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Uveal melanomas (UM) are detected earlier. Consequently, tumors are smaller, allowing for novel eye-preserving treatments. This reduces tumor tissue available for genomic profiling. Additionally, these small tumors can be hard to differentiate from nevi, creating the need for minimally invasive detection and prognostication. Metabolites show promise as minimally invasive detection by resembling the biological phenotype. In this pilot study, we determined metabolite patterns in the peripheral blood of UM patients (n = 113) and controls (n = 46) using untargeted metabolomics. Using a random forest classifier (RFC) and leave-one-out cross-validation, we confirmed discriminatory metabolite patterns in UM patients compared to controls with an area under the curve of the receiver operating characteristic of 0.99 in both positive and negative ion modes. The RFC and leave-one-out cross-validation did not reveal discriminatory metabolite patterns in high-risk versus low-risk of metastasizing in UM patients. Ten-time repeated analyses of the RFC and LOOCV using 50% randomly distributed samples showed similar results for UM patients versus controls and prognostic groups. Pathway analysis using annotated metabolites indicated dysregulation of several processes associated with malignancies. Consequently, minimally invasive metabolomics could potentially allow for screening as it distinguishes metabolite patterns that are putatively associated with oncogenic processes in the peripheral blood plasma of UM patients from controls at the time of diagnosis.

IntroductionConsensus is lacking on the optimal management of asymptomatic congenital pulmonary airway malformation (CPAM). For future studies, the CONNECT consortium (the COllaborative Neonatal Network for the first European CPAM Trial)—an international collaboration of specialised caregivers—has established consensus on a core outcome set of outcome parameters concerning respiratory insufficiency, surgical complications, mass effect and multifocal disease. These outcome parameters have been incorporated in the CONNECT trial, a randomised controlled trial which, in order to develop evidence-based practice, aims to compare conservative and surgical management of patients with an asymptomatic CPAM.Methods and analysisChildren are eligible for inclusion after the CPAM diagnosis has been confirmed on postnatal chest CT scan and they remain asymptomatic. On inclusion, children are randomised to receive either conservative or surgical management. Subsequently, children in both groups are enrolled into a standardised, 5-year follow-up programme with three visits, including a repeat chest CT scan at 2.5 years and a standardised exercise tolerance test at 5 years.The primary outcome is exercise tolerance at age 5 years, measured according to the Bruce treadmill protocol. Secondary outcome measures are molecular genetic diagnostics, validated questionnaires—on parental anxiety, quality of life and healthcare consumption—, repeated imaging and pulmonary morbidity during follow-up, as well as surgical complications and histopathology. This trial aims to end the continuous debate surrounding the optimal management of asymptomatic CPAM.Ethics and disseminationThis study is being conducted in accordance with the Declaration of Helsinki. The Medical Ethics Review Board of Erasmus University Medical Centre Rotterdam, The Netherlands, has approved this protocol (MEC-2022–0441). Results will be disseminated through peer-reviewed scientific journals and conference presentations.Trial registration numberNCT05701514.

Congenital diaphragmatic hernia (CDH) is a relatively common and severe birth defect with variable clinical outcome and associated malformations in up to 60% of patients. Mortality and morbidity remain high despite advances in pre-, intra-, and postnatal management. We review the current literature and give an overview about the genetics of CDH to provide guidelines for clinicians with respect to genetic diagnostics and counseling for families. Until recently, the common practice was (molecular) karyotyping or chromosome microarray if the CDH diagnosis is made prenatally with a 10% diagnostic yield. Undiagnosed patients can be reflexed to trio exome/genome sequencing with an additional diagnostic yield of 10 to 20%. Even with a genetic diagnosis, there can be a range of clinical outcomes. All families with a child with CDH with or without additional malformations should be offered genetic counseling and testing in a family-based trio approach.

Objective Maple syrup urine disease (MSUD; OMIM #248600) is an autosomal recessive metabolic disorder in the catabolism of branched-chain amino acids (leucine, isoleucine, and valine) and may be lethal if untreated in affected newborns. Methods Single-nucleotide polymorphism haplotyping and Sanger sequencing of BCKDHA, BCKDHB, and DBT genes were performed in a cohort of 10 MSUD patients. Results We identified a 16.6 Mb homozygous region harboring the DBT gene in an Iranian girl presenting with MSUD. Sanger sequencing revealed a pathogenic homozygous variant (NM_001918.3: c.1174A > C) in the DBT gene. We further found a controversial variant (rs12021720: c.1150 A > G) in the DBT gene. This substitution (p.Ser384Gly) is highly debated in literature. Bioinformatics and cosegregation analysis, along with identifying the real pathogenic variants (c.1174 A > C), lead to terminate these various interpretations of c.1150 A > G variant. Conclusion Our study introduced c.1150 A > G as a polymorphic variant, which is informative for variant databases and also helpful in molecular diagnosis.

