Functional copy-number alterations (fCNAs) are DNA copy-number changes with concordant differential gene expression. These are less likely to be bystander genetic lesions and could serve as robust and reproducible tumor biomarkers. To identify candidate fCNAs in neuroendocrine tumors (NETs), we integrated chromosomal microarray (CMA) and RNA-seq differential gene-expression data from 31 pancreatic (pNETs) and 33 small-bowel neuroendocrine tumors (sbNETs). Tumors were resected from 47 early-disease-progression (<24 months) and 17 late-disease-progression (>24 months) patients. Candidate fCNAs that accurately differentiated these groups in this discovery cohort were then replicated using fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin-embedded (FFPE) tissues in a larger validation cohort of 60 pNETs and 82 sbNETs (52 early- and 65 late-disease-progression samples). Logistic regression analysis revealed the predictive ability of these biomarkers, as well as the assay-performance metrics of sensitivity, specificity, and area under the curve. Our results indicate that copy-number changes at chromosomal loci 4p16.3, 7q31.2, 9p21.3, 17q12, 18q21.2, and 19q12 may be used as diagnostic and prognostic NET biomarkers. This involves a rapid, cost-effective approach to determine the primary tumor site for patients with metastatic liver NETs and to guide risk-stratified therapeutic decisions.

Figure S2. Representative IPA mechanistic network for signaling regulators predicted to contribute to the RABL6A knockdown phenotype.

Figure S3. Quantitative RT-PCR showing RABL6A depletion does not affect p21 mRNA expression

Figure S6. Down-regulation of p27 in arrested RABL6A knockdown cells promotes S phase entry.

Turner syndrome (TS) is a chromosomal disorder caused by complete or partial loss of the second sex chromosome and exhibits phenotypic heterogeneity, even after accounting for mosaicism and karyotypic variation. Congenital heart defects (CHD) are found in up to 45 percent of girls with TS and span a phenotypic continuum of obstructive left-sided lesions, with bicuspid aortic valve (BAV) being the most common. Several recent studies have demonstrated a genome-wide impact of X chromosome haploinsufficiency, including global hypomethylation and altered RNA expression. The presence of such broad changes to the TS epigenome and transcriptome led others to hypothesize that X chromosome haploinsufficiency sensitizes the TS genome, and several studies have demonstrated that a second genetic hit can modify disease susceptibility in TS. The objective of this study was to determine whether genetic variants in known heart developmental pathways act synergistically in this setting to increase the risk for CHD, specifically BAV, in TS. We analyzed 208 whole exomes from girls and women with TS and performed gene-based variant enrichment analysis and rare-variant association testing to identify variants associated with BAV in TS. Notably, rare variants in CRELD1 were significantly enriched in individuals with TS who had BAV compared to those with structurally normal hearts. CRELD1 is a protein that functions as a regulator of calcineurin/NFAT signaling, and rare variants in CRELD1 have been associated with both syndromic and non-syndromic CHD. This observation supports the hypothesis that genetic modifiers outside the X chromosome that lie in known heart development pathways may influence CHD risk in TS.

Table S1. List of differentially regulated genes caused by RABL6A knockdown (KD), relative to control (CON), in BON-1 PNET cells.

Figure S3. Quantitative RT-PCR showing RABL6A depletion does not affect p21 mRNA expression

Figure S1. Quantitative PCR measurement of human RABL6A DNA copy number in human PNET patient samples.

Figure S4. Quantitative RT-PCR showing effective silencing of Rb1 mRNA by Rb1 shRNAs in RABL6A knockdown cells.

Figure S5. Effect of RABL6A overexpression on Rb1 and p21 mRNA and protein expression

