Quality metrics for enhanced performance of an NGS panel using single-vial amplification technology.

AIMS: Targeted next-generation sequencing (NGS) panels, which identify genomic alterations, are the stronghold of molecular oncology laboratories. In spite of technological advances, the quantity and quality of DNA from formalin-fixed paraffin-embedded tissue and paucicellular specimens are barriers to successful sequencing. Here, we describe an NGS assay employing single tube stem-loop inhibition mediated amplification technology that delivers highly accurate results with low input DNA. Rigorous quality metrics, regular monitoring and in-depth validation make the test attractive for clinical laboratories. METHODS: The study used a customised NGS panel, targeting 48 genes across several solid tumour types. Validation, in accordance with guidelines from New York State, sequenced patient samples harbouring 136 known variants, including single-nucleotide variants (SNVs) and indels. Specimen types included formalin-fixed paraffin embedded blocks, core biopsies and cytology material. Neoplastic cellularity of the tumours ranged from 10% to 80%. RESULTS: The assay was highly specific and sensitive with excellent accuracy, reproducibility and repeatability/precision. Concordant results for identification of SNVs and indels were obtained from specimens with DNA input of 2-3 ng, tissue with 10% neoplastic cellularity and variant allelic frequencies of 2.5%-3%. Over 99% of the target areas are shown to achieve at least 500X coverage when parsed through two bioinformatics pipelines. With over 2000 clinical specimens analysed, the success of the panel for reporting of results is 95.3% CONCLUSIONS: The advanced technology enables accurate identification of clinically relevant variants with uniformity of coverage and an impressive turn-around-time. The overall workflow and cost-effectiveness provide added value.

A treatment-refractory aggressive MDS-MLD with multiple highly complex chromosome 5 intrachromosomal rearrangements: a case report.

BACKGROUND: A patient with a myelodysplastic neoplasm exhibited a karyotype with multiple complex chromosome 5 rearrangements. This patient appeared to have a catastrophic cytogenetic event that manifested as a treatment-refractory aggressive form of disease, which lead to patient demise within one year. Both the clinical presentation and disease course were unusual based on the medical history and morphologic findings. Such cases of myelodysplastic syndrome with multilineage dysplasia (MDS-MLD) with complex abnormalities are not reported in the literature. CASE PRESENTATION: The patient was a 62-year-old female who presented with pancytopenia and dyspnea. The morphologic appearance of the peripheral blood smear and bone marrow biopsy, along with flow cytometric findings, favored the diagnosis of MDS-MLD unclassifiable. Myelodysplastic syndrome (MDS) with multilineage dysplasia (MDS-MLD), is an MDS characterized by one or more cytopenias and dysplastic changes in two or more of the myeloid lineages (i.e., erythroid, granulocytic, and megakaryocytic). The bone marrow, in particular, showed prominent dysplasia, including the presence of atypical megakaryocytes with small hypolobated morphology reminiscent of those typically seen in MDS with isolated 5q deletion. Cytogenetic analysis, including interphase and metaphase FISH, karyotype and SNP chromosomal microarray were performed, as well as DNA sequencing studies. Cytogenetic analysis showed a very complex karyotype featuring multiple 5q intrachromosomal rearrangements including a pericentric inversion with multiple interspersed deletions and monosomy 7. FISH studies showed a partial deletion of the PDGFRß gene, and SNP chromosomal microarray and targeted panel-based sequencing identified biallelic loss of function of the TP53 gene. Based on the pathologic findings, the patient was treated for MDS but did not respond to either lenalidomide or azacitidine. CONCLUSION: The genetic changes described, in particular, the complex intrachromosomal rearrangements of chromosome 5, suggest the occurrence of a sudden catastrophic event that led to an aggressive course in the patient's disease. Conventional karyotyping, metaphase and interphase FISH, SNP chromosomal microarray and NGS helped to identify the complex genetic changes seen in this case. This highlights the importance of utilizing a multimodality approach to fully characterize complex chromosomal events that may significantly impact disease progression, treatment and survival.

Clinical exome sequencing for inherited retinal degenerations at a tertiary care center.

