EGFR internal tandem duplications in fusion-negative congenital and neonatal spindle cell tumors.

Next-generation sequencing (NGS) assays can sensitively detect somatic variation, and increasingly can enable the identification of complex structural rearrangements. A subset of infantile spindle cell sarcomas, particularly congenital mesoblastic nephromas with classic or mixed histology, have structural rearrangement in the form of internal tandem duplications (ITD) involving EGFR. We performed prospective analysis to identify EGFR ITD through clinical or research studies, as well as retrospective analysis to quantify the frequency of EGFR ITD in pediatric sarcomas. Within our institution, three tumors with EGFR ITD were prospectively identified, all occurring in patients less than 1 year of age at diagnosis, including two renal tumors and one mediastinal soft tissue tumor. These three cases exhibited both cellular and mixed cellular and classic histology. All patients had no evidence of disease progression off therapy, despite incomplete resection. To extend our analysis and quantify the frequency of EGFR ITD in pediatric sarcomas, we retrospectively analyzed a cohort of tumors (n = 90) that were previously negative for clinical RT-PCR-based fusion testing. We identified EGFR ITD in three analyzed cases, all in patients less than 1 year of age (n = 18; 3/18, 17%). Here we expand the spectrum of tumors with EGFR ITD to congenital soft tissue tumors and report an unusual example of an EGFR ITD in a tumor with cellular congenital mesoblastic nephroma histology. We also highlight the importance of appropriate test selection and bioinformatic analysis for identification of this genomic alteration that is unexpectedly common in congenital and infantile spindle cell tumors.

Refining prognosis in chronic lymphocytic leukemia with normal Fluorescence in situ hybridization results.

Fluorescence in situ hybridization (FISH) to detect the recurrent cytogenetics abnormalities deletion 13q, trisomy 12, deletion 11q, and deletion 17p is important for prognostication in chronic lymphocytic leukemia (CLL). A subset of patients are negative for each of these abnormalities (normal 12/13/11/17 FISH), and outcomes are heterogenous within this group. To elucidate variables important for prognostication in this subgroup we conducted a retrospective analysis of 280 treatment-naïve CLL patients with normal standard CLL FISH results. In a multivariable model, advanced Rai stage (p = 0.04, hazard ratio [HR] 1.24 (95% confidence interval [CI] 1.01-1.53)), unmutated immunoglobulin heavy chain gene (IGHV) (p < 0.0001, HR 5.59 (95% CI 3.63-8.62)) and IGH rearrangement by FISH (p = 0.02, HR 2.56 (95% CI 1.20-5.48)) were significantly associated with shorter time to first treatment. In a multivariable model for overall survival, increasing age at 5-year increments (p < 0.0001, HR 1.55 (95% CI 1.25-1.93)), unmutated IGHV (p = 0.01, HR 5.28 (95% CI 1.52-18.35)) and gain of REL (p = 0.01, HR 4.08 (5% CI 1.45-11.49)) were significantly associated with shorter survival. Our study identifies variables important for refining prognosis for CLL patients with normal standard CLL FISH results.

Loss of Baiap2l2 destabilizes the transducing stereocilia of cochlear hair cells and leads to deafness.

