study underscores the need of experimental assays for variants of unknown significance to improve pathogenicity classification, which is needed to better understand genotype-phenotype correlations, and in the long-term, hopefully also support the development of therapeutic strategies for patients with PRPH2-associated IRD.

Allele frequency of variants reported to cause adenine phosphoribosyltransferase deficiency

Adenine phosphoribosyltransferase deficiency is a rare, autosomal recessive disorder of purine metabolism that causes nephrolithiasis and progressive chronic kidney disease. The small number of reported cases indicates an extremely low prevalence, although it has been suggested that missed diagnoses may play a role. We assessed the prevalence of APRT deficiency based on the frequency of causally-related APRT sequence variants in a diverse set of large genomic databases. A thorough search was carried out for all APRT variants that have been confirmed as pathogenic under recessive mode of inheritance, and the frequency of the identified variants examined in six population genomic databases: the deCODE genetics database, the UK Biobank, the 100,000 Genomes Project, the Genome Aggregation Database, the Human Genetic Variation Database and the Korean Variant Archive. The estimated frequency of homozygous genotypes was calculated using the Hardy-Weinberg equation. Sixty-two pathogenic APRT variants were identified, including six novel variants. Most common were the missense variants c.407T>C (p.(Met136Thr)) in Japan and c.194A>T (p.(Asp65Val)) in Iceland, as well as the splice-site variant c.400 + 2dup (p.(Ala108Glufs*3)) in the European population. Twenty-nine variants were detected in at least one of the six genomic databases. The highest cumulative minor allele frequency (cMAF) of pathogenic variants outside of Japan and Iceland was observed in the Irish population (0.2%), though no APRT deficiency cases have been reported in Ireland. The large number of cases in Japan and Iceland is consistent with a founder effect in these populations. There is no evidence for widespread underdiagnosis based on the current analysis.