Inherited retinal degenerations are clinically and genetically heterogeneous diseases characterized by progressive deterioration of vision. This study aimed at assessing the diagnostic yield of exome sequencing (ES) for an unselected cohort of individuals with hereditary retinal disorders. It is a retrospective study of 357 unrelated affected individuals, diagnosed with retinal disorders who underwent clinical ES. Variants from ES were filtered, prioritized, and classified using the ACMG recommendations. Clinical diagnosis of the individuals included rod-cone dystrophy (60%), macular dystrophy (20%), cone-rod dystrophy (9%), cone dystrophy (4%) and other phenotypes (7%). Majority of the cases (74%) were singletons and 6% were trios. A confirmed molecular diagnosis was obtained in 24% of cases. In 6% of cases, two pathogenic variants were identified with phase unknown, bringing the potential molecular diagnostic rate to ~ 30%. Including the variants of uncertain significance (VUS), potentially significant findings were reported in 57% of cases. Among cases with a confirmed molecular diagnosis, variants in EYS, ABCA4, USH2A, KIZ, CERKL, DHDDS, PROM1, NR2E3, CNGB1, ABCC6, PRPH2, RHO, PRPF31, PRPF8, SNRNP200, RP1, CHM, RPGR were identified in more than one affected individual. Our results support the utility of clinical ES in the diagnosis of genetically heterogeneous retinal disorders.

Expanding the phenotype of ATP6AP1 deficiency.

Vacuolar ATPases (V-ATPases) are large multisubunit proton pumps conserved among all eukaryotic cells that are involved in diverse functions including acidification of membrane-bound intracellular compartments. The ATP6AP1 gene encodes an accessory subunit of the vacuolar (V)-ATPase protein pump. Pathogenic variants in ATP6AP1 have been described in association with a congenital disorder of glycosylation (CDG), which are highly variable, but often characterized by immunodeficiency, hepatopathy, and neurologic manifestations. Although the most striking and common clinical feature is hepatopathy, the phenotypic and genotypic spectrum of ATP6AP1-CDG continues to expand. Here, we report identical twins who presented with acute liver failure and jaundice. Prenatal features included cystic hygroma, atrial septal defect, and ventriculomegaly. Postnatal features included pectus carinatum, connective tissue abnormalities, and hypospadias. Whole-exome sequencing (WES) revealed a novel de novo in-frame deletion in the ATP6AP1 gene (c.230_232delACT;p.Tyr77del). Although both twins have the commonly reported clinical feature of hepatopathy seen in other individuals with ATP6AP1-CDG-related disorder, they do not have neurological sequelae. This report expands the phenotypic spectrum of ATP6AP1-CDG-related disorder with both probands exhibiting unique prenatal and postnatal features, including fetal ventriculomegaly, umbilical hernia, pectus carinatum, micropenis, and hypospadias. Furthermore, this case affirms that neurological features described in the initial case series on ATP6AP1-CDG do not appear to be central, whereas the prenatal and connective tissue manifestations may be more common than previously thought. This emphasizes the importance of long-term clinical follow-up and variant interpretation using current updated recommendations.

3q27.1 microdeletion causes prenatal and postnatal growth restriction and neurodevelopmental abnormalities.

BACKGROUND: Overlapping microdeletions of chromosome 3q26-3q28 have been reported in eight individuals. The common phenotype observed in these individuals include intrauterine growth restriction, short stature, microcephaly, feeding difficulties, facial dysmorphisms, limb abnormalities and developmental delay. The most striking clinical features shared among all reported cases is prenatal and postnatal growth restriction and neurodevelopmental abnormalities. CASE PRESENTATION: We identified two additional individuals with overlapping deletions and shared clinical features by high-resolution SNP oligonucleotide microarray, and refined the smallest region of overlap (SRO) to a 1.2 Mb genomic location in chromosome 3q27.1 by reviewing and comparing all published cases. We evaluated the SRO using ACMG/ClinGen current recommendations for classifying copy number variants (CNVs), and discussed the contribution of the genes deleted in the SRO to the abnormal phenotype observed in these individuals. CONCLUSIONS: This study provides further evidence supporting the existence of a novel 3q27.1 microdeletion syndrome and suggests that haploinsufficiency of potential candidate genes, DVL3, AP2M1, and PARL in the SRO in 3q27.1 is responsible for the phenotype.

