Utility of DNA, RNA, Protein, and Functional Approaches to Solve Cryptic Immunodeficiencies

Cousin, Margot A.; Smith, Matthew J.; Sigafoos, Ashley N.; Jin, Jay; Murphree, Marine I.; Boczek, Nicole J.; Blackburn, Patrick R.; Oliver, Gavin R.; Aleff, Ross A.; Clark, Karl J.; Wieben, Eric D.; Joshi, Avni Y.; Pichurin, Pavel N.; Abraham, Roshini S.; Klee, Eric W.

doi:10.1007/s10875-018-0499-6

Cited by 28 publications

(21 citation statements)

References 61 publications

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“…The detection of AT by NBS was first reported in 2012 in Swedish newborns (6) and subsequently in other jurisdictions where NBS for SCID has been implemented (31,32). We have shown here that AT, if detected early by TREC-based NBS, has a more profound immunological and neurological phenotype, and this intuitively might predict a more severe disease course.…”

Section: Discussionsupporting

confidence: 51%

Ataxia Telangiectasia Diagnosed on Newborn Screening–Case Cohort of 5 Years' Experience

et al. 2019

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Ataxia telangiectasia (AT) is a genetic condition caused by mutations involving ATM (Ataxia Telangiectasia Mutated). This gene is responsible for the expression of a DNA double stranded break repair kinase, the ATM protein kinase. The syndrome encompasses combined immunodeficiency and various degrees of neurological abnormalities and increased risk of malignancy. Typically, patients present early in life with delay in neurological milestones, but very infrequently, with life threatening infections typical of a profound T cell deficiency. It would therefore be unexpected to identify this condition immediately after birth using T cell receptor excision circle (TREC)-based newborn screening (NBS) for SCID. We sought to evaluate the frequency of AT detected by NBS, and to assess immunity as well as the genetic aberrations associated with this early presentation. Here, we describe the clinical, laboratory, and genetic features of patients diagnosed with AT through the Ontario NBS program for SCID, and followed in our center since its inception in 2013. Four patients were diagnosed with AT as a result of low TRECs on NBS. In each case, whole exome sequencing was diagnostic. All of our patients had compound heterozygous mutations involving the FRAP-ATM-TRRAP (FAT) domain of the ATM gene, which appears critical for kinase activity and is highly sensitive to mutagenesis. Our patients presented with profound lymphopenia involving both B and T cells. The ratio of naïve/memory CD45+RA/RO T cells population was variable. T cell repertoire showed decreased T cell diversity. Two out of four patients had decreased specific antibody response to vaccination and hypogammaglobulinemia requiring IVIG replacement. In two patients, profound decreased responses to phytohemagglutinin stimulation was observed. In the other two patients, the initial robust response declined with time. In summary, the rate of detection of AT through NBS had been surprisingly high at our center. One case was identified per year, while the total rate for SCID has been five new cases per year. This early detection may allow for better prospective evaluation of AT shortly after birth, and may assist in formulating early and more effective interventions both for the neurological as well as the immune abnormalities in this syndrome.

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Section: Discussionsupporting

confidence: 51%

Ataxia Telangiectasia Diagnosed on Newborn Screening–Case Cohort of 5 Years' Experience

et al. 2019

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“…The validation of our approach is the small number of candidate events that required manual review (5 in this case) and the ultimate discovery of two fusion events, each verified by clinical aCGH. We continue to pursue validation of fusion events in individuals with undiagnosed diseases (Cousin et al., ) and believe that sustained application of these methods will lead to resolution of further unsolved cases.…”

Section: Discussionmentioning

confidence: 99%

RNA‐Seq detects a SAMD12‐EXT1 fusion transcript and leads to the discovery of an EXT1 deletion in a child with multiple osteochondromas

