Dataset Information

Targeted Next Generation Sequencing and Diagnosis of Congenital Hemolytic Anemias: A Three Years Experience Monocentric Study.

ABSTRACT: Congenital hemolytic anemias (CHAs) are heterogeneous and rare disorders caused by alterations in structure, membrane transport, metabolism, or red blood cell production. The pathophysiology of these diseases, in particular the rarest, is often poorly understood, and easy-to-apply tools for diagnosis, clinical management, and patient stratification are still lacking. We report the 3-years monocentric experience with a 43 genes targeted Next Generation Sequencing (t-NGS) panel in diagnosis of CHAs; 122 patients from 105 unrelated families were investigated and the results compared with conventional laboratory pathway. Patients were divided in two groups: 1) cases diagnosed with hematologic investigations to be confirmed at molecular level, and 2) patients with unexplained anemia after extensive hematologic investigation. The overall sensitivity of t-NGS was 74 and 35% for families of groups 1 and 2, respectively. Inside this cohort of patients we identified 26 new pathogenic variants confirmed by functional evidence. The implementation of laboratory work-up with t-NGS increased the number of diagnoses in cases with unexplained anemia; cytoskeleton defects are well detected by conventional tools, deserving t-NGS to atypical cases; the diagnosis of Gardos channelopathy, some enzyme deficiencies, familial siterosterolemia, X-linked defects in females and other rare and ultra-rare diseases definitely benefits of t-NGS approaches.

SUBMITTER: Fermo E

PROVIDER: S-EPMC8176228 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Targeted Next Generation Sequencing and Diagnosis of Congenital Hemolytic Anemias: A Three Years Experience Monocentric Study.

Fermo Elisa E Vercellati Cristina C Marcello Anna Paola AP Keskin Ebru Yilmaz EY Perrotta Silverio S Zaninoni Anna A Brancaleoni Valentina V Zanella Alberto A Giannotta Juri A JA Barcellini Wilma W Bianchi Paola P

Frontiers in physiology 20210521

Congenital hemolytic anemias (CHAs) are heterogeneous and rare disorders caused by alterations in structure, membrane transport, metabolism, or red blood cell production. The pathophysiology of these diseases, in particular the rarest, is often poorly understood, and easy-to-apply tools for diagnosis, clinical management, and patient stratification are still lacking. We report the 3-years monocentric experience with a 43 genes targeted Next Generation Sequencing (t-NGS) panel in diagnosis of CHA ...[more]

PMID: 34093240

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Project description:Predominantly antibody deficiencies (PAD) are a heterogeneous group of disorders characterized by dysfunctional antibody production, low immunoglobulin levels in serum and impaired vaccine responses. The clinical picture is variable, ranging from mild symptoms to severe complications, which may include autoimmunity, gastrointestinal disease, allergy, and malignancies. If left untreated, PAD patients are at risk of enduring disease progression, irreversible organ damage, and reduced life expectancy. A timely diagnosis has been shown to significantly improve disease prognosis. Here, we report on our experience using targeted gene panel sequencing by employing Agilent's HaloPlex or SureSelect and Illumina's MiSeq technologies in a cohort of 291 individuals who presented with low or absent immunoglobulin levels in combination with or without other clinical features. In total, we have detected over 57 novel or previously reported relevant mutations in ADA, ADA2, BTK, CTLA4, LRBA, NFKB1, NFKB2, PIK3CD, STAT3, and TNFRSF13B. Overall, a genetic diagnosis could be made in 24.7% of the investigated patients. The percentage of coverage for the targeted regions ranged from 90% to 98% in this study. Moreover, functional assays were performed on a defined group of the patients carrying candidate variants in CTLA4, LRBA, NFKB1 and BTK, which confirmed their deleterious effect on protein expression and/or function. This study reiterates that the immunological heterogeneity of predominantly antibody deficiencies may have a diverse genetic origin, although certain clinical features may hint towards a specific group of defects. Employing targeted sequencing panels proves to be a very time- and cost-efficient, yet reliable, method for the establishment of a genetic diagnosis in individuals with PAD. However, in case of negative panel results, or if functional testing reveals inconspicuous observations in patients with a clear indication for genetic testing, further work-up including whole exome or whole genome sequencing should be considered.

Project description:BackgroundDefects in the development of the first and second pharyngeal arches and their derivatives result in abnormal formation of the craniofacial complex, consequently giving rise to facial dysostoses (FDs). FDs represent a group of rare and highly heterogeneous disease entities that encompass mandibulofacial dysostoses (MFDs) with normal extremities and acrofacial dysostoses (AFDs) with limb anomalies in addition to craniofacial defects.MethodsWe examined 11 families with variable clinical symptoms of FDs, in most of which only one member was affected. We applied two custom gene panels-first comprising 37 genes related to the genetic disorders of craniofacial development such as FDs (On-Demand AmpliSeq Thermo Fisher Scientific gene panel with two primer pools) and second composed of 61 genes and 11 single nucleotide variants (SNVs) known to be involved in the development of skull malformations, mainly in the form of craniosynostoses (SureSelect Agilent Technologies). Targeted next-generation sequencing (NGS) was performed using the Ion Torrent S5 platform. To confirm the presence of each detected variant, we have analyzed a genomic region of interest using Sanger sequencing.ResultsIn this paper, we summarized the results of custom targeted gene panel sequencing in the cohort of sixteen patients from 11 consecutive families affected by distinct forms of FDs. We have found three novel pathogenic variants in the TCOF1 gene-c.2145_2148dupAAAG p.(Ser717Lysfs ∗42), c.4370delA p.(Lys1457Argfs ∗118), c.83G>C p.(Arg28Pro) causing Treacher Collins syndrome type 1, two novel missense variants in the EFTUD2 gene-c.491A>G p.(Asp164Gly) and c.779T>A p.(Ile260Asn) in two female patients affected by acrofacial dysostosis Guion-Almeida type, one previously reported-c.403C>T (p.Arg135Cys), as well as one novel missense variant-c.128C>T p.(Pro43Leu) in the DHODH gene in the male patient with Miller syndrome and finally one known pathogenic variant c.574G>T p.(Glu192∗) in the SF3B4 gene in the patient with Nager syndrome.ConclusionOur study confirms the efficiency and clinical utility of the targeted gene panel sequencing and shows that this strategy is suitable and efficient in the molecular screening of variable forms of FDs.

Dataset Information

Targeted Next Generation Sequencing and Diagnosis of Congenital Hemolytic Anemias: A Three Years Experience Monocentric Study.

Publications

Targeted Next Generation Sequencing and Diagnosis of Congenital Hemolytic Anemias: A Three Years Experience Monocentric Study.

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