A novel de novo KDM5C variant in a female with global developmental delay and ataxia: a case report.

BACKGROUND: Pathogenic variants in KDM5C are a cause of X-linked intellectual disability in males. Other features in males include short stature, dysmorphic features, seizures and spasticity. In some instances, female relatives were noted to have learning difficulties and mild intellectual disabilities, but full phenotypic descriptions were often incomplete. Recently, detailed phenotypic features of five affected females with de novo variants were described. (Clin Genet 98:43-55, 2020) Four individuals had a protein truncating variant and 1 individual had a missense variant. All five individuals had developmental delay/intellectual disability and three neurological features. CASE PRESENTATION: Here we report a three-year-old female with global developmental delay, hypotonia and ataxia. Through whole exome sequencing, a de novo c.1516A > G (p.Met506Val) variant in KDM5C was identified. This missense variant is in the jumonji-C domain of this multi domain protein where other missense variants have been previously reported in KDM5C related disorder. The KDM5C gene is highly intolerant to functional variation which suggests its pathogenicity. The probands motor delays and language impairment is consistent with other reported female patients with de novo variants in KDM5C. However, other features reported in females (distinctive facial features, skeletal abnormalities, short stature and endocrine features) were absent. To the best of our knowledge, our proband is the first female patient reported with a diagnosis of ataxia. CONCLUSIONS: This case report provides evidence for an emerging and phenotypic variability that adds to the literature of the role of KDM5C in females with neurodevelopmental disorders as well as movement disorders.

ZTTK syndrome: Clinical and molecular findings of 15 cases and a review of the literature.

Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is caused by de novo loss-of-function variants in the SON gene (MIM #617140). This multisystemic disorder is characterized by intellectual disability, seizures, abnormal brain imaging, variable dysmorphic features, and various congenital anomalies. The wide application and increasing accessibility of whole exome sequencing (WES) has helped to identify new cases of ZTTK syndrome over the last few years. To date, there have been approximately 45 cases reported in the literature. Here, we describe 15 additional individuals with variants in the SON gene, including those with missense variants bringing the total number of known cases to 60. We have reviewed the clinical and molecular data of these new cases and all previously reported cases to further delineate the most common as well as emerging clinical findings related to this syndrome. Furthermore, we aim to delineate any genotype-phenotype correlations specifically for a recurring pathogenic four base pair deletion (c.5753_5756del) along with discussing the impact of missense variants seen in the SON gene.

Key considerations for comprehensive validation of an RNA fusion NGS panel.

OBJECTIVES: Validation of RNA-based NGS assays for the detection of therapeutically targetable gene fusions is challenging. Here, we report systematic validation and quality control monitoring of our targeted fusion panel for the detection of 17 clinically relevant fusion transcripts across several tumor types. We implemented this RNA Fusion Panel as a reflex test for tumors lacking DNA driver mutations. DESIGN: Forty-four formalin-fixed, paraffin-embedded (FFPE) or fresh-frozen lung, brain, soft tissue and skin tumors were used to determine the accuracy of the assay. Additional fusion-positive specimens and a calibrated reference standard were used to establish the precision, reproducibility and sensitivity of the assay. All aspects of the validation, including quality control metrics, were performed according to New York State guidelines. RESULTS: For the RNA fusion panel, accuracy, reproducibility and precision studies were above 99%. Reproducibility and sensitivity studies with the reference standard were helpful in identifying inconsistencies. The limit of detection for most RNA fusion transcripts was 50 copies. Application of the RNA fusion assay as a reflex test to 450 tumor samples lacking DNA driver mutations resulted in a 10% increase in diagnostic yield with minimal additional processing time. CONCLUSIONS: The validated RNA fusion panel provides clinical utility in therapy selection for patients with solid tumors. By using a sequential testing approach, the RNA fusion assay complements the DNA hotspot assay in identifying clinically relevant variants across many tumor types with minimal additional increase in processing time.