Background Uveal Melanoma (UM) micro-metastases can be present prior to diagnosis and relapse after treatment. Earlier detection resulted in an increased incidence of small (T1 and T2) tumors allowing for novel eye-preserving treatment strategies, but reducing available tumor tissue needed for prognostic genomic profiling. Thus, creating the need for minimal-invasive detection and novel prognostication methods. We determined whether tumor presence can be confirmed using metabolite patterns in blood plasma and evaluated if these patterns differ between high risk (BRCA1-associated protein-1, BAP1), intermediate risk (Splicing Factor 3b Subunit 1, SF3B1) and low risk (Eukaryotic Translation Initiation Factor 1A X-Linked, EIF1AX) mutated tumors. Methods In this retrospective observational study, samples of UM-patients in a discovery (n = 53) and replication (n = 42) set were compared to unaffected control-participants (n = 46) as well as across mutation-based subgroups. Peripheral blood plasma was collected prior to treatment. Metabolite profiles of patients and control-participants were generated as mass/charge (m/z) features using ultra-high performance liquid chromatography mass-spectrometry. After normalization, discriminatory feature patterns were determined using a random forest classifier and a leave-one-out cross-validation procedure. Results We detected differential metabolic patterns between UM-patients and control-participants with a sensitivity of 0.95 and 0.90 and a specificity of 0.98 and 0.98 in the positive and negative ion modes, respectively. Overall, the performance of the model for classifying the subgroups was insufficient in both the positive (merged dataset F1 scores: BAP1: 0.64, SF3B1: 0.37, and EIF1AX: 0.35) and negative (merged dataset F1 scores: BAP1: 0.60, SF3B1: 0.32, and EIF1AX: 0.36) ion modes, respectively. Pathway analysis using annotated metabolites indicated upregulation of tRNA charging, and glycine usage for the creatine biosynthesis. Purine ribonucleosides degradation and the super pathway of citrulline metabolism were downregulated in UM-patients. An increased salvage of bases or decreased purine degradation could indicate a higher energy consumption. Conclusion Minimally-invasive metabolomics has the potential to allow for minimally invasive screening as it distinguishes metabolite patterns, that are putatively associated with oncogenic processes, in peripheral blood derived plasma of UM-patients from control-participants at the time of diagnosis.

Unstructured abstractChromosome 8q gain is associated with poor prognosis. Here, we show that the predictive value of chromosome 8q gain depends on the mutation status and is true for BAP1 but not for SF3B1-mutated tumors.

Treatment of uveal melanoma (UM) patients with metastatic disease is unfortunately limited. Twenty percent of UM harbor a mutation in splicing factor gene SF3B1, suggesting that aberrant spliceosome functioning plays a vital role in tumorigenesis. Splicing inhibitors exploit the preferential sensitivity of spliceosome compromised leukemic cells to these compounds. We have studied the effect of splicing inhibitor E7107 using two UM cell lines and ex vivo cultured SF3B1 and BAP1 mutated primary UM tumor slices. These UM cell lines and ex vivo tumor slices were exposed for 24h to different concentrations of E7107. Tumor slices were stained with H&E and incubated with BAP1, MelanA, MIB-1 and caspase-3 antisera. E7107 exposed UM cell lines showed decreased cell viability and increased apoptosis with the largest effect sizes in SF3B1-mutated UM. A similar effect was observed upon exposure of E7107 on UM tumor slices. Additionally, RNA was isolated for transcriptome analysis and the type and number of alternative and aberrant transcripts was evaluated. Ninety-seven transcripts had a decrease in aberrant transcripts in all samples after E7107 exposure, and this effect was mostly in intron retention. This study indicates / suggests that mutated SF3B1 UM cells are more sensitive to splicing inhibitor E7107 compared to wild-type SF3B1 UM cells.Simple SummaryUveal melanoma (UM) is an aggressive malignancy of the eye. UM prognosis varies and depends on mutational status. Mutations in spliceosome gene Splicing Factor 3b subunit 1 (SF3B1) are in UM associated with late onset metastasis. Also in other malignancies, such as breast cancer or leukemia, SF3B1 mutations have been implied as drivers for tumor formation. Targeting the spliceosome with inhibitors is possible and would make SF3B1-mutated UM amenable for treatment. We investigated and demonstrated using two cell lines and ex-vivo culturing of 3 SF3B1-mutated tumors and 17 SF3B1-wildtype tumors that spliceosome inhibitor E7107 decreased the cell viability of both SF3B1-mutated and SF3B1-wildtype UM cell line, but significantly more in SF3B1-mutated UM cell line. Therefore, inhibiting the spliceosome has the highest therapeutic potential in SF3B1-mutated UM, but further research is recommended to determine the best suited strategy to minimize risk and maximize efficacy in a therapeutic setting.