KEY POINTS: Mechanoelectrical transduction at auditory hair cells requires highly specialized stereociliary bundles that project from their apical surface, forming a characteristic graded 'staircase' structure. The morphogenesis and maintenance of these stereociliary bundles is a tightly regulated process requiring the involvement of several actin-binding proteins, many of which are still unidentified. We identify a new stereociliary protein, the I-BAR protein BAIAP2L2, which localizes to the tips of the shorter transducing stereocilia in both inner and outer hair cells (IHCs and OHCs). We find that Baiap2l2 deficient mice lose their second and third rows of stereocilia, their mechanoelectrical transducer current, and develop progressive hearing loss, becoming deaf by 8 months of age. We demonstrate that BAIAP2L2 localization to stereocilia tips is dependent on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is a new key protein required for the maintenance of the transducing stereocilia in mature cochlear hair cells. ABSTRACT: The transduction of sound waves into electrical signals depends upon mechanosensitive stereociliary bundles that project from the apical surface of hair cells within the cochlea. The height and width of these actin-based stereocilia is tightly regulated throughout life to establish and maintain their characteristic staircase-like structure, which is essential for normal mechanoelectrical transduction. Here, we show that BAIAP2L2, a member of the I-BAR protein family, is a newly identified hair bundle protein that is localized to the tips of the shorter rows of transducing stereocilia in mouse cochlear hair cells. BAIAP2L2 was detected by immunohistochemistry from postnatal day 2.5 (P2.5) throughout adulthood. In Baiap2l2 deficient mice, outer hair cells (OHCs), but not inner hair cells (IHCs), began to lose their third row of stereocilia and showed a reduction in the size of the mechanoelectrical transducer current from just after P9. Over the following post-hearing weeks, the ordered staircase structure of the bundle progressively deteriorates, such that, by 8 months of age, both OHCs and IHCs of Baiap2l2 deficient mice have lost most of the second and third rows of stereocilia and become deaf. We also found that BAIAP2L2 interacts with other key stereociliary proteins involved in normal hair bundle morphogenesis, such as CDC42, RAC1, EPS8 and ESPNL. Furthermore, we show that BAIAP2L2 localization to the stereocilia tips depends on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is key to maintenance of the normal actin structure of the transducing stereocilia in mature mouse cochlear hair cells.

Activation of the RAS pathway through uncommon BRAF mutations in mucinous pancreatic cysts without KRAS mutation.

Diagnostic testing of pancreatic cyst fluid obtained by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has traditionally utilized elevated carcinoembryonic antigen (CEA) (≥192 ng/ml) and cytomorphologic examination to differentiate premalignant mucinous from benign pancreatic cystic lesions (PCLs). Molecular testing for KRAS/GNAS mutations has been shown to improve accuracy of detecting mucinous PCLs. Using a targeted next-generation sequencing (NGS) panel, we assess the status of PCL-associated mutations to improve understanding of the key diagnostic variables. Molecular analysis of cyst fluid was performed on 108 PCLs that had concurrent CEA and/or cytological analysis. A 48-gene NGS assay was utilized, which included genes commonly mutated in mucinous PCLs such as GNAS, KRAS, CDKN2A, and TP53. KRAS and/or GNAS mutations were seen in 59 of 68 (86.8%) cases with multimodality diagnosis of a mucinous PCL. Among 31 patients where surgical histopathology was available, the sensitivity, specificity, and diagnostic accuracy of NGS for the diagnosis of mucinous PCL was 88.5%, 100%, and 90.3%, respectively. Cytology with mucinous/atypical findings were found in only 29 of 62 cases (46.8%), with fluid CEA elevated in 33 of 58 cases (56.9%). Multiple KRAS mutations at different variant allele frequencies were seen in seven cases favoring multiclonal patterns, with six of them showing at least two separate PCLs by imaging. Among the 6 of 10 cases with GNAS + /KRAS- results, uncommon, non-V600E exon 11/15 hotspot BRAF mutations were identified. The expected high degree of accuracy of NGS detection of KRAS and/or GNAS mutations for mucinous-PCLs, as compared with CEA and cytological examination, was demonstrated. Multiple KRAS mutations correlated with multifocal cysts demonstrated by radiology. In IPMNs that lacked KRAS mutations, the concurring BRAF mutations with GNAS mutations supports an alternate mechanism of activation in the Ras pathway.

Somatic PIK3R1 variation as a cause of vascular malformations and overgrowth.