The interpretation of clinical chromosomal microarrays (CMAs) has historically relied on the relevance of identified copy number variants (CNVs) to the clinical phenotype. New interpretation guidelines are focused on standardizing pathogenicity classifications based on genomic location, gene content, and previous publications, rather than the immediate clinical relevance. Here we report on DISCRIMINATOR, which was developed to assign provisional pathogenicity classifications based on genomic location by integrating information on putative benign and pathogenic loci in the human genome. However, its application extends beyond that of a simple classifier. The novel utility of DISCRIMINATOR is its ability to operate on a cohort-level and easily integrate updated definitions of benign and pathogenic regions of the human genome. We used DISCRIMINATOR to assign provisional pathogenicity classifications (‘Benign’, ‘Secondary’, ‘Primary’ or ‘Non-Coding’) to 87,808 CNVs in 3,362 cases ascertained through clinical CMA testing. The majority of identified CNVs were provisionally classified as ‘Benign’ or ‘Non-Coding’ and consistent with their prevalence rates, 15q11.2, 16p11.2, and 22q11.2 were the most common ‘Primary’ CNVs detected. Targeted re-analysis led to the identification of several cases where DISCRIMINATOR identified a ‘Primary’ CNV within a case that had a non-Abnormal CMA test result, and several cases where only benign and/or non-coding CNVs were identified in reports with a ‘VUS’ CMA test result. Together these results show the utility of large-scale re-analysis of CMA data and how DISCRIMINATOR addresses this long-standing challenge.

Congenital heart disease (CHD) is one of the most common birth defects affecting about 1-2 % of newborns globally and is classified as syndromic or isolated based on the presence of extracardiac malformations. In addition to the type of cardiac abnormality and associated extracardiac malformations, the overall prognosis of CHD also depends on the accurate molecular diagnosis. The most common genetic etiologies resulting in CHD include chromosomal aneuploidies, recurrent microdeletions/microduplications, as well as copy number variants.

While copy number variants (CNVs) have been identified as an important cause of rare genetic disorders, they have also been identified in unaffected control populations, making clinical interpretation of these lesions challenging. Discriminating benign CNVs from those pathogenic for rare genetic disorders, therefore, relies on understanding what regions of the human genome are tolerant to copy number variation. Benign-Ex is a python-based program that uses information from databases of CNVs to generate one or more benign interval map(s) and then identifies the optimal map by computing the overlap with known pathogenic regions. We utilized Benign-Ex to identify the optimal set of benign intervals from two distinct CNV databases: Database of Genomic Variants (DGV) and Clinical Genome Resource (ClinGen). Benign-Ex called 41.1% of the genome benign using data from DGV and 37.6% of the genome benign using data from ClinGen. The benign regions from DGV and ClinGen were not spatially correlated, underscoring the importance of integrating both research and clinical databases for determining CNV benignity. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by R01DE021071 (MP and JM), the Stead Family Department of Pediatrics (AW, HM, BD) and the Interdisciplinary Genetics T32 Predoctoral Training Grant (T32 GM 008629; AW). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study used ONLY openly available human data that were obtained from the Database of Genomic Variants () and the Clinical Genome Resource via the UCSC Genome Table Browser () I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Benign-Ex is publicly available on GitHub at . Full simulation data is available upon request. All other data produced and analyzed in the present study are included in this published article and its supplementary information.

Hypothalamic hamartoma with gelastic seizures is a well-established cause of drug-resistant epilepsy in early life. The development of novel surgical techniques has permitted the genomic interrogation of hypothalamic hamartoma tissue. This has revealed causative mosaic variants within GLI3, OFD1 and other key regulators of the sonic-hedgehog pathway in a minority of cases. Sonic-hedgehog signalling proteins localize to the cellular organelle primary cilia. We therefore explored the hypothesis that cilia gene variants may underlie hitherto unsolved cases of sporadic hypothalamic hamartoma. We performed high-depth exome sequencing and chromosomal microarray on surgically resected hypothalamic hamartoma tissue and paired leukocyte-derived DNA from 27 patients. We searched for both germline and somatic variants under both dominant and bi-allelic genetic models. In hamartoma-derived DNA of seven patients we identified bi-allelic (one germline, one somatic) variants within one of four cilia genes-DYNC2I1, DYNC2H1, IFT140 or SMO. In eight patients, we identified single somatic variants in the previously established hypothalamic hamartoma disease genes GLI3 or OFD1. Overall, we established a plausible molecular cause for 15/27 (56%) patients. Here, we expand the genetic architecture beyond single variants within dominant disease genes that cause sporadic hypothalamic hamartoma to bi-allelic (one germline/one somatic) variants, implicate three novel cilia genes and reconceptualize the disorder as a ciliopathy.