ImportanceUveal Melanomas (UM) micro-metastasis can be present prior to diagnosis and relapse after treatment. Earlier detection resulted in an increased incidence of small (T1 and T2) tumors allowing for novel eye-preserving treatment strategies but reducing available tumor tissue needed for prognostic genomic profiling, creating the need for minimal-invasive detection and novel prognostication methods.ObjectiveTo determine whether tumor presence can be confirmed using metabolite patterns in blood plasma and to evaluate if these patterns differ between high risk (BRCA1-associated protein-1, BAP1), intermediate risk (Splicing Factor 3b Subunit 1, SF3B1) and low risk (Eukaryotic Translation Initiation Factor 1A X-Linked, EIF1AX) mutated tumors.DesignRetrospective observational study including discovery (n=53) and replication (n=42) convenience sample sets compared to unaffected control-participants (n=46) as well as across mutation-based subgroups.SettingPatients from two tertiary referral centers specialized in ocular oncology: The Rotterdam Eye Hospital and the Erasmus MC Cancer Institute were included.ParticipantsSex-matched controls and patients were included based on their prognostic relevant secondary driver mutations. Peripheral blood plasma was collected at diagnosis, prior to treatment. Exclusion criteria were the presence of other malignancies or co-occurrence of systemic diseases at time of diagnosis.Main outcome and measureMetabolite profiles of patients and control-participants were generated as mass/charge (m/z) features using ultra-high performance liquid chromatography mass-spectrometry. After normalization, discriminatory feature patterns were determined using a random forest classifier and leave-one-out cross-validation.ResultsWe detected differential metabolic patterns with a sensitivity of 0.95 and 0.90 and a specificity of 0.98 and 0.98 in the positive and negative ion modes, respectively. The accuracy of the model for classifying the subgroups was insufficient for the discovery (0.600 and 0.614 in the positive and negative ion modes, respectively) and replication cohort (0.544 and 0.672 in the positive and negative ion modes, respectively).Conclusion and relevanceMinimally invasive metabolomics does not discriminate between the prognostic relevant BAP1, SF3B1 and EIF1AX mutated UM-subgroups. However, this technique has the potential to allow for minimal invasive screening as it distinguishes metabolite patterns in peripheral blood derived plasma of UM-patients from control-participants.Key pointsQuestionCan we discriminate uveal melanoma patients and mutation subgroups from unaffected control-participants using the metabolome of peripheral blood plasma taken at time of diagnosis?FindingsIn this retrospective observational study, we find a low sensitivity and specificity to detect subgroups but a high sensitivity and specificity to discriminate patients from control-participants by measuring metabolite abundancy in plasma using ultra-high performance liquid chromatography mass-spectrometry and reach a receiver operating characteristic area under the curve of 0.993.MeaningThese results suggest that surveying the metabolome of uveal melanoma patients could aid in the minimal invasive detection of uveal melanoma.

Ocular melanoma consists of posterior uveal melanoma, iris melanoma and conjunctival melanoma. These malignancies derive from melanocytes in the uveal tract or conjunctiva. The genetic profiles of these different entities differ from each other. In uveal melanoma, GNAQ and GNA11 gene mutations are frequently found and prognosis is based on mutation status of BAP1, SF3B1 and EIF1AX genes. Iris melanoma, also originating from the uvea, has similarities to the genetic makeups of both posterior uveal melanoma (UM) and conjunctival melanoma since mutations in GNAQ and GNA11 are less common and genes involved in conjunctival melanoma such as BRAF have been described. The genetic spectrum of conjunctival melanoma, however, includes frequent mutations in the BRAF, NRAS and TERT promoter genes, which are found in cutaneous melanoma as well. The BRAF status of the tumor is not correlated to prognosis, whereas the TERT promoter gene mutations are. Clinical presentation, histopathological characteristics and copy number alterations are associated with survival in ocular melanoma. Tissue material is needed to classify ocular melanoma in the different subgroups, which creates a need for the use of noninvasive techniques to prognosticate patients who underwent eye preserving treatment.

__Background:__ Patients born with esophageal atresia (EA) have a higher incidence of infantile hypertrophic pyloric stenosis (IHPS), suggestive of a relationship. A shared etiology makes sense from a developmental perspective as both affected structures are foregut derived. A genetic component has been described for both conditions as single entities and EA and IHPS are variable components in several monogenetic syndromes. We hypothesized that defects disturbing foregut morphogenesis are responsible for this combination of malformations. __Methods:__ We investigated the genetic variation of 15 patients with both EA and IHPS with unaffected parents using exome sequencing and SNP arraybased genotyping, and compared the results to mouse transcriptome data of the developing foregut. __Results:__ We did not identify putatively deleterious de novo mutations or recessive variants. However, we detected rare inherited variants in EA or IHPS disease genes or in genes important in foregut morphogenesis, expressed at the proper developmental time-points. Two pathways were significantly enriched (p < 1 × 10−5): proliferation and differentiation of smooth muscle cells and self-renewal of satellite cells. __Conclusions:__ None of our findings could fully explain the combination of abnormalities on its own, which makes complex inheritance the most plausible genetic explanation, most likely in combination with mechanical and/or environmental factors. As we did not find one defining monogenetic cause for the EA/IHPS phenotype, the impact of the corrective surgery could should be further investigated.