Targeting SLMAP-ALK-a novel gene fusion in lung adenocarcinoma.

Assessment of ALK gene rearrangements is strongly recommended by the Molecular Testing Guideline for Selection of Lung Cancer Patients proposed by IASLC, AMP, and CAP at the time of diagnosis for patients with advanced stage disease. Non-small-cell lung cancer (NSCLC) with ALK gene rearrangements or the resulting fusion proteins have been, for the most part, successfully targeted with ALK tyrosine kinase inhibitors (TKIs). The most frequent rearrangement, the EML4-ALK oncogenic fusion, has more than 10 distinct variants, each with a discrete breakpoint in EML4 Recent studies have suggested that EML4-ALK variants may have differential responses to TKIs. Additionally, non-EML4-ALK fusions that result from ALK rearrangements with diverse 5' partners could possibly have varied biologic and clinical implications in their therapeutic responses and outcomes of patients with NSCLC. Existing literature documents at least 20 non-EML4 fusion partners for ALK, and the clinical responsiveness to crizotinib ranges from increased sensitivity to resistance. This underscores the importance of identifying the precise 5' fusion partner to ALK before initiation of therapy. Herein we report the identification of a novel SLMAP-ALK fusion in a patient with NSCLC.

Molecular Basis for Strain Variation in the Saccharomyces cerevisiae Adhesin Flo11p.

FLO11 encodes a yeast cell wall flocculin that mediates a variety of adhesive phenotypes in Saccharomyces cerevisiae. Flo11p is implicated in many developmental processes, including flocculation, formation of pseudohyphae, agar invasion, and formation of microbial mats and biofilms. However, Flo11p mediates different processes in different yeast strains. To investigate the mechanisms by which FLO11 determines these differences in colony morphology, flocculation, and invasion, we studied gene structure, function, and expression levels. Nonflocculent Saccharomyces cerevisiae Σ1278b cells exhibited significantly higher FLO11 mRNA expression, especially in the stationary phase, than highly flocculent S. cerevisiae var. diastaticus. The two strains varied in cell surface hydrophobicity, and Flo11p contributed significantly to surface hydrophobicity in S. cerevisiae var. diastaticus but not in strain Σ1278b. Sequencing of the FLO11 gene in S. cerevisiae var. diastaticus revealed strain-specific differences, including a 15-amino-acid insertion in the adhesion domain. Flo11p adhesion domains from strain Σ1278b and S. cerevisiae var. diastaticus were expressed and used to coat magnetic beads. The adhesion domain from each strain bound preferentially to homologous cells, and the preferences were independent of the cells in which the adhesion domains were produced. These results are consistent with the idea that strain-specific variations in the amino acid sequences in the adhesion domains cause different Flo11p flocculation activities. The results also imply that strain-specific differences in expression levels, posttranslational modifications, and allelic differences outside the adhesion domains have little effect on flocculation. IMPORTANCE As a nonmotile organism, Saccharomyces cerevisiae employs the cell surface flocculin Flo11/Muc1 as an important means of adapting to environmental change. However, there is a great deal of strain variation in the expression of Flo11-dependent phenotypes, including flocculation. In this study, we investigated the molecular basis of this strain-specific phenotypic variability. Our data indicate that strain-specific differences in the level of flocculation result from significant sequence differences in the FLO11 alleles and do not depend on quantitative differences in FLO11 expression or on surface hydrophobicity. We further have shown that beads coated with amino-terminal domain peptide bind preferentially to homologous cells. These data show that variability in the structure of the Flo11 adhesion domain may thus be an important determinant of membership in microbial communities and hence may drive selection and evolution.

DNA Methylation Profiling at Single-Base Resolution Reveals Gestational Folic Acid Supplementation Influences the Epigenome of Mouse Offspring Cerebellum.

