Dominant negative variants in IKZF2 cause ICHAD syndrome, a new disorder characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay.

BACKGROUND: Helios (encoded by IKZF2), a member of the Ikaros family of transcription factors, is a zinc finger protein involved in embryogenesis and immune function. Although predominantly recognised for its role in the development and function of T lymphocytes, particularly the CD4+ regulatory T cells (Tregs), the expression and function of Helios extends beyond the immune system. During embryogenesis, Helios is expressed in a wide range of tissues, making genetic variants that disrupt the function of Helios strong candidates for causing widespread immune-related and developmental abnormalities in humans. METHODS: We performed detailed phenotypic, genomic and functional investigations on two unrelated individuals with a phenotype of immune dysregulation combined with syndromic features including craniofacial differences, sensorineural hearing loss and congenital abnormalities. RESULTS: Genome sequencing revealed de novo heterozygous variants that alter the critical DNA-binding zinc fingers (ZFs) of Helios. Proband 1 had a tandem duplication of ZFs 2 and 3 in the DNA-binding domain of Helios (p.Gly136_Ser191dup) and Proband 2 had a missense variant impacting one of the key residues for specific base recognition and DNA interaction in ZF2 of Helios (p.Gly153Arg). Functional studies confirmed that both these variant proteins are expressed and that they interfere with the ability of the wild-type Helios protein to perform its canonical function-repressing IL2 transcription activity-in a dominant negative manner. CONCLUSION: This study is the first to describe dominant negative IKZF2 variants. These variants cause a novel genetic syndrome characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay.

Genetic testing in monogenic early-onset atrial fibrillation.

A substantial proportion of atrial fibrillation (AF) cases cannot be explained by acquired AF risk factors. Limited guidelines exist that support routine genetic testing. We aim to determine the prevalence of likely pathogenic and pathogenic variants from AF genes with robust evidence in a well phenotyped early-onset AF population. We performed whole exome sequencing on 200 early-onset AF patients. Variants from exome sequencing in affected individuals were filtered in a multi-step process, prior to undergoing clinical classification using current ACMG/AMP guidelines. 200 AF individuals were recruited from St. Paul's Hospital and London Health Sciences Centre who were ≤ 60 years of age and without any acquired AF risk factors at the time of AF diagnosis. 94 of these AF individuals had very early-onset AF ( ≤ 45). Mean age of AF onset was 43.6 ± 9.4 years, 167 (83.5%) were male and 58 (29.0%) had a confirmed family history. There was a 3.0% diagnostic yield for identifying a likely pathogenic or pathogenic variant across AF genes with robust gene-to-disease association evidence. This study demonstrates the current diagnostic yield for identifying a monogenic cause for AF in a well-phenotyped early-onset AF cohort. Our findings suggest a potential clinical utility for offering different screening and treatment regimens in AF patients with an underlying monogenic defect. However, further work is needed to dissect the additional monogenic and polygenic determinants for patients without a genetic explanation for their AF despite the presence of specific genetic indicators such as young age of onset and/or positive family history.

Genome-wide sequencing as a first-tier screening test for short tandem repeat expansions.

BACKGROUND: Screening for short tandem repeat (STR) expansions in next-generation sequencing data can enable diagnosis, optimal clinical management/treatment, and accurate genetic counseling of patients with repeat expansion disorders. We aimed to develop an efficient computational workflow for reliable detection of STR expansions in next-generation sequencing data and demonstrate its clinical utility. METHODS: We characterized the performance of eight STR analysis methods (lobSTR, HipSTR, RepeatSeq, ExpansionHunter, TREDPARSE, GangSTR, STRetch, and exSTRa) on next-generation sequencing datasets of samples with known disease-causing full-mutation STR expansions and genomes simulated to harbor repeat expansions at selected loci and optimized their sensitivity. We then used a machine learning decision tree classifier to identify an optimal combination of methods for full-mutation detection. In Burrows-Wheeler Aligner (BWA)-aligned genomes, the ensemble approach of using ExpansionHunter, STRetch, and exSTRa performed the best (precision = 82%, recall = 100%, F1-score = 90%). We applied this pipeline to screen 301 families of children with suspected genetic disorders. RESULTS: We identified 10 individuals with full-mutations in the AR, ATXN1, ATXN8, DMPK, FXN, or HTT disease STR locus in the analyzed families. Additional candidates identified in our analysis include two probands with borderline ATXN2 expansions between the established repeat size range for reduced-penetrance and full-penetrance full-mutation and seven individuals with FMR1 CGG repeats in the intermediate/premutation repeat size range. In 67 probands with a prior negative clinical PCR test for the FMR1, FXN, or DMPK disease STR locus, or the spinocerebellar ataxia disease STR panel, our pipeline did not falsely identify aberrant expansion. We performed clinical PCR tests on seven (out of 10) full-mutation samples identified by our pipeline and confirmed the expansion status in all, showing absolute concordance between our bioinformatics and molecular findings. CONCLUSIONS: We have successfully demonstrated the application of a well-optimized bioinformatics pipeline that promotes the utility of genome-wide sequencing as a first-tier screening test to detect expansions of known disease STRs. Interrogating clinical next-generation sequencing data for pathogenic STR expansions using our ensemble pipeline can improve diagnostic yield and enhance clinical outcomes for patients with repeat expansion disorders.