Oliver

Blackburn

Ellingson

et al. 2019

Molec Gen & Gen Med

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Background We describe a patient presenting with pachygyria, epilepsy, developmental delay, short stature, failure to thrive, facial dysmorphisms, and multiple osteochondromas. Methods The patient underwent extensive genetic testing and analysis in an attempt to diagnose the cause of his condition. Clinical testing included metaphase karyotyping, array comparative genomic hybridization, direct sequencing and multiplex ligation‐dependent probe amplification and trio‐based exome sequencing. Subsequently, research‐based whole transcriptome sequencing was conducted to determine whether it might shed light on the undiagnosed phenotype. Results Clinical exome sequencing of patient and parent samples revealed a maternally inherited splice‐site variant in the doublecortin ( DCX ) gene that was classified as likely pathogenic and diagnostic of the patient's neurological phenotype. Clinical array comparative genome hybridization analysis revealed a 16p13.3 deletion that could not be linked to the patient phenotype based on affected genes. Further clinical testing to determine the cause of the patient's multiple osteochondromas was unrevealing despite extensive profiling of the most likely causative genes, EXT 1 and EXT 2 , including mutation screening by direct sequence analysis and multiplex ligation‐dependent probe amplification. Whole transcriptome sequencing identified a SAMD 12‐ EXT 1 fusion transcript that could have resulted from a chromosomal deletion, leading to the loss of EXT 1 function. Re‐review of the clinical array comparative genomic hybridization results indicated a possible unreported mosaic deletion affecting the SAMD 12 and EXT 1 genes that corresponded precisely to the introns predicted to be affected by a fusion‐causing deletion. The existence of the mosaic deletion was subsequently confirmed clinically by an increased density copy number array and orthogonal methodologies Conclusions While mosaic mutations and deletions of EXT 1 and EXT 2 have been reported in the context of multiple osteochondromas, to our knowledge, this is the first time that transcriptomics technologies have been used to diagnose a patient via fusion transcript analysis in the congenital disease setting.

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“…Blood-derived RNA was obtained by collecting peripheral whole blood in PAXgene blood RNA tubes and using the QIAcube system (Qiagen) according to the manufacturer’s protocol for RNA extraction. RNA was isolated from fibroblasts as previously described [34].…”

Section: Methodsmentioning

confidence: 99%

A tailored approach to fusion transcript identification increases diagnosis of rare inherited disease

et al. 2019

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BackgroundRNA sequencing has been proposed as a means of increasing diagnostic rates in studies of undiagnosed rare inherited disease. Recent studies have reported diagnostic improvements in the range of 7.5–35% by profiling splicing, gene expression quantification and allele specific expression. To-date however, no study has systematically assessed the presence of gene-fusion transcripts in cases of germline disease. Fusion transcripts are routinely identified in cancer studies and are increasingly recognized as having diagnostic, prognostic or therapeutic relevance. Isolated reports exist of fusion transcripts being detected in cases of developmental and neurological phenotypes, and thus, systematic application of fusion detection to germline conditions may further increase diagnostic rates. However, current fusion detection methods are unsuited to the investigation of germline disease due to performance biases arising from their development using tumor, cell-line or in-silico data.MethodsWe describe a tailored approach to fusion candidate identification and prioritization in a cohort of 47 undiagnosed, suspected inherited disease patients. We modify an existing fusion transcript detection algorithm by eliminating its cell line-derived filtering steps, and instead, prioritize candidates using a custom workflow that integrates genomic and transcriptomic sequence alignment, biological and technical annotations, customized categorization logic, and phenotypic prioritization.ResultsWe demonstrate that our approach to fusion transcript identification and prioritization detects genuine fusion events excluded by standard analyses and efficiently removes phenotypically unimportant candidates and false positive events, resulting in a reduced candidate list enriched for events with potential phenotypic relevance. We describe the successful genetic resolution of two previously undiagnosed disease cases through the detection of pathogenic fusion transcripts. Furthermore, we report the experimental validation of five additional cases of fusion transcripts with potential phenotypic relevance.ConclusionsThe approach we describe can be implemented to enable the detection of phenotypically relevant fusion transcripts in studies of rare inherited disease. Fusion transcript detection has the potential to increase diagnostic rates in rare inherited disease and should be included in RNA-based analytical pipelines aimed at genetic diagnosis.

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Utility of DNA, RNA, Protein, and Functional Approaches to Solve Cryptic Immunodeficiencies

Abstract: We identified a novel ATM-breaking chromosome 11 inversion in trans with a pathogenic indel (compound heterozygote) resulting in non-functional ATM protein, consistent with a diagnosis of AT. Utilization of several molecular and functional assays allowed successful resolution of this case.

Cited by 28 publications

References 61 publications

Ataxia Telangiectasia Diagnosed on Newborn Screening–Case Cohort of 5 Years' Experience

Ataxia Telangiectasia Diagnosed on Newborn Screening–Case Cohort of 5 Years' Experience

RNA‐Seq detects a SAMD12‐EXT1 fusion transcript and leads to the discovery of an EXT1 deletion in a child with multiple osteochondromas

A tailored approach to fusion transcript identification increases diagnosis of rare inherited disease

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