It is becoming increasingly more evident that lifestyle, environmental factors, and maternal nutrition during gestation can influence the epigenome of the developing fetus and thus modulate the physiological outcome. Variations in the intake of maternal nutrients affecting one-carbon metabolism may influence brain development and exert long-term effects on the health of the progeny. In this study, we investigated whether supplementation with high maternal folic acid during gestation alters DNA methylation and gene expression in the cerebellum of mouse offspring. We used reduced representation bisulfite sequencing to analyze the DNA methylation profile at the single-base resolution level. The genome-wide DNA methylation analysis revealed that supplementation with higher maternal folic acid resulted in distinct methylation patterns (P < 0.05) of CpG and non-CpG sites in the cerebellum of offspring. Such variations of methylation and gene expression in the cerebellum of offspring were highly sex-specific, including several genes of the neuronal pathways. These findings demonstrate that alterations in the level of maternal folic acid during gestation can influence methylation and gene expression in the cerebellum of offspring. Such changes in the offspring epigenome may alter neurodevelopment and influence the functional outcome of neurologic and psychiatric diseases.

High Gestational Folic Acid Supplementation Alters Expression of Imprinted and Candidate Autism Susceptibility Genes in a sex-Specific Manner in Mouse Offspring.

Maternal nutrients play critical roles in modulating epigenetic events and exert long-term influences on the progeny's health. Folic acid (FA) supplementation during pregnancy has decreased the incidence of neural tube defects in newborns, but the influence of high doses of maternal FA supplementation on infants' brain development is unclear. The present study was aimed at investigating the effects of a high dose of gestational FA on the expression of genes in the cerebral hemispheres (CHs) of 1-day-old pups. One week prior to mating and throughout the entire period of gestation, female C57BL/6J mice were fed a diet, containing FA at either 2 mg/kg (control diet (CD)) or 20 mg/kg (high maternal folic acid (HMFA)). At postnatal day 1, pups from different dams were sacrificed and CH tissues were collected. Quantitative RT-PCR and Western blot analysis confirmed sex-specific alterations in the expression of several genes that modulate various cellular functions (P < 0.05) in pups from the HMFA group. Genomic DNA methylation analysis showed no difference in the level of overall methylation in pups from the HMFA group. These findings demonstrate that HMFA supplementation alters offsprings' CH gene expression in a sex-specific manner. These changes may influence infants' brain development.

Lifestyle, pregnancy and epigenetic effects.

Rapidly growing evidences link maternal lifestyle and prenatal factors with serious health consequences and diseases later in life. Extensive epidemiological studies have identified a number of factors such as diet, stress, gestational diabetes, exposure to tobacco and alcohol during gestation as influencing normal fetal development. In light of recent discoveries, epigenetic mechanisms such as alteration of DNA methylation, chromatin modifications and modulation of gene expression during gestation are believed to possibly account for various types of plasticity such as neural tube defects, autism spectrum disorder, congenital heart defects, oral clefts, allergies and cancer. The purpose of this article is to review a number of published studies to fill the gap in our understanding of how maternal lifestyle and intrauterine environment influence molecular modifications in the offspring, with an emphasis on epigenetic alterations. To support these associations, we highlighted laboratory studies of rodents and epidemiological studies of human based on sampling population cohorts.

Microarray analysis reveals higher gestational folic Acid alters expression of genes in the cerebellum of mice offspring-a pilot study.

Folate is a water-soluble vitamin that is critical for nucleotide synthesis and can modulate methylation of DNA by altering one-carbon metabolism. Previous studies have shown that folate status during pregnancy is associated with various congenital defects including the risk of aberrant neural tube closure. Maternal exposure to a methyl supplemented diet also can alter DNA methylation and gene expression, which may influence the phenotype of offspring. We investigated if higher gestational folic acid (FA) in the diet dysregulates the expression of genes in the cerebellum of offspring in C57BL/6 J mice. One week before gestation and throughout the pregnancy, groups of dams were supplemented with FA either at 2 mg/kg or 20 mg/kg of diet. Microarray analysis was used to investigate the genome wide gene expression profile in the cerebellum from day old pups. Our results revealed that exposure to the higher dose FA diet during gestation dysregulated expression of several genes in the cerebellum of both male and female pups. Several transcription factors, imprinted genes, neuro-developmental genes and genes associated with autism spectrum disorder exhibited altered expression levels. These findings suggest that higher gestational FA potentially dysregulates gene expression in the offspring brain and such changes may adversely alter fetal programming and overall brain development.