Genetic Landscapes of Relapsed and Refractory Diffuse Large B-Cell Lymphomas.

PURPOSE: Relapsed or refractory diffuse large B-cell lymphoma (rrDLBCL) is fatal in 90% of patients, and yet little is known about its biology. EXPERIMENTAL DESIGN: Using exome sequencing, we characterized the mutation profiles of 38 rrDLBCL biopsies obtained at the time of progression after immunochemotherapy. To identify genes that may be associated with relapse, we compared the mutation frequency in samples obtained at relapse to an unrelated cohort of 138 diagnostic DLBCLs and separately amplified specific mutations in their matched diagnostic samples to identify clonal expansions. RESULTS: On the basis of a higher frequency at relapse and evidence for clonal selection, TP53, FOXO1, MLL3 (KMT2C), CCND3, NFKBIZ, and STAT6 emerged as top candidate genes implicated in therapeutic resistance. We observed individual examples of clonal expansions affecting genes whose mutations had not been previously associated with DLBCL including two regulators of NF-κB: NFKBIE and NFKBIZ We detected mutations that may be affect sensitivity to novel therapeutics, such as MYD88 and CD79B mutations, in 31% and 23% of patients with activated B-cell-type of rrDLBCL, respectively. We also identified recurrent STAT6 mutations affecting D419 in 36% of patients with the germinal center B (GCB) cell rrDLBCL. These were associated with activated JAK/STAT signaling, increased phospho-STAT6 protein expression and increased expression of STAT6 target genes. CONCLUSIONS: This work improves our understanding of therapeutic resistance in rrDLBCL and has identified novel therapeutic opportunities especially for the high-risk patients with GCB-type rrDLBCL. Clin Cancer Res; 22(9); 2290-300. ©2015 AACR.

Comprehensive genetic analysis identifies a pathognomonic NAB2/STAT6 fusion gene, nonrandom secondary genomic imbalances, and a characteristic gene expression profile in solitary fibrous tumor.

Solitary fibrous tumor (SFT) is a mesenchymal neoplasm displaying variable morphologic and clinical features. To identify pathogenetically important genetic rearrangements, 44 SFTs were analyzed using a variety of techniques. Chromosome banding and fluorescence in situ hybridization (FISH) showed recurrent breakpoints in 12q13, clustering near the NAB2 and STAT6 genes, and single nucleotide polymorphism array analysis disclosed frequent deletions affecting STAT6. Quantitative real-time PCR revealed high expression levels of the 5'-end of NAB2 and the 3'-end of STAT6, which at deep sequencing of enriched DNA corresponded to NAB2/STAT6 fusions. Subsequent reverse-transcriptase PCR (RT-PCR) analysis identified a NAB2/STAT6 fusion in 37/41 cases, confirming that this fusion gene underlies the pathogenesis of SFT. The hypothesis that the NAB2/STAT6 fusions will result in altered properties of the transcriptional co-repressor NAB2--a key regulator of the early growth response 1 (EGR1) transcription factor - was corroborated by global gene expression analysis; SFTs showed deregulated expression of EGR1 target genes, as well as of other, developmentally important genes. We also identified several nonrandom secondary changes, notably loss of material from 13q and 14q. As neither chromosome banding nor FISH analysis identify more than a minor fraction of the fusion-positive cases, and because multiple primer combinations are required to identify all possible fusion transcripts by RT-PCR, alternative diagnostic markers might instead be found among deregulated genes identified at global gene expression analysis. Indeed, using immunohistochemistry on tissue microarrays, the top up-regulated gene, GRIA2, was found to be differentially expressed also at the protein level.

SNP array and FISH findings in two pleomorphic hyalinizing angiectatic tumors.

Pleomorphic hyalinizing angiectatic tumor (PHAT) is a rare soft tissue tumor of intermediate malignancy and uncertain cellular origin and lineage of differentiation. Although PHAT is still poorly characterized at the genetic level, there is a potential genetic overlap with two other soft tissue tumors: myxoinflammatory fibroblastic sarcoma (MIFS) and hemosiderotic fibrolipomatous tumor (HFLT); MIFS and HFLT share a characteristic t(1;10)(p22;q24) with breakpoints in the TGFBR3 locus on chromosome 1 and near the MGEA5 locus on chromosome 10. Recently, a PHAT with a similar t(1;10) was reported, suggesting a genetic link between MIFS/HFLT and PHAT. To ascertain whether PHAT is also associated with this translocation, two cases were subjected to single nucleotide polymorphism (SNP) array and fluorescence in situ hybridization analyses. Neither PHAT showed a t(1;10) or other types of rearrangement of the TGFBR3 or MGEA5 loci. Both tumors showed imbalances in the SNP array analysis, but none was shared. Thus, the results indicate that PHAT is genetically distinguishable from MIFS and HFLT, but further studies are needed to identify the salient genetic pathways involved in PHAT development.