PURPOSE: Somatic activating variants in the PI3K-AKT pathway cause vascular malformations with and without overgrowth. We previously reported an individual with capillary and lymphatic malformation harboring a pathogenic somatic variant in PIK3R1, which encodes three PI3K complex regulatory subunits. Here, we investigate PIK3R1 in a large cohort with vascular anomalies and identify an additional 16 individuals with somatic mosaic variants in PIK3R1. METHODS: Affected tissue from individuals with vascular lesions and overgrowth recruited from a multisite collaborative network was studied. Next-generation sequencing targeting coding regions of cell-signaling and cancer-associated genes was performed followed by assessment of variant pathogenicity. RESULTS: The phenotypic and variant spectrum associated with somatic variation in PIK3R1 is reported herein. Variants occurred in the inter-SH2 or N-terminal SH2 domains of all three PIK3R1 protein products. Phenotypic features overlapped those of the PIK3CA-related overgrowth spectrum (PROS). These overlapping features included mixed vascular malformations, sandal toe gap deformity with macrodactyly, lymphatic malformations, venous ectasias, and overgrowth of soft tissue or bone. CONCLUSION: Somatic PIK3R1 variants sharing attributes with cancer-associated variants cause complex vascular malformations and overgrowth. The PIK3R1-associated phenotypic spectrum overlaps with PROS. These data extend understanding of the diverse phenotypic spectrum attributable to genetic variation in the PI3K-AKT pathway.

Complete sequencing of the SMN2 gene in SMA patients detects SMN gene deletion junctions and variants in SMN2 that modify the SMA phenotype.

Spinal muscular atrophy (SMA) is a progressive motor neuron disease caused by loss or mutation of the survival motor neuron 1 (SMN1) gene and retention of SMN2. We performed targeted capture and sequencing of the SMN2, CFTR, and PLS3 genes in 217 SMA patients. We identified a 6.3 kilobase deletion that occurred in both SMN1 and SMN2 (SMN1/2) and removed exons 7 and 8. The deletion junction was flanked by a 21 bp repeat that occurred 15 times in the SMN1/2 gene. We screened for its presence in 466 individuals with the known SMN1 and SMN2 copy numbers. In individuals with 1 SMN1 and 0 SMN2 copies, the deletion occurred in 63% of cases. We modeled the deletion junction frequency and determined that the deletion occurred in both SMN1 and SMN2. We have identified the first deletion junction where the deletion removes exons 7 and 8 of SMN1/2. As it occurred in SMN1, it is a pathogenic mutation. We called variants in the PLS3 and SMN2 genes, and tested for association with mild or severe exception patients. The variants A-44G, A-549G, and C-1897T in intron 6 of SMN2 were significantly associated with mild exception patients, but no PLS3 variants correlated with severity. The variants occurred in 14 out of 58 of our mild exception patients, indicating that mild exception patients with an intact SMN2 gene and without modifying variants occur. This sample set can be used in the association analysis of candidate genes outside of SMN2 that modify the SMA phenotype.

Correlation of actin crosslinker and capper expression levels with stereocilia growth phases.

During development of the chick cochlea, actin crosslinkers and barbed-end cappers presumably influence growth and remodeling of the actin paracrystal of hair cell stereocilia. We used mass spectrometry to identify and quantify major actin-associated proteins of the cochlear sensory epithelium from E14 to E21, when stereocilia widen and lengthen. Tight actin crosslinkers (i.e. fascins, plastins, and espin) are expressed dynamically during cochlear epithelium development between E7 and E21, with FSCN2 replacing FSCN1 and plastins remaining low in abundance. Capping protein, a barbed-end actin capper, is located at stereocilia tips; it is abundant during growth phase II, when stereocilia have ceased elongating and are increasing in diameter. Capping protein levels then decline during growth phase III, when stereocilia reinitiate barbed-end elongation. Although actin crosslinkers are readily detected by electron microscopy in developing chick cochlea stereocilia, quantitative mass spectrometry of stereocilia isolated from E21 chick cochlea indicated that tight crosslinkers are present there in stoichiometric ratios relative to actin that are much lower than their ratios for vestibular stereocilia. These results demonstrate the value of quantitation of global protein expression in chick cochlea during stereocilia development.