Folic acid supplementation in pregnancy and implications in health and disease.

Maternal exposure to dietary factors during pregnancy can influence embryonic development and may modulate the phenotype of offspring through epigenetic programming. Folate is critical for nucleotide synthesis, and preconceptional intake of dietary folic acid (FA) is credited with reduced incidences of neural tube defects in infants. While fortification of grains with FA resulted in a positive public-health outcome, concern has been raised for the need for further investigation of unintended consequences and potential health hazards arising from excessive FA intakes, especially following reports that FA may exert epigenetic effects. The objective of this article is to discuss the role of FA in human health and to review the benefits, concerns and epigenetic effects of maternal FA on the basis of recent findings that are important to design future studies.

Increasing maternal or post-weaning folic acid alters gene expression and moderately changes behavior in the offspring.

BACKGROUND: Studies have indicated that altered maternal micronutrients and vitamins influence the development of newborns and altered nutrient exposure throughout the lifetime may have potential health effects and increased susceptibility to chronic diseases. In recent years, folic acid (FA) exposure has significantly increased as a result of mandatory FA fortification and supplementation during pregnancy. Since FA modulates DNA methylation and affects gene expression, we investigated whether the amount of FA ingested during gestation alters gene expression in the newborn cerebral hemisphere, and if the increased exposure to FA during gestation and throughout the lifetime alters behavior in C57BL/6J mice. METHODS: Dams were fed FA either at 0.4 mg or 4 mg/kg diet throughout the pregnancy and the resulting pups were maintained on the diet throughout experimentation. Newborn pups brain cerebral hemispheres were used for microarray analysis. To confirm alteration of several genes, quantitative RT-PCR (qRT-PCR) and Western blot analyses were performed. In addition, various behavior assessments were conducted on neonatal and adult offspring. RESULTS: Results from microarray analysis suggest that the higher dose of FA supplementation during gestation alters the expression of a number of genes in the newborns' cerebral hemispheres, including many involved in development. QRT-PCR confirmed alterations of nine genes including down-regulation of Cpn2, Htr4, Zfp353, Vgll2 and up-regulation of Xist, Nkx6-3, Leprel1, Nfix, Slc17a7. The alterations in the expression of Slc17a7 and Vgll2 were confirmed at the protein level. Pups exposed to the higher dose of FA exhibited increased ultrasonic vocalizations, greater anxiety-like behavior and hyperactivity. These findings suggest that although FA plays a significant role in mammalian cellular machinery, there may be a loss of benefit from higher amounts of FA. Unregulated high FA supplementation during pregnancy and throughout the life course may have lasting effects, with alterations in brain development resulting in changes in behavior.

Single-base resolution of mouse offspring brain methylome reveals epigenome modifications caused by gestational folic acid.

BACKGROUND: Epigenetic modifications, such as cytosine methylation in CpG-rich regions, regulate multiple functions in mammalian development. Maternal nutrients affecting one-carbon metabolism during gestation can exert long-term effects on the health of the progeny. Using C57BL/6 J mice, we investigated whether the amount of ingested maternal folic acid (FA) during gestation impacted DNA methylation in the offspring's cerebral hemispheres. Reduced representation bisulfite sequencing at single-base resolution was performed to analyze genome-wide DNA methylation profiles. RESULTS: We identified widespread differences in the methylation patterns of CpG and non-CpG sites of key developmental genes, including imprinted and candidate autism susceptibility genes (P <0.05). Such differential methylation of the CpG and non-CpG sites may use different mechanisms to alter gene expressions. Quantitative real time reverse transcription-polymerase chain reaction confirmed altered expression of several genes. CONCLUSIONS: These finding demonstrate that high maternal FA during gestation induces substantial alteration in methylation pattern and gene expression of several genes in the cerebral hemispheres of the offspring, and such changes may influence the overall development. Our findings provide a foundation for future studies to explore the influence of gestational FA on genetic/epigenetic susceptibility to altered development and disease in offspring.