Evaluation of pathogenic variants detected in high homology regions of the PMS2 gene. How effective is long-range PCR?

Introduction: Lynch syndrome (LS) is an inherited cancer predisposition syndrome characterized by a high risk of colorectal and extracolonic tumors. Germline pathogenic variants (GPV) in the PMS2 gene are associated with <15% of all cases. The PMS2CL pseudogene presents high homology with PMS2, challenging molecular diagnosis by next-generation sequencing (NGS). Due to the high methodological complexity required to distinguish variants between PMS2 and PMS2CL, most laboratories do not clearly report the origin of this molecular finding. Objective: The aim of this study was to confirm the GPVs detected by NGS in regions of high homology segments of the PMS2 gene in a Brazilian sample. Methods: An orthogonal and gold standard long-range PCR (LR-PCR) methodology to separate variants detected in the PMS2 gene from those detected in the pseudogene. Results: A total of 74 samples with a PMS2 GPV detected by NGS in exons with high homology with PMS2CL pseudogene were evaluated. The most common was NM_000535.6:c.2182_2184delinsG, which was previously described as deleterious mutation in a study of African-American patients with LS and has been widely reported by laboratories as a pathogenic variant associated with the LS phenotype. Of all GPVs identified, only 6.8% were confirmed by LR-PCR. Conversely, more than 90% of GPV were not confirmed after LR-PCR, and the diagnosis of LS was ruled out by molecular mechanisms associated with PMS2. Conclusion: In conclusion, the use of LR-PCR was demonstrated to be a reliable approach for accurate molecular analysis of PMS2 variants in segments with high homology with PMS2CL. We highlight that our laboratory is a pioneer in routine diagnostic complementation of the PMS2 gene in Brazil, directly contributing to a more assertive molecular diagnosis and adequate genetic counseling for these patients and their families.

Copy number variation in pituitary stalk interruption syndrome: A large case series of sporadic non-syndromic patients and literature review.

Abnormal hypothalamic/posterior pituitary development appears to be a major determinant of pituitary stalk interruption syndrome (PSIS). The observation of familial cases and associated congenital abnormalities suggests a genetic basis. Single-gene mutations explain less than 5% of the cases, and whole exome sequencing has shown heterogeneous results. The present study aimed to assess copy number variation (CNV) using array-based comparative genomic hybridization (aCGH) in patients with non-syndromic PSIS and comprehensively review data from the literature on CNV analysis in congenital hypopituitarism (CH) patients. Twenty-one patients with sporadic CH from our outpatient clinics presented with ectopic posterior pituitary (EPP) and no central nervous system abnormalities on magnetic resonance image (MRI) or any other malformations on physical examination at presentation were enrolled in the study. aCGH using a whole-genome customized 400K oligonucleotide platform was performed in our patients. For the literature review, we searched for case reports of patients with CH and CNV detected by either karyotype or aCGH reported in PubMed up to November 2021. Thirty-five distinct rare CNVs were observed in 18 patients (86%) and two of them (6%) were classified as pathogenic: one deletion of 1.8 Mb in chromosome 17 (17q12) and one deletion of 15 Mb in chromosome 18 (18p11.32p11.21), each one in a distinct patient. In the literature review, 67 pathogenic CNVs were published in 83 patients with CH, including the present study. Most of these patients had EPP (78% out of the 45 evaluated by sellar MRI) and were syndromic (70%). The most frequently affected chromosomes were X, 18, 20 and 1. Our study has found that CNV can be a mechanism of genetic abnormality in non-syndromic patients with CH and EPP. In future studies, one or more genes in those CNVs, both pathogenic and variant of uncertain significance, may be considered as good candidate genes.

Diagnostic power and clinical impact of exome sequencing in a cohort of 500 patients with rare diseases.

Rare diseases comprise a diverse group of conditions, most of which involve genetic causes. We describe the variable spectrum of findings and clinical impacts of exome sequencing (ES) in a cohort of 500 patients with rare diseases. In total, 164 primary findings were reported in 158 patients, representing an overall diagnostic yield of 31.6%. Most of the findings (61.6%) corresponded to autosomal dominant conditions, followed by autosomal recessive (25.6%) and X-linked (12.8%) conditions. These patients harbored 195 variants, among which 43.6% are novel in the literature. The rate of molecular diagnosis was considerably higher for prenatal samples (67%; 4/6), younger children (44%; 24/55), consanguinity (50%; 3/6), gastrointestinal/liver disease (44%; 16/36) and syndromic/malformative conditions (41%; 72/175). For 15.6% of the cohort patients, we observed a direct potential for the redirection of care with targeted therapy, tumor screening, medication adjustment and monitoring for disease-specific complications. Secondary findings were reported in 37 patients (7.4%). Based on cost-effectiveness studies in the literature, we speculate that the reports of secondary findings may influence an increase of 123.2 years in the life expectancy for our cohort, or 0.246 years/cohort patient. ES is a powerful method to identify the molecular bases of monogenic disorders and redirect clinical care.