FindDNAFusion: An Analytical Pipeline with Multiple Software Tools Improves Detection of Cancer-Associated Gene Fusions from Genomic DNA.

Detection of cancer-associated gene fusions is crucial for diagnosis, prognosis, and treatment selection. Many bioinformatics tools are available for the detection of fusion transcripts by RNA sequencing, but there are fewer well-validated software tools for DNA next-generation sequencing (NGS). A 542-gene solid tumor NGS panel was designed, with exonic probes supplemented with intronic bait probes against genes commonly involved in oncogenic fusions, with a focus on lung cancer. Three software tools for the detecting gene fusions in this DNA-NGS panel were selected and evaluated. The performance of these tools was compared after a pilot study, and each was configured for optimal batch analysis and detection rate. A blacklist for filtering common tool-specific artifacts, and criteria for selecting clinically reportable fusions, were established. Visualization tools for annotating and confirming somatic fusions were applied. Subsequently, a full clinical validation was used for comparing the results to those from in situ hybridization and/or RNA sequencing. With JuLI, Factera, and GeneFuse, 94.1%, 88.2%, and 66.7% of expected fusions were detected, respectively. With a combinatorial pipeline (termed FindDNAFusion), developed by integrating fusion-calling tools with methods for fusion filtering, annotating, and flagging reportable calls, the accuracy of detection of intron-tiled genes was improved to 98.0%. FindDNAFusion is an accurate and efficient tool in detecting somatic fusions in DNA-NGS panels with intron-tiled bait probes when RNA is unavailable.

Analytic validation of an FGFR -focused cell-free DNA liquid biopsy assay (FGFR-Dx).

Commercial liquid biopsy assays are routinely used by oncologists to monitor disease response and resistance to therapy. Additionally, in cases where tumor tissue is not available, clinicians may rely on cell-free DNA (cfDNA) testing as a surrogate for comprehensive tumor testing. While some gene rearrangements are well detected, current commercial liquid biopsy assays exhibit low sensitivity for fibroblast growth factor receptor ( FGFR ) rearrangements. FGFRs are altered in ∼2.5% of all cancers, including FGFR2 rearrangements in 10% of intrahepatic cholangiocarcinoma and FGFR3 point mutations and rearrangements in 10-15% of urothelial carcinoma. Therefore, we developed and analytically validated FGFR-Dx, an FGFR -focused cfDNA assay with improved sensitivity for FGFR rearrangements. FGFR-Dx comprehensively targets the introns in FGFR1-3 previously shown to be involved in gene fusions as well as all coding exons. Custom FGFR synthetic reference standards representing both single nucleotide variants (SNVs) and gene rearrangements were utilized at a range of variant frequencies and revealed a detection limit of 0.5% with sensitivities of 97.2% and 92.9% for SNVs and rearrangements, respectively. Furthermore, FGFR-Dx detected rearrangements and identified the intronic breakpoints from cfDNA collected from 13 of 15 patients with known FGFR fusions.

FGFR2-fusions define a clinically actionable molecular subset of pancreatic cancer.

Genomic alterations in fibroblast growth factor receptor (FGFR) genes are present in a small number of metastatic pancreatic ductal adenocarcinomas (PDAC) and may represent an emerging subgroup of patients likely to benefit from FGFR targeted therapies. Here we present four FGFR2 fusion-positive metastatic PDAC patients who exhibited durable responses or disease control to FGFR kinase inhibitors. Utilizing our custom FGFR focused cell-free DNA assay, FGFR-Dx, we serially monitored variant allele fractions of FGFR2 fusions during FGFR inhibitor treatment and observed dynamic changes correlating with clinical responses. Genomic analysis of 30,229 comprehensively profiled pancreatic cancers revealed FGFR1-3 fusions in 245 cases, an incidence of 0.81%. FGFR fusions were generally mutually exclusive from other known oncogenes. Our findings provide clinical evidence for identifying and treating FGFR2 fusion-positive PDAC patients with FGFR targeted therapy.

