Diagnostic Sequencing Research Articles

New variant strain of Streptococcus canis with Lancefield group C isolated from canine otitis externa

Every basic course in microbiology teaches us, Streptococcus canis always tests positive for Lancefield group G. Surprisingly, we identified a strain of S. canis with Lancefield group C, cultured from a dog with otitis externa after lateral ear canal resection. Whole genome sequencing data and analysis points towards a horizontal gene transfer event between S. canis and S. dysgalactiae. Although these species are closely related, gene transfer in this region of the genome of S. canis has not been described before. The value of technologies as MALDI-TOF MS and sequencing in microbiological diagnostics will grow as more diverse streptococci arise that do not always conform anymore to the classical Lancefield group typing.

Diagnostic strategies for muscular dystrophies: a cross-sectional study

Background: Muscular dystrophies are a wide heterogeneity group of neuromuscular diseases that very often constitutes a challenge for clinicians to perform an adequate diagnosis. Many patients remain underdiagnosed o misdiagnosed consequently affecting their prognosis and quality of life. Therefore, we aimed to establish clinical and molecular characteristics of patients with increased CPK levels and muscular dystrophies in our region to facilitate diagnosis and follow-up on patients with suspected muscular dystrophies. Methods: A cross-sectional study was made using a retrospective search of patients attended in Comfamiliar Risaralda between 2010 and 2021. The study included patients from both genders and all ages who presented with a diagnosis of polymyositis, myoclonus, myopathy, and muscular dystrophy between 2010 and 2022 in Comfamiliar Risaralda. Patients with CPK levels lower than 500 U/L were excluded. Results: A database analysis was carried out from 2010 to 2022 of 5219 patients treated in a fourth-level care institution in the Eje Cafetero region, finding 221 patients filtered by a diagnosis of myopathy, myoclonus, polymyositis, and dystrophy. We found a combined prevalence of all muscular dystrophies of 4.2 per 100.000 habitants, Duchenne muscular dystrophy of 0.6 per 100.000 habitants, limb-girdle muscular dystrophy of 0.6 per 100.000 habitants, facioscapulohumeral dystrophy of 0.5 per 100.000 habitants, Bethem dystrophy, type 2 Emery Dreifuss muscular dystrophy and merosin-deficient muscular dystrophy of 0.1 per 100.000 habitants. A diagnostic sequence was elaborated from clinical and paraclinical features found in our patients. Conclusions: Although muscular dystrophies consist of a heterogeneous group of neuromuscular diseases, there are still clinical and paraclinical features that can help physicians to detect any particular case and perform a good approach and follow-up. Our diagnostic sequence will facilitate physicians to determine any particular muscular dystrophy.

Morphological and molecular characterization, including parasitic and pathogenic studies of a new spherical cyst nematode species, Globodera vulgaris n. sp. (Nematoda: Heteroderidae), associated with potatoes in China.

In this study, a new spherical cyst nematode belonging to the genus Globodera, herein described as Globodera vulgaris n. sp., was extracted from the roots and rhizosphere soil of potato and circumjacent weeds belonging to different families in three provinces in southwest China. The new species was characterized by 8-24 ridges between the anus and fenestra and an average Granek's ratio of 2.8-3.8 in cysts, a head with 3-4 annules and dorsal knob anteriorly projected and ventral knobs round or anteriorly projected in second stage juveniles, and a head with 3-5 annules, spicule short and average length less than 30.0 μm, with a developed velum in males. Pathogenicity tests showed that G. vulgaris n. sp. infected potato but did not damage or affect the potato yield compared to control, and it parasitized tomato with a low reproduction rate (RF < 1) while did not parasitize tobacco (RF = 0). The new species was close to G. rostochiensis based on molecular diagnostic marker sequences and constructed phylogenetic analysis, that is internal transcribed spacers of ribosomal DNA (ITS), large-subunit rDNA (LSU) and small-subunit rDNA (SSU). However, the new species exhibited differences from G. rostochiensis in terms of morphological characteristics, a wide host preference, lack of damage to hosts, and egg hatching rate induced by hatching factors.