JAK2-mutated acute myeloid leukemia: comparison of next-generation sequencing (NGS) and single nucleotide polymorphism array (SNPa) findings between two cases.

JAK2 mutations are rare in de novo acute myeloid leukemia (AML), and JAK2-mutated acute myeloid leukemia (AML) patients usually have a previous history of myeloproliferative neoplasms (MPNs). Current advances in laboratory techniques, such as single nucleotide polymorphism array (SNPa) and next-generation sequencing (NGS), have facilitated new insight into the molecular basis of hematologic diseases. Herein, we present two cases of JAK2-mutated AML in which both SNPa and NGS methods added valuable information. Both cases had leukemogenic collaboration, namely, copy-neutral loss of heterozygosity (CN-LOH), detected on chromosome 9. One of the cases exhibited both JAK2 and IDH2 mutations, most likely having originated as an MPN with leukemic transformation, while the other case was classified as a de novo AML with JAK2, CEBPA, and FLT3 mutations.

JAK2-mutated acute myeloid leukemia: comparison of next-generation sequencing (NGS) and single nucleotide polymorphism array (SNPa) findings between two cases

JAK2 mutations are rare in de novo acute myeloid leukemia (AML), and JAK2-mutated acute myeloid leukemia (AML) patients usually have a previous history of myeloproliferative neoplasms (MPNs). Current advances in laboratory techniques, such as single nucleotide polymorphism array (SNPa) and next-generation sequencing (NGS), have facilitated new insight into the molecular basis of hematologic diseases. Herein, we present two cases of JAK2-mutated AML in which both SNPa and NGS methods added valuable information. Both cases had leukemogenic collaboration, namely, copy-neutral loss of heterozygosity (CN-LOH), detected on chromosome 9. One of the cases exhibited both JAK2 and IDH2 mutations, most likely having originated as an MPN with leukemic transformation, while the other case was classified as a de novo AML with JAK2, CEBPA, and FLT3 mutations.

Mutational profiling of acute myeloid leukemia with normal cytogenetics in Brazilian patients: the value of next-generation sequencing for genomic classification.

Karyotype (KT) aberrations are important prognostic factors for acute myeloid leukemia (AML); however, around 50% of cases present normal results. Single nucleotide polymorphism array can detect chromosomal gains, losses or uniparental disomy that are invisible to KT, thus improving patients' risk assessment. However, when both tests are normal, important driver mutations can be detected by the use of next-generation sequencing (NGS). Fourteen adult patients with AML with normal cytogenetics were investigated by NGS for 19 AML-related genes. Every patient presented at least one mutation: DNMT3A in nine patients; IDH2 in six; IDH1 in three; NRAS and NPM1 in two; and TET2, ASXL1, PTPN11, and RUNX1 in one patient. No mutations were found in FLT3, KIT, JAK2, CEBPA, GATA2, TP53, BRAF, CBL, KRAS, and WT1 genes. Twelve patients (86%) had at least one mutation in genes related with DNA methylation (DNMT3A, IDH1, IDH2, and TET2), which is involved in regulation of gene expression and genomic stability. All patients could be reclassified based on genomic status and nine had their prognosis modified. In summary, NGS offers insights into the molecular pathogenesis and biology of cytogenetically normal AML in Brazilian patients, indicating that the prognosis could be further stratified by different mutation combinations. This study shows a different frequency of mutations in Brazilian population that should be confirmed.

Development and validation of a variant detection workflow for BRCA1 and BRCA2 genes and its clinical application based on the Ion Torrent technology.

BACKGROUND: Breast cancer is the most common among women worldwide, and ovarian cancer is the most difficult gynecological tumor to diagnose and with the lowest chance of cure. Mutations in BRCA1 and BRCA2 genes increase the risk of ovarian cancer by 60% and breast cancer by up to 80% in women. Molecular tests allow a better orientation for patients carrying these mutations, affecting prophylaxis, treatment, and genetic counseling. RESULTS: Here, we evaluated the performance of a panel for BRCA1 and BRCA2, using the Ion Torrent PGM (Life Technologies) platform in a customized workflow and multiplex ligation-dependent probe amplification for detection of mutations, insertions, and deletions in these genes. We validated the panel with 26 samples previously analyzed by Myriad Genetics Laboratory, and our workflow showed 95.6% sensitivity and 100% agreement with Myriad reports, with 85% sensitivity on the positive control sample from NIST. We also screened 68 clinical samples and found 22 distinct mutations. CONCLUSIONS: The selection of a robust methodology for sample preparation and sequencing, together with bioinformatics tools optimized for the data analysis, enabled the development of a very sensitive test with high reproducibility. We also highlight the need to explore the limitations of the NGS technique and the strategies to overcome them in a clinically confident manner.