Uveal melanoma (UM) is the second most frequent type of melanoma. Therapeutic options for UM favor minimally invasive techniques such as irradiation for vision preservation. As a consequence, no tumor material is obtained. Without available tissue, molecular analyses for gene expression, mutation or copy number analysis cannot be performed. Thus, proper patient stratification is impossible and patients’ uncertainty about their prognosis rises. Minimally invasive techniques have been studied for prognostication in UM. Blood-based biomarker analysis has become more common in recent years; however, no clinically standardized protocol exists. This review summarizes insights in biomarker analysis, addressing new insights in circulating tumor cells, circulating tumor DNA, extracellular vesicles, proteomics, and metabolomics. Additionally, medical imaging can play a significant role in staging, surveillance, and prognostication of UM and is addressed in this review. We propose that combining multiple minimally invasive modalities using tumor biomarkers should be the way forward and warrant more attention in the coming years.

Approximately 25% of all uveal melanoma (UM) contain driver mutations in the gene encoding the spliceosome factor SF3B1, and whilst patients with such SF3B1 mutations generally have an intermediate risk on developing metastatic disease, a third of these patients develop early metastasis within 5 years after diagnosis. We therefore investigated whether clinical and/or genetic variables could be indicative of short progression-free survival (PFS < 60 months) or long PFS (PFS ≥ 60 months) for SF3B1-mutated (SF3B1mut) UM patients. We collected 146 SF3B1mut UM from our Rotterdam Ocular Melanoma Studygroup (ROMS) database and external published datasets. After stratification of all SF3B1mut UM using short PFS vs. long PFS, only largest tumor diameter (LTD) was significantly larger (mean: 17.7 mm (±2.8 SD) in the short PFS SF3B1mut group vs. the long PFS group (mean: 14.7 (±3.7 SD, p = 0.001). Combined ROMS and The Cancer Genome Atlas (TCGA) transcriptomic data were evaluated, and we identified SF3B1mut-specific canonical transcripts (e.g., a low expression of ABHD6 indicative for early-onset metastatic disease) or distinct expression of SF3B1mut UM aberrant transcripts, indicative of early- or late-onset or no metastatic SF3B1mut UM.

Simple Summary We investigated the increased prevalence of Barrett's esophagus in adults with esophageal atresia. A higher polygenic risk score and disturbances in inflammatory, stress response and oncological pathways upon acid exposure suggest a genetic susceptibility and increased induction of inflammatory processes. Although further research is required to explore this hypothesis, this could be a first-step into selecting patients that are more at risk to develop Barrett's esophagus and/or esophageal carcinoma. Currently, an endoscopic screening and surveillance program is in practice in our institution for patients born with esophageal atresia, to early detect (pre)malignant lesions. Since recurrent endoscopies can be a burden for the patient, selecting patients by for example genetic susceptibility would allow to only include those at risk in future practice. The prevalence of Barrett's esophagus (BE) in adults born with esophageal atresia (EA) is four times higher than in the general population and presents at a younger age (34 vs. 60 years). This is (partly) a consequence of chronic gastroesophageal reflux. Given the overlap between genes and pathways involved in foregut and BE development, we hypothesized that EA patients have an intrinsic predisposition to develop BE. Transcriptomes of Esophageal biopsies of EA patients with BE (n = 19, EA/BE); EA patients without BE (n = 44, EA-only) and BE patients without EA (n = 10, BE-only) were compared by RNA expression profiling. Subsequently, we simulated a reflux episode by exposing fibroblasts of 3 EA patients and 3 controls to acidic conditions. Transcriptome responses were compared to the differential expressed transcripts in the biopsies. Predisposing single nucleotide polymorphisms, associated with BE, were slightly increased in EA/BE versus BE-only patients. RNA expression profiling and pathway enrichment analysis revealed differences in retinoic acid metabolism and downstream signaling pathways and inflammatory, stress response and oncological processes. There was a similar effect on retinoic acid signaling and immune response in EA patients upon acid exposure. These results indicate that epithelial tissue homeostasis in EA patients is more prone to acidic disturbances.

Contains fulltext : 282474.pdf (Publisher’s version ) (Open Access) High myopia [refractive error ≤ -6 diopters (D)] is a heterogeneous condition, and without clear accompanying features, it can be difficult to pinpoint a genetic cause. This observational study aimed to evaluate the utility of whole exome sequencing (WES) using an eye disorder gene panel in European patients with high myopia. Patients with high myopia were recruited by ophthalmologists and clinical geneticists. Clinical features were categorized into isolated high myopia, high myopia with other ocular involvement or with systemic involvement. WES was performed and an eye disorder gene panel of ~500 genes was evaluated. Hundred and thirteen patients with high myopia [mean (SD) refractive error - 11.8D (5.2)] were included. Of these, 53% were children younger than 12 years of age (53%), 13.3% were aged 12-18 years and 34% were adults (aged > 18 years). Twenty-three out of 113 patients (20%) received a genetic diagnosis of which 11 patients displayed additional ocular or systemic involvement. Pathogenic variants were identified in retinal dystrophy genes (e.g. GUCY2D and CACNA1F), connective tissue disease genes (e.g. COL18A1 and COL2A1), non-syndromic high myopia genes (ARR3), ocular development genes (e.g. PAX6) and other genes (ASPH and CNNM4). In 20% of our high myopic study population, WES using an eye gene panel enabled us to diagnose the genetic cause for this disorder. Eye genes known to cause retinal dystrophy, developmental or syndromic disorders can cause high myopia without apparent clinical features of other pathology.