Reclassification of the Etiology of Infant Mortality With Whole-Genome Sequencing

ABSTRACT Infant mortality rates are quite high even in developed countries; in the United States, infant deaths occur approximately 1 in 200 live births. The leading cause of infant mortality is congenital malformation or chromosomal abnormality, which cause roughly 20% of infant deaths, followed by other causes such as pregnancy complications, preterm birth or low birth weight, and sudden infant death syndrome. Improvements in treatments for certain conditions such as pregnancy complications or prematurity have previously resulted in a reduction in mortality due to these causes. Although the same may be true of congenital malformations or chromosomal abnormalities, the relationship between these factors and infant mortality has not been closely examined. Previous studies have largely been retrospective in nature and varied greatly among method and records used, perpetuating inaccuracies and imprecision. This study aimed to clarify and add to previous literature by examining the relationship between single-locus genetic disease (Mendelian genetic disease) and overall infant mortality. This study applied diagnostic whole-genome sequencing (WGS) to infants in intensive care to diagnose diseases of unknown etiology in a single hospital system between 2015 and 2020. The study was retrospective and included review of medical records and death certificates, as well as results from WGS. Where possible, WGS information was obtained on living infants and their parents, and where that was not possible, it was obtained postmortem. Death certificates and causes of death were also examined and compared with WGS results. In the final sample, 112 infants who died underwent WGS either premortem or postmortem. WGS identified genetic diseases in 46 (41%) of these infants. Of the genetic diseases identified, 83% had previous evidence supporting an association with childhood mortality, and evidence was gathered from previous literature that could improve outcomes for 30%. Death certificate comparisons showed that of 45 infants where WGS showed underlying genetic disease, 62% were not reported on the certificate or attributed to the cause of death. In addition to the 112 infants who died, the study population included 434 infants who survived and also had WGS performed. No significant differences were found between the groups in sex, race, or ethnicity. Types of genetic disease varied between infants who died and those who survived, with only 4% being found in both. Certain diseases were more likely to occur in infants who survived, and others were more likely to occur in infants who died. Specific etiology had some prognostic value, positively predicting either death or survival. These results indicate that first, after examination of WGS in comparison with death certificates, genetic diseases are chronically underreported. In addition, WGS showed that etiology of leading causes of infant mortality could be fundamentally different than previously thought. WGS is also not currently a widespread treatment, and although steps are being taken to make it more widely accessible, there are still many changes that need to be made. This study is limited by its small size, and may have limited generalizability to other patient populations. Future research should characterize the optimal breadth of WGS testing and implementation, as well as the potential for WGS to reduce infant mortality by identifying treatable disorders.

Incidence and predictors of anthracycline-related left ventricular dysfunction in acute myeloid leukemia

IntroductionAnthracycline-related left ventricular dysfunction (ARLVD) is a concern in patients with acute myeloid leukemia (AML) undergoing anthracyclinecontaining induction chemotherapy. However, the incidence of ARLVD in the modern era of routine pretreatment left ventricular ejection fraction (LVEF) echocardiographic assessment, as well as the clinical and genetic predictors of ARLVD are not well understood. MethodsConsecutive adult patients with AML receiving anthracycline-containing induction chemotherapy at the Dana-Farber Cancer Institute from 2014 to 2022 were studied. Inclusion criteria included availability of a pre and post chemotherapy echocardiogram to assess the LVEF, pre-treatment LVEF > 50 %, as well as comprehensive diagnostic next generation sequencing assessing for the presence of myeloid mutations. The primary endpoint was the incidence of ARLVD defined as LVEF < 50 % post-induction. ResultsOut of 419 patients meeting inclusion criteria, 34 (8%) patients developed ARLVD. Among the 122/419 patients who did not undergo planned allogeneic stem cell transplantation (allo-SCT), ARLVD was the deciding factor for ineligibility in 4 patients (1%). Baseline cardiovascular comorbidities (hypertension, diabetes mellitus, hyperlipidemia, smoking and coronary artery disease) and cumulative anthracycline dose were not predictive of post-induction ARLVD. However, the presence of a JAK2 mutation (but not other myeloid mutations) was associated with an increased risk of ARLVD in multivariable analysis (OR 8.34, 95 % CI 1.55–39.3, p = 0.007). DiscussionIn a group of AML patients with normal LVEF prior to anthracycline-containing induction chemotherapy, ARLVD was infrequent and did not commonly preclude post-remission allo-SCT consolidation. Genetic predictors of ARLVD require further investigation in a larger patient cohort.