Abstract Background Reports of interstitial duplication of chromosome 20q11 are rare with only nine published patients to date. Methods We performed karyotype and chromosomal microarray analysis on a peripheral blood sample for our patient and reviewed the genes in the region to provide genotype–phenotype correlation. Results Clinical features of the patient include minor dysmorphic facial features, shorthands and feet, bilateral conductive hearing loss, global developmental delay, and behavioral issues with attention deficit hyperactivity disorder. Together with previously published cases of 20q11 duplication, we show that patients with overlapping duplications share a similar clinical phenotype of dysmorphic craniofacial features and developmental delay. Conclusion We report an 8‐year‐old girl with a 9.1 Mb interstitial duplication of chromosome 20q11.22q13.11. Our observations suggest that a novel duplication syndrome and documentation of similar cases will further help clarify the phenotype.

Abstract Purpose: Malignant peripheral nerve sheath tumors (MPNST) are deadly sarcomas that lack effective therapies. In most MPNSTs, the retinoblastoma (RB1) tumor suppressor is disabled by hyperactivation of cyclin-dependent kinases (CDK), commonly through loss of CDK-inhibitory proteins such as p27(Kip1). RABL6A is an inhibitor of RB1 whose role in MPNSTs is unknown. To gain insight into MPNST development and establish new treatment options, we investigated RABL6A-RB1 signaling and CDK inhibitor–based therapy in MPNSTs. Experimental Design: We examined patient-matched MPNSTs and precursor lesions by RNA sequencing (RNA-Seq) and IHC. Molecular and biological effects of silencing RABL6A and/or p27 in MPNST lines and normal human Schwann cells were determined. Tumor-suppressive effects of CDK inhibitors were measured in MPNST cells and orthotopic tumors. Results: RABL6A was dramatically upregulated in human MPNSTs compared with precursor lesions, which correlated inversely with p27 levels. Silencing RABL6A caused MPNST cell death and G1 arrest that coincided with p27 upregulation, CDK downregulation, and RB1 activation. The growth-suppressive effects of RABL6A loss, and its regulation of RB1, were largely rescued by p27 depletion. Importantly, reactivation of RB1 using a CDK4/6 inhibitor (palbociclib) killed MPNST cells in vitro in an RABL6A-dependent manner and suppressed MPNST growth in vivo. Low-dose combination of drugs targeting multiple RB1 kinases (CDK4/6, CDK2) had enhanced antitumorigenic activity associated with potential MPNST cell redifferentiation. Conclusions: RABL6A is a new driver of MPNST pathogenesis that acts in part through p27-RB1 inactivation. Our results suggest RB1 targeted therapy with multiple pathway drugs may effectively treat MPNSTs.

BACKGROUND:The 22q11.2 deletion syndrome (22q11.2DS) is the most common contiguous microdeletion affecting humans and exhibits extreme phenotypic heterogeneity. Patients can manifest any combination of comorbidities including congenital heart disease, hypoparathyroidism, cleft palate, kidney abnormalities, neurodevelopmental disorders, and immune dysfunction. Immunodeficiency is present in the majority of patients with 22q11.2DS and is the second leading cause of death in these patients. Knowing the genetic determinants of immune dysfunction will aid in prognostication and potentially novel treatments. METHODS:We performed exome sequencing and gene-based variant association analysis on 31 deeply phenotyped individuals with the canonical 3Mb 22q11.2 deletion to identify what genes outside the 22q11.2 locus may be modifying the immune dysregulated phenotype. Immunophenotyping was performed using preexisting medical data and a novel scoring system developed from numerous clinical laboratory values including immunoglobulin levels, lymphocyte transformation to antigens (LTA), lymphocyte transformation to mitogens (LTM), and peripheral blood flow cytometry. Immunophenotypic scoring was validated against newborn screening T-cell receptor excision circle (TREC) results. RESULTS:Rare DNA variants in transcriptional regulators involved in retinoic acid signaling (NCOR2, OMIM *600848 and EP300, OMIM *602700) were found to be associated with immunophenotype. CONCLUSION:The expression of TBX1, which seems to confer the major phenotypic features of 22q11.2DS, is regulated via retinoic acid signaling, and alterations in retinoic acid signaling during embryonic development can lead to phenocopies of 22q11.2DS. These observations support the hypothesis that genetic modifiers outside the microdeletion locus may influence the immune function in 22q11.2DS patients.