Thoracic aortic aneurysm is a potentially life-threatening disease with a strong genetic contribution. Despite identification of multiple genes involved in aneurysm formation, little is known about the specific underlying mechanisms that drive the pathological changes in the aortic wall. The aim of our study was to unravel the molecular mechanisms underlying aneurysm formation in Marfan syndrome (MFS). We collected aortic wall samples from FBN1 variant-positive MFS patients (n = 6) and healthy donor hearts (n = 5). Messenger RNA (mRNA) expression levels were measured by RNA sequencing and compared between MFS patients and controls, and between haploinsufficient (HI) and dominant negative (DN) FBN1 variants. Immunohistochemical staining, proteomics and cellular respiration experiments were used to confirm our findings. FBN1 mRNA expression levels were highly variable in MFS patients and did not significantly differ from controls. Moreover, we did not identify a distinctive TGF-β gene expression signature in MFS patients. On the contrary, differential gene and protein expression analysis, as well as vascular smooth muscle cell respiration measurements, pointed toward inflammation and mitochondrial dysfunction. Our findings confirm that inflammatory and mitochondrial pathways play important roles in the pathophysiological processes underlying MFS-related aortic disease, providing new therapeutic options.

In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results. This work was supported by the Solve-RD project (European Union’s Horizon 2020 grant agreement No 779257) to MS, SB, OR and CG, and the National Centre for medical genomics (www.ncmg.cz, CZ.02.1.01/0.0/0.0/16_026/000844, LM2018132 (MSMT.cz) and 00064203/6003 (MZCR.cz) to MM). OR received financial support from Illumina for implementing whole genome sequencing into clinical diagnostics (Ge-Med project)

Congenital diaphragmatic hernia (CDH) is a life-threatening birth defect that presents as either an isolated diaphragm defect or as part of a complex disorder with a wide array of anomalies (complex CDH). Some patients with complex CDH display distinct craniofacial anomalies such as craniofrontonasal dysplasia or craniosynostosis, defined by the premature closure of cranial sutures. Using clinical whole exome sequencing (WES), we found a BCL11B missense variant in a patient with a left-sided congenital diaphragmatic hernia as well as sagittal suture craniosynostosis. We applied targeted sequencing of BCL11B in patients with craniosynostosis or with a combination of craniosynostosis and CDH. This resulted in three additional BCL11B missense mutations in patients with craniosynostosis. The phenotype of the patient with both CDH as well as craniosynostosis was similar to the phenotype of previously reported patients with BCL11B missense mutations. Although these findings imply that both craniosynostosis as well as CDH may be associated with BCL11B mutations, further studies are required to establish whether BCL11B variants are causative mutations for both conditions or if our finding was coincidental.

Contains fulltext : 245512.pdf (Publisher’s version ) (Open Access) Patients with Hirschsprung disease (HSCR) do not always receive a genetic diagnosis after routine screening in clinical practice. One of the reasons for this could be that the causal mutation is not present in the cell types that are usually tested-whole blood, dermal fibroblasts or saliva-but is only in the affected tissue. Such mutations are called somatic, and can occur in a given cell at any stage of development after conception. They will then be present in all subsequent daughter cells. Here, we investigated the presence of somatic mutations in HSCR patients. For this, whole-exome sequencing and copy number analysis were performed in DNA isolated from purified enteric neural crest cells (ENCCs) and blood or fibroblasts of the same patient. Variants identified were subsequently validated by Sanger sequencing. Several somatic variants were identified in all patients, but causative mutations for HSCR were not specifically identified in the ENCCs of these patients. Larger copy number variants were also not found to be specific to ENCCs. Therefore, we believe that somatic mutations are unlikely to be identified, if causative for HSCR. Here, we postulate various modes of development following the occurrence of a somatic mutation, to describe the challenges in detecting such mutations, and hypothesize how somatic mutations may contribute to 'missing heritability' in developmental defects.