Mutations in Grxcr1 are the basis for inner ear dysfunction in the pirouette mouse.

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.

Human male infertility caused by mutations in the CATSPER1 channel protein.

Male infertility, a common barrier that prevents successful conception, is a reproductive difficulty affecting 15% of couples. Heritable forms of nonsyndromic male infertility can arise from single-gene defects as well as chromosomal abnormalities. Although no CATSPER gene has been identified as causative for human male infertility, male mice deficient for members of the CatSper gene family are infertile. In this study, we used routine semen analysis to identify two consanguineous Iranian families segregating autosomal-recessive male infertility. Autozygosity by descent was demonstrated in both families for a approximately 11 cM region on chromosome 11q13.1, flanked by markers D11S1765 and D11S4139. This region contains the human CATSPER1 gene. Denaturing high-performance liquid chromatography (DHPLC) and bidirectional sequence analysis of CATSPER1 in affected family members revealed two separate insertion mutations (c.539-540insT and c.948-949insATGGC) that are predicted to lead to frameshifts and premature stop codons (p.Lys180LysfsX8 and p.Asp317MetfsX18). CATSPER1 is one of four members of the sperm-specific CATSPER voltage-gated calcium channel family known to be essential for normal male fertility in mice. These results suggest that CATSPER1 is also essential for normal male fertility in humans.

The clinical utility of a risk-modifying SNP to detect carriers for spinal muscular atrophy with increased sensitivity.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by biallelic inactivation of the survival motor neuron 1 (SMN1) gene. With a prevalence of ~1 in 11,000 live births (carrier frequency of ~1:50), SMA is one of the most common severe childhood-onset diseases; therefore, current guidelines recommend pan-ethnic carrier screening for SMA before or during pregnancy. Routine SMN1 copy number assessment detects ~96% of all SMA carriers, but not the remaining 4% who harbor two copies of SMN1 arrayed in -cis [2 + 0]. The c.*3+80T>G risk-modifying SNP positively correlates with this chromosomal configuration and may be used to modify the residual risk of being a carrier for SMA. METHODS: One year after incorporating the detection of the c.*3+80>G risk-modifying SNP into our routine SMA carrier screen, we perform a retrospective chart review to evaluate its frequency and utilization in the prenatal clinic. RESULTS: In comparison with classic carriers for SMA, study data show that individuals with two copies of SMN1 and the risk modifier were counseled less frequently about their increased risk of being a carrier for SMA. CONCLUSION: Incorporating the c.*3+80T>G risk-modifying SNP is important for detecting carriers for SMA with a higher clinical sensitivity.

Two Iranian families with a novel mutation in GJB2 causing autosomal dominant nonsyndromic hearing loss.

Mutations in GJB2, encoding connexin 26 (Cx26), cause both autosomal dominant and autosomal recessive nonsyndromic hearing loss (ARNSHL) at the DFNA3 and DFNB1 loci, respectively. Most of the over 100 described GJB2 mutations cause ARNSHL. Only a minority has been associated with autosomal dominant hearing loss. In this study, we present two families with autosomal dominant nonsyndromic hearing loss caused by a novel mutation in GJB2 (p.Asp46Asn). Both families were ascertained from the same village in northern Iran consistent with a founder effect. This finding implicates the D46N missense mutation in Cx26 as a common cause of deafness in this part of Iran mandating mutation screening of GJB2 for D46N in all persons with hearing loss who originate from this geographic region.

Genetic Characterization of Pediatric Sarcomas by Targeted RNA Sequencing.