MRI sequence optimisation methods to identify cranial nerve course for radiotherapy planning.

Magnetic resonance imaging (MRI) is being increasingly used to improve radiation therapy planning by allowing visualisation of organs at risk that cannot be well-defined on computed tomography (CT). Diagnostic sequences are increasingly being adapted for radiation therapy planning, such as the use of heavily T2-weighted 3D SPACE (Sampling Perfection with Application optimised Contrasts using different flip angle Evolution) sequence for cranial nerve identification in head and neck tumour treatment planning. A 3D isotropic T2 SPACE sequence used for cranial nerve identification was adapted for radiation therapy purposes. Distortion was minimised using a spin-echo-based sequence, 3D distortion correction, isocentre scanning and an increased readout bandwidth. Radiation therapy positioning was accounted for by utilising two small flex, 4-channel coils. The protocol was validated for cranial nerve identification in clinical applications and distortion minimisation using an MRI QA phantom. Normal anatomy of the cranial nerves CI-CIX, were presented, along with a selection of clinical applications and abnormal anatomy. The usefulness of cranial nerve identification is discussed for several case studies, particularly in proximity to tumours extending into the base of skull region. In-house testing validated that higher bandwidths of 600 Hz resulted in minimal displacement well below 1 mm. The use of MRI for radiation therapy planning allows for greater individualisation and prediction of patient outcomes. Dose reduction to cranial nerves can decrease late side effects such as cranial neuropathy. In addition to current applications, future directions include further applications of this technology for radiation therapy treatments.

Assessing Diversity in Newborn Genomic Sequencing Research Recruitment: Race/Ethnicity and Primary Spoken Language Variation in Eligibility, Enrollment, and Reasons for Declining

Diagnostic genomic research has the potential to directly benefit participants. This study sought to identify barriers to equitable enrollment of acutely ill newborns into a diagnostic genomic sequencing research study. We reviewed the 16-month recruitment process of a diagnostic genomic research study enrolling newborns admitted to the neonatal intensive care unit at a regional pediatric hospital that primarily serves English- and Spanish-speaking families. Differences in eligibility, enrollment, and reasons for not enrolling were examined as functions of race/ethnicity and primary spoken language. Of the 1248 newborns admitted to the neonatal intensive care unit, 46% (n=580) were eligible, and 17% (n=213) were enrolled. Of the 16 languages represented among the newborns' families, 4 (25%) had translated consent documents. Speaking a language other than English or Spanish increased a newborn's likelihood of being ineligible by 5.9 times (P < 0.001) after controlling for race/ethnicity. The main reason for ineligibility was documented as the clinical team declined having their patient recruited (41% [51 of 125]). This reason significantly affected families who spoke languages other than English or Spanish and was able to be remediated with training of the research staff. Stress (20% [18 of 90]) and the study intervention(s) (20% [18 of 90]) were the main reasons given for not enrolling. This analysis of eligibility, enrollment, and reasons for not enrolling in a diagnostic genomic research study found that recruitment generally did not differ as a function of a newborn's race/ethnicity. However, differences were observed depending on the parent's primary spoken language. Regular monitoring and training can improve equitable enrollment into diagnostic genomic research. There are also opportunities at the federal level to improve access to those with limited English proficiency and thus decrease disparities in representation in research participation.

