Mendelian neurodevelopmental disorders customarily present with complex and overlapping symptoms, complicating the clinical diagnosis. Individuals with a growing number of the so-called rare ...disorders exhibit unique, disorder-specific DNA methylation patterns, consequent to the underlying gene defects. Besides providing insights to the pathophysiology and molecular biology of these disorders, we can use these epigenetic patterns as functional biomarkers for the screening and diagnosis of these conditions. This review summarizes our current understanding of DNA methylation episignatures in rare disorders and describes the underlying technology and analytical approaches. We discuss the computational parameters, including statistical and machine learning methods, used for the screening and classification of genetic variants of uncertain clinical significance. Describing the rationale and principles applied to the specific computational models that are used to develop and adapt the DNA methylation episignatures for the diagnosis of rare disorders, we highlight the opportunities and challenges in this emerging branch of diagnostic medicine.
Personalized targeted therapies have transformed management of several solid tumors. Timely and accurate detection of clinically relevant genetic variants in tumor is central to the implementation of ...molecular targeted therapies. To facilitate precise molecular testing in solid tumors, targeted next-generation sequencing (NGS) assays have emerged as a valuable tool. In this study, we provide an overview of the technical validation, diagnostic yields, and spectrum of variants observed in 3,164 solid tumor samples that were tested as part of the standard clinical diagnostic assessment in an academic healthcare institution over a period of 2 years.
The Ion Ampliseq™ Cancer Hotspot Panel v2 assay (ThermoFisher) that targets ~2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes was validated, and a total of 3,164 tumor DNA samples were tested in 2 years. A total of 500 tumor samples were tested by the comprehensive panel containing all the 50 genes. Other samples, including 1,375 lung cancer, 692 colon cancer, 462 melanoma, and 135 brain cancer, were tested by tumor-specific targeted subpanels including a few clinically actionable genes.
Of 3,164 patient samples, 2,016 (63.7%) tested positive for at least one clinically relevant variant. Of 500 samples tested by a comprehensive panel, 290 had a clinically relevant variant with TP53, KRAS, and PIK3CA being the most frequently mutated genes. The diagnostic yields in major tumor types were as follows: breast (58.4%), colorectal (77.6%), lung (60.4%), pancreatic (84.6%), endometrial (72.4%), ovary (57.1%), and thyroid (73.9%). Tumor-specific targeted subpanels also demonstrated high diagnostic yields: lung (69%), colon (61.2%), melanoma (69.7%), and brain (20.7%). Co-occurrence of mutations in more than one gene was frequently observed.
The findings of our study demonstrate the feasibility of integrating an NGS-based gene panel screen as part of a standard diagnostic protocol for solid tumor assessment. High diagnostic rates enable significant clinical impact including improved diagnosis, prognosis, and clinical management in patients with solid tumors.
Wiedemann-Steiner syndrome (WDSTS) is a Mendelian syndromic intellectual disability (ID) condition associated with hypertrichosis cubiti, short stature, and characteristic facies caused by pathogenic ...variants in the
gene. Clinical features can be inconclusive in mild and unusual WDSTS presentations with variable ID (mild to severe), facies (typical or not) and other associated malformations (bone, cerebral, renal, cardiac and ophthalmological anomalies). Interpretation and classification of rare
variants can be challenging. A genome-wide DNA methylation episignature for
-related syndrome could allow functional classification of variants and provide insights into the pathophysiology of WDSTS. Therefore, we assessed genome-wide DNA methylation profiles in a cohort of 60 patients with clinical diagnosis for WDSTS or Kabuki and identified a unique highly sensitive and specific DNA methylation episignature as a molecular biomarker of WDSTS. WDSTS episignature enabled classification of variants of uncertain significance in the
gene as well as confirmation of diagnosis in patients with clinical presentation of WDSTS without known genetic variants. The changes in the methylation profile resulting from
mutations involve global reduction in methylation in various genes, including homeobox gene promoters. These findings provide novel insights into the molecular etiology of WDSTS and explain the broad phenotypic spectrum of the disease.