This study reports the role played by the mutation status of Uveal Melanoma (UM) in relation to hepatic metastatic patterns as seen on imaging modalities. Radiological images were obtained from 123 patients treated at the Erasmus Medical Center Rotterdam or the Rotterdam Eye Hospital. Radiological images were derived from either computed tomography or magnetic resonance imaging. Hepatic metastatic patterns were classified by counting the number of metastases found in the liver. Miliary metastatic pattern (innumerable small metastases in the entire liver) was analyzed separately. Mutation status was determined in 85 patients. Median disease-free survival (DFS) and survival with metastases differed significantly between each of the metastatic patterns (respectively, p = 0.009, p &lt, 0.001), both in favor of patients with less hepatic metastases. The mutation status of the primary tumor was not correlated with any hepatic tumor profiles (p = 0.296). Of the patients who had a solitary metastasis (n = 18), 11 originated from a primary BAP1-mutated tumors and one from a primary SF3B1-mutated tumor. Of the patients who had a miliary metastasis pattern (n = 24), 17 had a primary BAP1-mutated tumor and two had a primary SF3B1-mutated tumor. Chromosome 8p loss was significantly more in patients with more metastases (p = 0.045). Moreover, the primary UMs of patients with miliary metastases harbored more chromosome 8p and 1p loss, compared to patients with single solitary metastasis (p = 0.035 and p = 0.026, respectively). In conclusion, our study shows that there is an inverse correlation of the number of metastasis with the DFS and metastasized survival, indicating separate growth patterns. We also revealed that the number and type of metastases is irrelevant to the prognostic mutation status of the tumor, showing that both BAP1- and SF3B1-mutated UM can result in solitary and miliary metastases, indicating that other processes lay ground to the different metastatic patterns.

Simple Summary The most lethal tumor in the eye is metastatic uveal melanomas, while the most common cause of irreversible blindness is glaucoma. Glaucoma is treated by prescribing intraocular pressure-lowering drugs. Theoretically, these drugs may affect the risk of metastasis of intraocular tumors (uveal melanomas). Using data of a long-running and ongoing study on uveal melanomas, we found that eye drops that lower the intraocular pressure by stimulating outflow of fluid (aqueous humor) may increase the risk of metastasis, and subsequent mortality. Therefore, in patients at risk or suspect for uveal melanoma, we recommend choosing ophthalmic drugs with a working mechanism that is not based on the increase of outflow of aqueous humor from the eye. Abstract Background: There has been speculation that IOP-lowering medication, which increases aqueous humor outflow, increases the risk of metastatic uveal melanoma (UM). This hypothesis has not been studied previously but is relevant for UM patients who use IOP-lowering medication. The aim of the current study is to assess the association between the use of intraocular pressure (IOP)-lowering medication and the risk of metastatic UM, and mortality. Methods: A retrospective cohort study, in which patients from the Rotterdam Ocular Melanoma Study were included from 1986 onwards. Medical records were evaluated for use of IOP-lowering medication at baseline (i.e., before diagnosis). For each IOP-lowering medication, we divided patients into two groups for comparison (e.g., patients with alpha2-agonist use and patients without alpha2-agonist use). All patients underwent regular ophthalmic examinations and routine screening for metastasis. Survival analyses were initiated to compare groups in each IOP-lowering medication group. In addition, secondary analyses were performed to examine the association between IOP and the development of metastatic UM, and mortality. Results: A total of 707 patients were included of whom 13 patients used prostaglandin or pilocarpine at baseline. For alpha2-agonist, beta-blocker, carbonic anhydrase inhibitor, and oral IOP-lowering medication these were 4, 14, 11, and 12 patients, respectively. The risk of metastatic UM (choroid and ciliary body melanoma) among the prostaglandin/pilocarpine users was significantly higher than controls (HR [95% CI]: 4.840 [1.452–16.133]). Mortality did not differ significantly among the IOP-lowering medications groups, except for the prostaglandin or pilocarpine group (HR [95% CI]: 7.528 [1.836–30.867]). If we combined all IOP-lowering medication that increase aqueous humor outflow, the risk (HR [95% CI]) of metastatic UM and mortality was 6.344 (1.615–24.918) and 9.743 (2.475–38.353), respectively. There was an association between IOP and mortality, but not for the onset of metastatic UM. Conclusion: The use of topical prostaglandin or pilocarpine may increase the risk of metastatic UM and mortality compared to patients without prostaglandin or pilocarpine use. Therefore, use of IOP-lowering medication which increases aqueous humor outflow, should be avoided in patients with (presumed) UM.

Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) is the most common congenital malformation of the upper digestive tract. This study represents the first genome-wide association study (GWAS) to identify risk loci for EA/TEF. We used a European case-control sample comprising 764 EA/TEF patients and 5,778 controls and observed genome-wide significant associations at three loci. On chromosome 10q21 within the gene