RNA Sequencing of Urine-Derived Cells for the Characterisation and Diagnosis of Osteogenesis Imperfecta.

DNA sequencing is a reliable tool for identifying genetic variants in osteogenesis imperfecta (OI), but cannot always establish pathogenicity, particularly in variants altering splicing. RNA sequencing can provide functional evidence of the effect of a variant on the transcript but requires cells expressing the relevant genes. Here we used urine-derived cells (UDC) to characterize genetic variants in patients with suspected or confirmed OI and provide evidence on the pathogenicity of variants of uncertain significance (VUS). Urine samples were obtained from 45 children and adolescents; UDS culture was successful in 40 of these participants (age range 4 to 20 years 21 females), including 18 participants with OI or suspected OI who had a candidate variant or VUS on DNA sequencing. RNA was extracted from UDC and sequenced on an Illumina NextSeq550 device. Principal component analysis showed that the gene expression profiles of UDC and fibroblasts (based on GTEx consortium data) clustered close together, and had less variability than those of whole blood cells. Transcript abundance was sufficient for analysis by RNA sequencing (defined as a median gene expression level of ≥ 10 transcripts per million) for 25 of the 32 bone fragility genes (78%) that were included in our diagnostic DNA sequencing panel. These results were similar to GTEx data for fibroblasts. Abnormal splicing was identified in 7 of the 8 participants with pathogenic or likely pathogenic variants in the splice region or deeper within the intron. Abnormal splicing was also observed in 2 VUS (COL1A1 c.2829+5G>A and COL1A2 c.693+6T>G), but no splice abnormality was seen in 3 other VUS. Abnormal deletions and duplications could also be observed in UDC transcripts. In conclusion, UDC are suitable for RNA transcript analysis in patients with suspected OI and can provide functional evidence for pathogenicity, in particular of variants affecting splicing. This article is protected by copyright. All rights reserved.

#6812 ON THE TOP PHARMACOGENETICS STUDY EXTRACTED FROM EXOME SEQUENCING DONE FOR CHRONIC KIDNEY DISEASES OF UNKNOW ORIGIN

Abstract Background and Aims A recent study from a large cohort published by the Ubiquitous Pharmacogenomic Consortium demonstrates the interest of pre-emptive pharmacogenetic panel testing, with a 30% reduction rate of clinically detectable side-effects. Our laboratory routinely performs diagnostic exome sequencing (ES) for adult patient with kidney disease and more than 2000 patients have been analyzed to date. Patient with chronic kidney diseases (CKD) could benefit from pharmacogenetics information, since they are often polymedicated and efficacy or safety of their medications are known to be influenced by the genetic background. We studied if meaningful pharmacogenetics information could be delivered from exome sequencing data provided for CKD, either retrospectively or prospectively. Method We selected 214 variants of pharmacogenetics relevance, based on evidence and frequency criteria. We evaluated their depth of coverage on 300 patients randomly selected in our CKD cohort. Results Eleven of those 214 variants were not covered by ES. Three of 11 variants were in UGT1A1 and were excluded from analysis since this information is only relevant for Irinotecan treatment. The remaining 8 variants fall in CYP2C19, CYP3A5, CYP3A4, CYP2D6, HLA B (B15:02), HLA A (A31:01), IFNL3, VKORC1 genes: we completed exome data by targeted sanger sequencing for these variants. One challenging region is CYP2D6 given high sequence homology with CYP2D7 and CYP2D8: to assess if we could reliably call variants in this region, we used 7 additional Coriell samples, as 8 others were also assessed with an orthogonal technique. We were able to properly detect variants in these complex regions in all cases, including hybrid genes. Conclusion Our preliminary data show that exome sequencing, initially ordered for CKD, could also be used to deliver pharmacogenetic meaningful information, by adding a few targeted genotyping assays. We currently evaluate the clinical utility and impact of pharmacogenetics in our CKD cohort prospectively.