Different Donors Mesenchymal Stromal Cells Secretomes Reveal Heterogeneous Profile of Relevance for Therapeutic Use.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by null mutations in the dystrophin gene. Although the primary defect is the deficiency of muscle dystrophin, secondary events, including chronic inflammation, fibrosis, and muscle regeneration failure are thought to actively contribute to disease progression. Despite several advances, there is still no effective therapy for DMD. Therefore, the potential regenerative capacities, and immune-privileged properties of mesenchymal stromal cells (MSCs), have been the focus of intense investigation in different animal models aiming the treatment of these disorders. However, these studies have shown different outcomes according to the sources from which MSCs were obtained, which raise the question whether stem cells from distinct sources have comparable clinical effects. Here, we analyzed the protein content of the secretome of MSCs, isolated from three different sources (adipose tissue, skeletal muscle, and uterine tubes), obtained from five donors and evaluated their in vitro properties when cocultured with DMD myoblasts. All MSC lineages showed pathways enrichment related to protein metabolic process, oxidation-reduction process, cell proliferation, and regulation of apoptosis. We found that MSCs secretome proteins and their effect in vitro vary significantly according to the tissue and donors, including opposite effects in apoptosis assay, indicating the importance of characterizing MSC secretome profile before its use in animal and clinical trials. Despite the individual differences a pool of conditioned media from all MSCs lineages was able to delay apoptosis and enhance migration when in contact with DMD myoblasts.

CALR mutations screening in wild type JAK2(V617F) and MPL(W515K/L) Brazilian myeloproliferative neoplasm patients.

Some myeloproliferative neoplasm (MPN) patients harbor JAK2(V617F) mutation, and CALR mutations were recently discovered in wild type (WT) JAK2(V617F). We evaluated the frequency and type of CALR mutations, and clinical and hematological characteristics in WT JAK2(V617F) and MPL(W515K/L) MPN patients. Sixty-five patients were included: 21 with primary myelofibrosis (PMF), 21 with myelofibrosis post-essential thrombocythemia (MPET) and 23 with essential thrombocythemia (ET). Screening for JAK2(V617F) and MPL(W515K/L) were performed using real-time PCR, while CALR mutations were analyzed by fragment analysis and Sanger sequencing. JAK2(V617F) was the most frequent mutation (54.5%) and one patient (1.5%) harbored MPL(W515L). CALR mutations were present in 38.1% of PMF, 12.5% of ET and 33.3% of MPET patients. Five types of CALR mutations were detected, among which type 1 (32.1%) and type 2 (21.4%) were found to be the most common. A novel CALR mutation in a PMF patient was found. Patients carrying CALR mutations had higher platelet count and less presence of splenomegaly than JAK2(V617F), while triple negatives had higher C-reactive protein levels than CALR mutant carriers. Screening for CALR mutations and its correlation with clinical features could be useful for the characterization of MPN patients and result in its incorporation into a new prognostic score.

Molecular genetic tests for JAK2V617F, Exon12_JAK2 and MPLW515K/L are highly informative in the evaluation of patients suspected to have BCR-ABL1-negative myeloproliferative neoplasms.

Polycythaemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (MF), are the most common myeloproliferative neoplasms (MPN) in patients without the BCR-ABL1 gene rearrangement. They are caused by clonal expansion of haematopoietic stem cells and share, as a diagnostic criterion, the identification of JAK2V617F mutation. Classically, when other clinical criteria are present, a JAK2V617F negative case requires the analysis of Exon12_JAK2 for the diagnosis of PV, and of MPL515K/L mutations for the diagnosis of ET and MF. Here, we evaluated 78 samples from Brazilian patients suspected to have MPN, without stratification for PV, ET or MF. We found that 28 (35.9%) are JAK2V617F carriers; from the 50 remaining samples, one (2%) showed an Exon12_JAK2 mutation, and another (2%) was positive for MPLW515L mutation. In summary, the investigation of JAK2V617F, Exon12_JAK2 and MPLW515K/L was relevant for the diagnosis of 38.4% of patients suspected to have BCR-ABL1-negative MPN, suggesting that molecular genetic tests are useful for a quick and unequivocal diagnosis of MPN.

A multi-exonic SPG4 duplication underlies sex-dependent penetrance of hereditary spastic paraplegia in a large Brazilian pedigree.

SPG4 mutations are the most frequent cause of autosomal-dominant hereditary spastic paraplegia (HSP). SPG4 HSP is characterized by large inter- and intrafamilial variability in age at onset (AAO) and disease severity. The broad spectrum of SPG4 mutations has recently been further extended by the finding of large genomic deletions in SPG4-linked pedigrees negative for 'small' mutations. We had previously reported a very large pedigree, linked to the SPG4 locus with many affected members, which showed gender difference in clinical manifestation. Screening for copy number aberrations revealed the first case of a multi-exonic duplication (exon10_12dup) in the SPG4 gene. The mutation leads to a premature stop codon, suggesting that the protein product is not functional. The analysis of 30 individuals who carry the mutation showed that males have on average an earlier AAO and are more severely affected. The present family suggests that this HSP pathogenesis may be modulated by factors related to individual background and gender as observed for other autosomal dominant conditions, such as facio-scapulohumeral muscular dystrophy or amyloidosis. Understanding why some individuals, particularly women, are 'partially protected' from the effects of this and other pathogenic mutations is of utmost importance.