Congenital diaphragmatic hernia (CDH) is a congenital structural anomaly in which the diaphragm has not developed properly. It may occur either as an isolated anomaly or with additional anomalies. It is thought to be a multifactorial disease in which genetic factors could either substantially contribute to or directly result in the developmental defect. Patients with aneuploidies, pathogenic variants or de novo Copy Number Variations (CNVs) impacting specific genes and loci develop CDH typically in the form of a monogenetic syndrome. These patients often have other associated anatomical malformations. In patients without a known monogenetic syndrome, an increased genetic burden of de novo coding variants contributes to disease development. In early years, genetic evaluation was based on karyotyping and SNP-array. Today, genomes are commonly analyzed with next generation sequencing (NGS) based approaches. While more potential pathogenic variants are being detected, analysis of the data presents a bottleneck—largely due to the lack of full appreciation of the functional consequence and/or relevance of the detected variant. The exact heritability of CDH is still unknown. Damaging de novo alterations are associated with the more severe and complex phenotypes and worse clinical outcome. Phenotypic, genetic—and likely mechanistic—variability hampers individualpatient diagnosis, short and long-term morbidity prediction and subsequent care strategies. Detailed phenotyping, clinical follow-up at regular intervals and detailed registries are needed to find associations between long-term morbidity, genetic alterations, and clinical parameters. Since CDH is a relatively rare disorder with only a few recurrent changes large cohorts of patients are needed to identify genetic associations. Retrospective whole genome sequencing of historical patient cohorts using will yield valuable data from which today's patients and parents will profit Trio whole genome sequencing has an excellent potential for future re-analysis and data-sharing increasing the chance to provide a genetic diagnosis and predict clinical prognosis. In this review, we explore the pitfalls and challenges in the analysis and interpretation of genetic information, present what is currently known and what still needs further study, and propose strategies to reap the benefits of genetic screening.

Tracheoesophageal Fistula (TOF) is a congenital anomaly for which the cause is unknown in the majority of patients. OA/TOF is a variable feature in many (often mono-) genetic syndromes. Research using animal models targeting genes involved in candidate pathways often result in tracheoesophageal phenotypes. However, there is limited overlap in the genes implicated by animal models and those found in OA/TOF-related syndromic anomalies. Knowledge on affected pathways in animal models is accumulating, but our understanding on these pathways in patients lags behind. If an affected pathway is associated with both animals and patients, the mechanisms linking the genetic mutation, affected cell types or cellular defect, and the phenotype are often not well understood. The locus heterogeneity and the uncertainty of the exact heritability of OA/TOF results in a relative low diagnostic yield. OA/TOF is a sporadic finding with a low familial recurrence rate. As parents are usually unaffected, de novo dominant mutations seems to be a plausible explanation. The survival rates of patients born with OA/TOF have increased substantially and these patients start families; thus, the detection and a proper interpretation of these dominant inherited pathogenic variants are of great importance for these patients and for our understanding of OA/TOF aetiology.

Hirschsprung disease (HSCR) is a complex genetic disease characterized by absence of ganglia in the intestine. HSCR etiology can be explained by a unique combination of genetic alterations: rare coding variants, predisposing haplotypes and Copy Number Variation (CNV). Approximately 18% of patients have additional anatomical malformations or neurological symptoms (HSCR-AAM). Pinpointing the responsible culprits within a CNV is challenging as often many genes are affected. Therefore, we selected candidate genes based on gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics. Next, we used a zebrafish model to investigate whether loss of these genes affects enteric neuron development in vivo. This study included three groups of patients, two groups without coding variants in disease associated genes: HSCR-AAM and HSCR patients without associated anomalies (HSCR-isolated). The third group consisted of all HSCR patients in which a confirmed pathogenic rare coding variant was identified. We compared these patient groups to unaffected controls. Predisposing haplotypes were determined, confirming that every HSCR subgroup had increased contributions of predisposing haplotypes, but their contribution was highest in isolated HSCR patients without RET coding variants. CNV profiling proved that specifically HSCR-AAM patients had larger Copy Number (CN) losses. Gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics were used to determine plausible candidate genes located within CN losses. Validation in zebrafish using CRISPR/Cas9 targeting confirmed the contribution of UFD1L, TBX2, SLC8A1, and MAPK8 to ENS development. In addition, we revealed epistasis between reduced Ret and Gnl1 expression and between reduced Ret and Tubb5 expression in vivo. Rare large CN losses—often de novo—contribute to HSCR in HSCR-AAM patients. We proved the involvement of six genes in enteric nervous system development and Hirschsprung disease., Author summary Hirschsprung disease is a congenital disorder characterized by the absence of intestinal neurons in the distal part of the intestine. It is a complex genetic disorder in which multiple variations in our genome combined, result in disease. One of these variations are Copy Number Variations (CNVs): large segments of our genome that are duplicated or deleted. Patients often have Hirschsprung disease without other symptoms. However, a proportion of patients has additional associated anatomical malformations and neurological symptoms. We found that CNVs, present in patients with associated anomalies, are more often larger compared to unaffected controls or Hirschsprung patients without other symptoms. Furthermore, Copy Number (CN) losses are enriched for constrained coding regions (CCR; genes usually not impacted by genomic alterations in unaffected controls) of which the expression is higher in the developing intestinal neurons compared to the intestine. We modelled loss of these candidate genes in zebrafish by disrupting the zebrafish orthologues by genome editing. For several genes this resulted in changes in intestinal neuron development, reminiscent of HSCR observed in patients. The results presented here highlight the importance of Copy Number profiling, zebrafish validation and evaluating all CCR expressed in developing intestinal neurons during diagnostic evaluation.