Diagnostic genomic sequencing in critically ill children.

Rare genetic diseases are a major cause of severe illnesses and deaths in new-borns and infants. Disease manifestation in critically ill children may be atypical or incomplete, making a monogenetic disease difficult to diagnose clinically. Rapid exome or genome ("genomic") sequencing in critically ill children demonstrated profound diagnostic and clinical value, and there is growing evidence that the faster a molecular diagnosis is established in such children, the more likely clinical management is influenced positively. An early molecular diagnosis enables treatment of critically ill children with precision medicine, has the potential to improve patient outcome and leads to healthcare cost savings. In this review, we outline the status quo of rapid genomic sequencing and possible future implications.

THE STRUCTURES, PROPERTIES, SYNTHESIS, AND INNOVATIVE APPLICATIONS OF GOLD NANOPARTICLES IN LABORATORY MEDICINE: A REVIEW

Nanoparticles technology has gained a great deal of interest in medical and non-medical fields. Gold nanoparticles (AuNPs) which are one of the most attractive metal nanoparticles are widely used in many biomedical fields either for therapeutic or diagnostic purposes. The properties of AuNPs, such as their stability, high biocompatibility, large surface area, relatively inert element, and tunable chemical and physical characteristics have enabled a wide range of applications and discoveries. The AuNPs can be synthesised chemical, physical, or biological synthesis. It is prepared through top-down or bottom-up methods depending on the starting material preparation. AuNPs are used in assays to detect diagnostic markers and specific nucleic acid sequence. Additionally, it is also used in assays to improve bacterial species identification. This review article provides a general overview of the structures, properties, synthesis, and various innovative applications of AuNPs in laboratory medicine such as in chemical pathology, immunology, haematology, molecular, cytogenetics, and transfusion medicine laboratories.

Criticism and bibliography: Zmitrovich I. V., Perelygin V. V., Zharikov M. V. Nomenclature and rank correlation of higher taxa of eukaryotes. Мoscow: INFRA-M, 2022. 183 p. ISBN 978-5-16-018531-6. (Book series “Folia Cryptogamica Petropolitana”. № 8)

The monograph titled "Nomenclature and Rank Correlation of Eukaryotic Higher Taxa," written by I.V. Zmitrovich, V.V. Perelygin, and M.V. Zharikov, has a unique feature - it is addressed to both theoretical biologists dealing with problems of systematics and practitioners who develop issues of industrial microbiology, bioengineering, and biomedicine. It unites these areas of biology through systematics, which, as academician A.L. Takhtajan said, is the basis and pinnacle of biology.&#x0D; Modern progress in molecular biology allows for the precise determination of the relationship between organisms based on the diagnostic nucleotide sequences of important genome regions, leading to important discoveries. For example, Pneumocystis carinii, which was long considered a protozoan and classified as a sporozoan, turned out to be a fungus related to some yeast groups. This information was demanded by pharmacologists who know which antibiotics are effective against fungal infections. However, an excessive flow of scientific information can hinder the systematization and classification according to the existing paradigm of information hysteresis. Changes in systematics happen so quickly that Internet classifiers cannot keep up with them. In addition, there are many competing versions of classification, which makes it difficult to find the optimal solution for a user who does not follow the latest developments in systematics. This can be time-consuming and resource-intensive, making the classification process more complicated.&#x0D; The authors of this monograph aim to systematize all new data expressed in recent years in the field of eukaryotic classification, to determine the consensus of experts' opinions, and to identify those moments on which consensus has not been reached. This mainly concerns the taxonomic rank of groups, which reflects the degree of their differences. To determine the rank of large groups, it is necessary to carefully consider the topology of the enormous eukaryotic tree from different perspectives. Each research group working in different areas of this tree research takes into account its overall topology, but the amount of information is so large that there is not always enough time for a complete and detailed review. However, the authors of this monograph conducted such a review, conveying to the reader the rank structure of the system of eukaryotes, the development of which is based on modern phylogenetic classification.&#x0D; It is important to note that the next stage of the authors' work on systematics will be further research on the development of algorithms for managing microbial communities in different environmental conditions with different sets of limiting factors to implement the useful properties of microorganisms with different secretory activity. The book contains an extensive auxiliary apparatus, including interesting notes discussing various issues such as the priority of domestic researchers in discovering the antibiotic activity of Penicillium, the "Florida hypothesis" of the origin of fungi, and a detailed presentation of the group of fungus-like protists oomycetes.