Background. Thrombocytosis is a common reason for referral to Hematology. Differentiating between secondary causes of thrombocytosis and essential thrombocythemia (ET) is often clinically ...challenging. A practical diagnostic approach to identify secondary thrombocytosis could reduce overinvestigation such as next generation sequencing (NGS) panel. Methods and Results. All adult patients with thrombocytosis (≥450 × 109/L) who underwent molecular testing at a single tertiary care centre between January 1, 2018 and May 31, 2021 were evaluated. Clinical and laboratory variables were compared between patients with secondary thrombocytosis vs. ET. Clinical variables included smoking, thrombosis, splenectomy, active malignancy, chronic inflammatory disease, and iron deficiency anemia. Laboratory variables included complete blood count (CBC), ferritin, and myeloid mutations detected by NGS. The overall yield of molecular testing was 52.4%; 92.1% of which were mutations in JAK2, CALR, and/or MPL. Clinical factors predictive of ET included history of arterial thrombosis (p<0.05); active malignancy, chronic inflammatory disease, splenectomy, and iron deficiency were associated with secondary thrombocytosis (p<0.05). A diagnosis of ET was associated with higher hemoglobin, mean corpuscular volume (MCV), red cell distribution width (RDW), and mean platelet volume (MPV), while secondary thrombocytosis was associated with higher body mass index, white blood cells, and neutrophils (p<0.01). Conclusion. A practical approach to investigating patients with persistent thrombocytosis based on clinical characteristics such as active malignancy, chronic inflammatory disease, splenectomy, and iron deficiency may assist in accurately identifying patients more likely to have secondary causes of thrombocytosis and reduce overinvestigation, particularly costly molecular testing.
Myelodysplastic neoplasms (MDS) with ring sideroblasts (RS) are diagnosed via bone marrow aspiration in the presence of either (i) ≥15% RS or (ii) 5-14% RS and an
mutation. In the MEDALIST trial and ...in an interim analysis of the COMMANDS trial, lower-risk MDS-RS patients had decreased transfusion dependency with luspatercept treatment. A total of 6817 patients with suspected hematologic malignancies underwent molecular testing using a next-generation-sequencing-based genetic assay and 395 MDS patients, seen at our centre from 1 January 2018 to 31 May 2023, were reviewed. Of these, we identified 39 evaluable patients as having lower-risk MDS with
mutations: there were 20 (51.3%) males and 19 (48.7%) females, with a median age of 77 years (range of 57 to 92). Nineteen (48.7%) patients had an isolated
mutation with a mean variant allele frequency of 35.2% +/- 8.1%, ranging from 7.4% to 46.0%. There were 29 (74.4%) patients with ≥15% RS, 6 (15.4%) with 5 to 14% RS, one (2.6%) with 1% RS, and 3 (7.7%) with no RS. Our study suggests that a quarter of patients would be missed based on the morphologic criterion of only using RS greater than 15% and supports the revised 2022 definitions of the World Health Organization (WHO) and International Consensus Classification (ICC), which shift toward molecularly defined subtypes of MDS and appropriate testing.
Since the identification of
and exon 12 mutations as driver mutations in polycythemia vera (PV) in 2005, molecular testing of these mutations for patients with erythrocytosis has become a routine ...clinical practice. However, the incidence of myeloid mutations other than the common
mutation in unselected patients referred for elevated hemoglobin is not well studied. This study aimed to characterize the mutational landscape in a real-world population of patients referred for erythrocytosis using a targeted next-generation sequencing (NGS)-based assay.
A total of 529 patients (hemoglobin levels >160 g/L in females or >165 g/L in males) were assessed between January 2018 and May 2021 for genetic variants using the Oncomine Myeloid Research Assay (ThermoFisher Scientific, Waltham, MA, USA) targeting 40 key genes with diagnostic and prognostic implications in hematological conditions (17 full genes and 23 genes with clinically relevant "hotspot" regions) and a panel of 29 fusion driver genes (>600 fusion partners).
mutations were detected in 10.9% (58/529) of patients, with 57 patients positive for
, while one patient had a
exon 12 mutation. Additional mutations were detected in 34.5% (20/58) of
-positive patients:
(11; 19%),
(2;3.4%),
(2; 3.4%),
(2; 3.4%),
(1; 1.7%),
(1; 1.7%), and
(1; 1.7%). Diagnosis of PV was suspected in 2
-negative patients based on the 2016 World Health Organization (WHO) diagnostic criteria. Notably, one patient carried mutations in the
and
genes, and the other patient carried mutations in the
and
genes. Three
-negative patients with elevated hemoglobin who tested positive for
fusion were diagnosed with chronic myeloid leukemia (CML) and excluded from further analysis. The remaining 466
-negative patients were diagnosed with secondary erythrocytosis and mutations were found in 6% (28/466) of these cases.
Mutations other than
mutations were frequently identified in patients referred for erythrocytosis, with mutations in the
and
genes being detected in 34.5% of
-positive PV patients. The presence of additional mutations, such as
in this population has implications for prognosis. Both the incidence and mutation type identified in patients with secondary erythrocytosis likely reflects incidental, age-associated clonal hematopoiesis of indeterminate potential (CHIP).
Background
Hereditary cancer predisposition syndromes account for approximately 10% of cancer cases. Next generation sequencing (NGS) based multi-gene targeted panels is now a frontline approach to ...identify pathogenic mutations in cancer predisposition genes in high-risk families. Recent evolvement of NGS technologies have allowed simultaneous detection of sequence and copy number variants (CNVs) using a single platform. In this study, we have analyzed frequency and nature of sequence variants and CNVs, in a Canadian cohort of patients, suspected with hereditary cancer syndrome, referred for genetic testing following specific genetic testing guidelines based on patient’s personal and/or family history of cancer.