Somatic variants, primarily fusion genes and single-nucleotide variants (SNVs) or insertions/deletions (indels), are prevalent among sarcomas. In many cases, accurate diagnosis of these tumors incorporates genetic findings that may also carry prognostic or therapeutic significance. Using the anchored multiplex PCR-based FusionPlex system, a custom RNA sequencing panel was developed that simultaneously detects fusion genes, SNVs, and indels in 112 genes found to be recurrently mutated in solid tumors. Using this assay, a retrospective analysis was conducted to identify somatic variants that may have assisted with classifying a cohort of 90 previously uncharacterized primarily pediatric sarcoma specimens. In total, somatic variants were identified in 45.5% (41/90) of the samples tested, including 22 cases with fusion genes and 19 cases with SNVs or indels. In addition, two of these findings represent novel alterations: a WHSC1L1/NCOA2 fusion and a novel in-frame deletion in the NRAS gene (NM_002524: c.174_176delAGC p.Ala59del). These sequencing results, taken in context with the available clinical data, indicate a potential change in the initial diagnosis, prognosis, or management in 27 of the 90 cases. This study presents a custom RNA sequencing assay that detects fusion genes and SNVs in tandem and has the ability to identify novel fusion partners. These features highlight the advantages associated with utilizing anchored multiplex PCR technology for the rapid and highly sensitive detection of somatic variants.

A forward genetics screen in mice identifies recessive deafness traits and reveals that pejvakin is essential for outer hair cell function.

Deafness is the most common form of sensory impairment in the human population and is frequently caused by recessive mutations. To obtain animal models for recessive forms of deafness and to identify genes that control the development and function of the auditory sense organs, we performed a forward genetics screen in mice. We identified 13 mouse lines with defects in auditory function and six lines with auditory and vestibular defects. We mapped several of the affected genetic loci and identified point mutations in four genes. Interestingly, all identified genes are expressed in mechanosensory hair cells and required for their function. One mutation maps to the pejvakin gene, which encodes a new member of the gasdermin protein family. Previous studies have described two missense mutations in the human pejvakin gene that cause nonsyndromic recessive deafness (DFNB59) by affecting the function of auditory neurons. In contrast, the pejvakin allele described here introduces a premature stop codon, causes outer hair cell defects, and leads to progressive hearing loss. We also identified a novel allele of the human pejvakin gene in an Iranian pedigree that is afflicted with progressive hearing loss. Our findings suggest that the mechanisms of pathogenesis associated with pejvakin mutations are more diverse than previously appreciated. More generally, our findings demonstrate that recessive screens in mice are powerful tools for identifying genes that control the development and function of mechanosensory hair cells and cause deafness in humans, as well as generating animal models for disease.

TRPV6, TRPM6 and TRPM7 Do Not Contribute to Hair-Cell Mechanotransduction.

Hair cells of the inner ear transduce mechanical stimuli like sound or head movements into electrical signals, which are propagated to the central nervous system. The hair-cell mechanotransduction channel remains unidentified. We tested whether three transient receptor channel (TRP) family members, TRPV6, TRPM6 and TRPM7, were necessary for transduction. TRPV6 interacted with USH1C (harmonin), a scaffolding protein that participates in transduction. Using a cysteine-substitution knock-in mouse line and methanethiosulfonate (MTS) reagents selective for this allele, we found that inhibition of TRPV6 had no effect on transduction in mouse cochlear hair cells. TRPM6 and TRPM7 each interacted with the tip-link component PCDH15 in cultured eukaryotic cells, which suggested they might be part of the transduction complex. Cochlear hair cell transduction was not affected by manipulations of Mg2+, however, which normally perturbs TRPM6 and TRPM7. To definitively examine the role of these two channels in transduction, we showed that deletion of either or both of their genes selectively in hair cells had no effect on auditory function. We suggest that TRPV6, TRPM6 and TRPM7 are unlikely to be the pore-forming subunit of the hair-cell transduction channel.