The role of androgen therapy in acquired aplastic anemia and other bone marrow failure syndromes.

Bone marrow failure syndromes are a heterogeneous group of diseases. With the major advancements in diagnostic tools and sequencing techniques, these diseases may be better classified and therapies may be further tailored. Androgens, a historic group of drugs, were found to stimulate hematopoiesis by enhancing the responsiveness of progenitors. These agents have been used for decades to treat different forms of bone marrow failure. With the availability of more effective pathways to treat BMF, androgens are less used currently. Nevertheless, this group of drugs may serve BMF patients where standard therapy is contraindicated or not available. In this article, we review the published literature addressing the use of androgens in BMF patients and we make recommendations on how to best use this class of drugs within the current therapeutic landscape.

Precision Oncology in Gastrointestinal Stromal Tumors

GIST (gastrointestinal stromal tumors) represent 20% of sarcomatous tumors and 1-2% of primary gastrointestinal cancers. They have an excellent prognosis when localized and resectable, though their prognosis is poor in the metastatic setting, with limited options after the second line until recently. Four lines are now standard in KIT-mutated GIST and one in PDGFRA-mutated GIST. An exponential growth of new treatments is expected in this era of molecular diagnostic techniques and systematic sequencing. Currently, the main challenge remains the emergence of resistance linked to secondary mutations caused by selective pressure induced by TKIs. Repeating biopsies to tailor treatments might be a step in the right direction, and liquid biopsies at progression may offer a non-invasive alternative. New molecules with wider KIT inhibition are under investigation and could change the catalog and the sequence of existing treatments. Combination therapies may also be an approach to overcome current resistance mechanisms. Here, we review the current epidemiology and biology of GIST and discuss future management options, with an emphasis on genome-oriented therapies.

Is Penguin Circovirus Circulating Only in the Antarctic Circle? Lack of Viral Detection in Namibia.

The known host range of circoviruses is continuously expanding because of more intensive diagnostic activities and advanced sequencing tools. Recently, a new circovirus (penguin circovirus (PenCV)) was identified in the guano and cloacal samples collected from Adélie penguins (Pygoscelis adeliae) and chinstrap penguins (Pygoscelis antarcticus) in Antarctica. Although the virus was detected in several asymptomatic subjects, a potential association with feather disease was speculated. To investigate the occurrence and implications of PenCV in other penguin species located outside of Antarctica, a broad survey was undertaken in African penguins (Spheniscus demersus) on two islands off the southern Namibian coast. For this purpose, specific molecular biology assays were developed and validated. None of the 151 blood samples tested positive for PenCV. Several reasons could explain the lack of PenCV positive samples. African penguins and Pygoscelis species are separated by approximately 6000 km, so there is almost no opportunity for transmission. Similarly, host susceptibility to PenCV might be penguin genus-specific. Overall, the present study found no evidence of PenCV in African penguin colonies in Namibia. Further dedicated studies are required to assess the relevance of PenCV among different penguin species.