Simple Summary Uveal melanoma (UM) is a rare ocular malignancy that often gives rise to metastases. Tumours with an inflammatory phenotype have an especially bad prognosis. As an increased HLA expression and the presence of tumour-infiltrating lymphocytes and macrophages may be regulated by miRNAs, we set out to investigate whether any miRNAs are associated with inflammatory parameters in this malignancy. Some miRNAs were increased in UM with a high HLA expression and high T cell numbers, while others were decreased, showing two opposing patterns; however, both patterns were related to the tumour’s chromosome 3/BAP1 status. We conclude that specific miRNAs are related to the inflammatory phenotype and that these are differentially expressed between disomy 3/BAP1-positive versus monosomy 3/BAP1-negative UM. Abstract MicroRNAs are known to play a role in the regulation of inflammation. As a high HLA Class I expression is associated with a bad prognosis in UM, we set out to determine whether any miRNAs were related to a high HLA Class I expression and inflammation. We also determined whether such miRNAs were related to the UM’s genetic status. The expression of 125 miRNAs was determined in 64 primary UM from Leiden. Similarly, the mRNA expression of HLA-A, HLA-B, TAP1, BAP1, and immune cell markers was obtained. Expression levels of 24 of the 125 miRNAs correlated with expression of at least three out of four HLA Class I probes. Four miRNAs showed a positive correlation with HLA expression and infiltration with leukocytes, 20 a negative pattern. In the first group, high miRNA levels correlated with chromosome 3 loss/reduced BAP1 mRNA expression, in the second group low miRNA levels. The positive associations between miRNA-22 and miRNA-155 with HLA Class I were confirmed in the TCGA study and Rotterdam cohort, and with TAP1 in the Rotterdam data set; the negative associations between miRNA-125b2 and miRNA-211 and HLA-A, TAP1, and CD4 were confirmed in the Rotterdam set. We demonstrate two patterns: miRNAs can either be related to a high or a low HLA Class I/TAP1 expression and the presence of infiltrating lymphocytes and macrophages. However, both patterns were associated with chromosome 3/BAP1 status, which suggests a role for BAP1 loss in the regulation of HLA expression and inflammation in UM through miRNAs.

The aim of this study was exploration of the genetic background of conjunctival melanoma (CM) and correlation with recurrent and metastatic disease. Twenty-eight CM from the Rotterdam Ocular Melanoma Study group were collected and DNA was isolated from the formalin-fixed paraffin embedded tissue. Targeted next-generation sequencing was performed using a panel covering GNAQ, GNA11, EIF1AX, BAP1, BRAF, NRAS, c-KIT, PTEN, SF3B1, and TERT genes. Recurrences and metastasis were present in eight (29%) and nine (32%) CM cases, respectively. TERT promoter mutations were most common (54%), but BRAF (46%), NRAS (21%), BAP1 (18%), PTEN (14%), c-KIT (7%), and SF3B1 (4%) mutations were also observed. No mutations in GNAQ, GNA11, and EIF1AX were found. None of the mutations was significantly associated with recurrent disease. Presence of a TERT promoter mutation was associated with metastatic disease (p-value = 0.008). Based on our molecular findings, CM comprises a separate entity within melanoma, although there are overlapping molecular features with uveal melanoma, such as the presence of BAP1 and SF3B1 mutations. This warrants careful interpretation of molecular data, in the light of clinical findings. About three quarter of CM contain drug-targetable mutations, and TERT promoter mutations are correlated to metastatic disease in CM.

The aim of this study was exploration of the genetic background of conjunctival melanoma (CM) and correlation with recurrent and metastatic disease. Twenty-eight CM from the Rotterdam Ocular Melanoma Study group were collected and DNA was isolated from the formalin-fixed paraffin embedded tissue. Targeted next-generation sequencing was performed using a panel covering GNAQ, GNA11, EIF1AX, BAP1, BRAF, NRAS, c-KIT, PTEN, SF3B1, and TERT genes. Recurrences and metastasis were present in eight (29%) and nine (32%) CM cases, respectively. TERT promoter mutations were most common (54%), but BRAF (46%), NRAS (21%), BAP1 (18%), PTEN (14%), c-KIT (7%), and SF3B1 (4%) mutations were also observed. No mutations in GNAQ, GNA11, and EIF1AX were found. None of the mutations was significantly associated with recurrent disease. Presence of a TERT promoter mutation was associated with metastatic disease (p-value = 0.008). Based on our molecular findings, CM comprises a separate entity within melanoma, although there are overlapping molecular features with uveal melanoma, such as the presence of BAP1 and SF3B1 mutations. This warrants careful interpretation of molecular data, in the light of clinical findings. About three quarter of CM contain drug-targetable mutations, and TERT promoter mutations are correlated to metastatic disease in CM.