Methods
A 2870 patients were subjected to a single NGS based multi-gene targeted hereditary cancer panel testing algorithm to identify sequence variants and CNVs in cancer predisposition genes at our reference laboratory in Southwestern Ontario. CNVs identified by NGS were confirmed by alternative techniques like Multiplex ligation-dependent probe amplification (MLPA).
Results
A 15% (431/2870) patients had a pathogenic variant and 36% (1032/2870) had a variant of unknown significance (VUS), in a cancer susceptibility gene. A total of 287 unique pathogenic variant were identified, out of which 23 (8%) were novel. CNVs identified by NGS based approach accounted for 9.5% (27/287) of pathogenic variants, confirmed by alternate techniques with high accuracy.
Conclusion
This study emphasizes the utility of NGS based targeted testing approach to identify both sequence and CNVs in patients suspected with hereditary cancer syndromes in clinical setting and expands the mutational spectrum of high and moderate penetrance cancer predisposition genes.
The MRN complex consisting of MRE11A-RAD50-NBS1 proteins is involved in the repair of double-strand breaks, and mutations in genes coding for the MRN complex have been identified in families with ...breast and ovarian cancer.
In a
negative family with positive history of breast and endometrial cancer, next-generation sequencing-based panel testing identified a mutation in the
gene (NM_005590 c.1090C>T: p.Arg364Ter). This mutation results in a shorter mutated protein lacking 2 DNA binding domains (the GAR domain and the RAD50 binding site), abolishing the function of protein.
This case provides insight into the role of the
gene in causing breast cancer susceptibility in families, and supports the use of multigene panel testing in cases with hereditary predisposition to breast cancer.
Congenital prothrombin deficiency is an extremely rare, autosomal recessive bleeding disorder with a prevalence of 1 in 2 million individuals. Here, we report a case of congenital prothrombin ...deficiency with two concurrent mutations in the prothrombin gene (F2), affecting the heavy B chain. The patient presented with a history of multiple bleeding events in his youth that are mostly trauma associated, with a family history of prothrombin deficiency. Laboratory analysis showed a prolonged activated partial thromboplastin time and a prothrombin activity level of 5%. Genetic analysis of the F2 gene identified two heterozygous variants; one is a previously reported pathogenic deletion (c.1814_1815del; p.His605Argfs*13), and the other is a novel missense variant (c.1147C>T; p.Arg383Trp). In silico analysis predicted that p.Arg383Trp is likely to be disease causing, as it affects one of the anion‐binding exosites‐I of the B chain. This case highlights the significance of molecular findings in confirming the diagnosis of patients with congenital prothrombin deficiency.
Atypical hemolytic uremic syndrome (aHUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Complement and coagulation gene variants have been associated ...with aHUS susceptibility. We assessed the diagnostic yield of a next-generation sequencing (NGS) panel in a large cohort of Canadian patients with suspected aHUS. Molecular testing was performed on peripheral blood DNA samples from 167 patients, collected between May 2019 and December 2021, using a clinically validated NGS pipeline. Coding exons with 20 base pairs of flanking intronic regions for 21 aHUS-associated or candidate genes were enriched using a custom hybridization protocol. All sequence and copy number variants were assessed and classified following American College of Medical Genetics guidelines. Molecular diagnostic results were reported for four variants in three individuals (1.8%). Twenty-seven variants of unknown significance were identified in 25 (15%) patients, and 34 unique variants in candidate genes were identified in 28 individuals. An illustrative patient case describing two genetic alterations in complement genes is presented, highlighting that variable expressivity and incomplete penetrance must be considered when interpreting genetic data in patients with complement-mediated disease, alongside the potential additive effects of genetic variants on aHUS pathophysiology. In this cohort of patients with suspected aHUS, using clinical pipelines for genetic testing and variant classification, pathogenic/likely pathogenic variants occurred in a very small percentage of patients. Our results highlight the ongoing challenges in variant classification following NGS panel testing in patients with suspected aHUS, alongside the need for clear testing guidance in the clinical setting.
Key messages
• Clinical molecular testing for disease associated genes in aHUS is challenging.
• Challenges include patient selection criteria, test validation, and interpretation.
• Most variants were of uncertain significance (31.7% of patients; VUS + candidates).
• Their clinical significance may be elucidated as more evidence becomes available.
• Low molecular diagnostic rate (1.8%), perhaps due to strict classification criteria.
• Case study identified two likely pathogenic variants; one each in
MCP/CD46
and
CFI
.
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