Understanding the Clinical Utility of Genome Sequencing in Critically Ill Newborns

Diagnostic genome sequencing (GS) in newborns may have many benefits. More accurate diagnosis could spur the development of innovative genomic therapies. A precise diagnosis could help doctors and parents anticipate clinical problems and inform a family's future reproductive choices. However, the integration of GS into neonatal care remains associated with a variety of ethical controversies, including concerns about informed consent, about interpreting uncertain results, about resource allocation and whether access to genomic services could exacerbate health disparities, and about the effect of genome diagnostics on people with disabilities. There also remains significant uncertainty about which babies should be tested and when and how the potential benefits of GS ought to be measured. Probably related to these challenges, some payors have been reluctant to cover the cost of GS for critically ill newborns. Much of the reluctance appears to turn on questions about the clinical benefit associated with GS and whether and for whom GS will be cost-effective. These situations point to the urgent need for careful assessments of the clinical utility of GS in critically ill infants. In this paper, we critically examine the ways in which the clinical utility of GS has been evaluated in this patient population. We focus on "change of management" (COM), a widely used measure of clinical utility for diagnostic GS. We suggest that this measure is often ambiguous because not all COMs can be attributed to genomic results and because not all COMs lead to patient benefit. Finally, we suggest ways that measurement of clinical utility could be improved.

Analytical Performance of a Highly Sensitive System to Detect Gene Variants Using Next-Generation Sequencing for Lung Cancer Companion Diagnostics

The recent increase in the number of molecular targeted agents for lung cancer has led to the demand for the simultaneous testing of multiple genes. Although gene panels using next-generation sequencing (NGS) are ideal, conventional panels require a high tumor content, and biopsy samples often do not meet this requirement. We developed a new NGS panel, called compact panel, characterized by high sensitivity, with detection limits for mutations of 0.14%, 0.20%, 0.48%, 0.24%, and 0.20% for EGFR exon 19 deletion, L858R, T790M, BRAF V600E, and KRAS G12C, respectively. Mutation detection also had a high quantitative ability, with correlation coefficients ranging from 0.966 to 0.992. The threshold for fusion detection was 1%. The panel exhibited good concordance with the approved tests. The identity rates were as follows: EGFR positive, 100% (95% confidence interval, 95.5-100); EGFR negative, 90.9 (82.2-96.3); BRAF positive, 100 (59.0-100); BRAF negative, 100 (94.9-100); KRAS G12C positive, 100 (92.7-100); KRAS G12C negative, 100 (93.0-100); ALK positive, 96.7 (83.8-99.9); ALK negative, 98.4 (97.2-99.2); ROS1 positive, 100 (66.4-100); ROS1 negative, 99.0 (94.6-100); MET positive, 98.0 (89.0-99.9); MET negative 100 (92.8-100); RET positive, 93.8 (69.8-100); RET negative, 100 (94.9-100). The analytical performance showed that the panel could handle various types of biopsy samples obtained by routine clinical practice without requiring strict pathological monitoring, as in the case of conventional NGS panels.

Opt-in for secondary findings as part of diagnostic whole-exome sequencing: Real-life experience from an international diagnostic laboratory.

Discussion about the risks and benefits of offering secondary findings as part of genome-wide diagnostics lacks real-life data. We studied the opt-in decisions of patients/families referred to whole exome study (WES) in Blueprint Genetics (BpG), a genetic testing company with customers in over 70 countries to receive secondary findings. Based on the American College of Medical Genetics (ACMG) recommendations for reporting secondary findings, BpG offered testing of specific actionable genes without additional charge for specimens submitted to WES diagnostics. Individuals could opt-in for a secondary findings analysis by using a separate electronic consent form. Data from BpG database of electronic consent forms was used for the analysis. During the selected study period there were 3263 WES referrals, from which 2012 were index patients. About half of the individuals (50.4%) opted in to receiving secondary findings. Of patients who opted in, a secondary finding was detected for 2.7%, similar to other studies. We detected huge differences relating to opt-in between individuals from different countries; for instance, 90% of the 41 patients and their family members in Romania opted to receive secondary findings, while none of the 98 patients in Luxembourg chose that option. Differences between sexes or between children and adults were small. This data offers one view to the interest of patients and family members to opt in to receiving secondary findings. Research is needed to understand the influence of factors like age, education etc. and possible participation in pre-test counseling to receiving/not receiving secondary findings.

A Novel Alu Element Insertion in ATM Induces Exon Skipping in Suspected HBOC Patients

The vast majority of patients at risk of hereditary breast and/or ovarian cancer (HBOC) syndrome remain without a molecular diagnosis after routine genetic testing. One type of genomic alteration that is commonly missed by diagnostic pipelines is mobile element insertions (MEIs). Here, we reanalyzed multigene panel data from suspected HBOC patients using the MEI detection tool Mobster. A novel Alu element insertion in ATM intron 54 (ATM:c.8010+30_8010+31insAluYa5) was identified as a potential contributing factor in seven patients. Transcript analysis of patient-derived RNA from three heterozygous carriers revealed exon 54 skipping in 38% of total ATM transcripts. To manifest the direct association between the Alu element insertion and the aberrant splice pattern, HEK293T and MCF7 cells were transfected with wild-type or Alu element-carrying minigene constructs. On average, 77% of plasmid-derived transcripts lacked exon 54 in the presence of the Alu element insertion compared to only 4.7% of transcripts expressed by the wild-type minigene. These results strongly suggest ATM:c.8010+30_8010+31insAluYa5 as the main driver of ATM exon 54 skipping. Since this exon loss is predicted to cause a frameshift and a premature stop codon, mutant transcripts are unlikely to translate into functional proteins. Based on its estimated frequency of up to 0.05% in control populations, we propose to consider ATM:c.8010+30_8010+31insAluYa5 in suspected HBOC patients and to clarify its role in carcinogenesis through future epidemiological and functional analyses. Generally, the implementation of MEI detection tools in diagnostic sequencing pipelines could increase the diagnostic yield, as MEIs are likely underestimated contributors to genetic diseases.

Kidney Disease Associated With Mono-allelic COL4A3 and COL4A4 Variants: A Case Series of 17 Families.

Mono-allelic variants in COL4A3 and COL4A4 (COL4A3/COL4A4) havebeen identified in a spectrum of glomerular basement membrane nephropathies, including thin basement membrane nephropathy and autosomal dominant Alport syndrome. With the increasing useof next generation sequencing, mono-allelic COL4A3/COL4A4 variants are detected morefrequently, but phenotypic heterogeneity impedes counseling. We aimed to investigate the phenotypic spectrum, kidney biopsy results, and segregation patterns of patients with mono-allelic COL4A3/COL4A4 variants identified by whole exome sequencing. Case series. We evaluated clinical andpathologic characteristics of 17 Dutch index patients with mono-allelic variants in COL4A3/COL4A4 detected by diagnostic whole exome sequencing and 25 affected family members with variants confirmed by Sanger sequencing. Eight different mono-allelic COL4A3/COL4A4 variants were identified across members of 11 families, comprising 7 glycine substituted missense variants and 1 frameshift variant. All index patients had microscopic hematuria at clinical presentation (median age 43 years) and 14 had (micro)albuminuria/proteinuria. All family members showed co-segregation of the variant with at least hematuria. At end of follow-up of all 42 individuals (median age 54 years), 16/42 patients had kidney function impairment, of whom 6 had kidney failure.Reports of kidney biopsies of 14 patients described thin basement membrane nephropathy, focal segmental glomerulosclerosis, minimal change lesions, and Alport syndrome. Electron microscopy images of 7 patients showed a significantly thinner glomerular basement membrane compared with images of patients with idiopathic focal segmental glomerulosclerosis and other hereditary glomerular diseases. No genotype-phenotype correlations could be established. Retrospective design, ascertainment bias toward severe kidney phenotypes, and familial hematuria. This study confirms the wide phenotypic spectrum associated with mono-allelic COL4A3/COL4A4 variants, extending from isolated microscopic hematuria to kidney failure with high intra- and interfamilial variability.