Annelies de Klein

Mutations in a TGF-β Ligand, TGFB3, Cause Syndromic Aortic Aneurysms and Dissections

Background Aneurysms affecting the aorta are a common condition associated with high mortality as a result of aortic dissection or rupture. Investigations of the pathogenic mechanisms involved in syndromic types of thoracic aortic aneurysms, such as Marfan and Loeys-Dietz syndromes, have revealed an important contribution of disturbed transforming growth factor (TGF)-β signaling. Objectives This study sought to discover a novel gene causing syndromic aortic aneurysms in order to unravel the underlying pathogenesis. Methods We combined genome-wide linkage analysis, exome sequencing, and candidate gene Sanger sequencing in a total of 470 index cases with thoracic aortic aneurysms. Extensive cardiological examination, including physical examination, electrocardiography, and transthoracic echocardiography was performed. In adults, imaging of the entire aorta using computed tomography or magnetic resonance imaging was done. Results Here, we report on 43 patients from 11 families with syndromic presentations of aortic aneurysms caused by TGFB3 mutations. We demonstrate that TGFB3 mutations are associated with significant cardiovascular involvement, including thoracic/abdominal aortic aneurysm and dissection, and mitral valve disease. Other systemic features overlap clinically with Loeys-Dietz, Shprintzen-Goldberg, and Marfan syndromes, including cleft palate, bifid uvula, skeletal overgrowth, cervical spine instability and clubfoot deformity. In line with previous observations in aortic wall tissues of patients with mutations in effectors of TGF-β signaling (TGFBR1/2, SMAD3, and TGFB2), we confirm a paradoxical up-regulation of both canonical and noncanonical TGF-β signaling in association with up-regulation of the expression of TGF-β ligands. Conclusions Our findings emphasize the broad clinical variability associated with TGFB3 mutations and highlight the importance of early recognition of the disease because of high cardiovascular risk.

Prognostic parameters in uveal melanoma and their association with BAP1 expression

Aim To determine whether BAP1 gene and protein expression associates with different prognostic parameters in uveal melanoma and whether BAP1 expression correctly identifies patients as being at risk for metastases, following enucleation of the primary tumour. Methods Thirty cases of uveal melanoma obtained by enucleation between 1999 and 2004 were analysed for a variety of prognostic markers, including histological characteristics, chromosome aberrations obtained by fluorescence in situ hybridisation (FISH) and single nucleotide polymorphism (SNP) analysis and gene expression profiling. These parameters were compared with BAP1 gene expression and BAP1 immunostaining. Results The presence of monosomy of chromosome 3 as identified by the different chromosome 3 tests showed significantly increased HRs (FISH on isolated nuclei cut-off 30%: HR 11.6, p=0.002; SNP analysis: HR 20.3, p=0.004) for death due to metastasis. The gene expression profile class 2, based on the 15-gene expression profile, similarly provided a significantly increased HR for a poor outcome (HR 8.5, p=0.005). Lower BAP1 gene expression and negative BAP1 immunostaining (50% of 28 tumours were immunonegative) were both associated with these markers for prognostication: FISH cut-off 30% monosomy 3 (BAP1 gene expression: p=0.037; BAP1 immunostaining: p=0.001), SNP-monosomy 3 (BAP1 gene expression: p=0.008; BAP1 immunostaining: p=0.002) and class 2 profile (BAP1 gene expression: p<0.001; BAP1 immunostaining: p=0.001) and were themselves associated with an increased risk of death due to metastasis (BAP1 gene expression dichotomised: HR 8.7, p=0.006; BAP1 immunostaining: HR 4.0, p=0.010). Conclusions Loss of BAP1 expression associated well with all of the methods currently used for prognostication and was itself predictive of death due to metastasis in uveal melanoma after enucleation, thereby emphasising the importance of further research on the role of BAP1 in uveal melanoma.

Low grade mosaic for a complex supernumerary ring chromosome 18 in an adult patient with multiple congenital anomalies

BackgroundSeveral cases have been reported of patients with a ring chromosome 18 replacing one of the normal chromosomes 18. Less common are patients with a supernumerary ring chromosomes 18. High resolution whole genome examination in patients with multiple congenital abnormalities might reveal cytogenetic abnormalities of an unexpected complexity.ResultsWe report a 24 years old male patient with lower spinal anomalies, hypospadia, bifid scrotum, cryptorchism, anal atresia, kidney stones, urethra anomalies, radial dysplasia, and a hypoplastic thumb. Some of the anomalies overlap with the VACTERL association. Chromosome analysis of cultured peripheral blood lymphocytes revealed an additional ring chromosome in 13% of the metaphases. Both parents had a normal karyotype, demonstrating the de novo origin of this ring chromosome. FISH analysis using whole chromosome paints showed that the additional chromosomal material was derived from chromosome 18. Chromosome analysis of cultured fibroblasts revealed only one cell with the supernumerary ring chromosome in the 400 analyzed. To characterize the ring chromosome in more detail peripheral blood derived DNA was analyzed using SNP-arrays. The array results indicated a 5 Mb gain of the pericentromeric region of chromosome 18q10-q11.2. FISH analysis using BAC-probes located in the region indicated the presence of 6 signals on the r(18) chromosome. In addition, microsatellite analysis demonstrated that the unique supernumerary ring chromosome was paternally derived and both normal copies showed biparental disomy.ConclusionsWe report on an adult patient with multiple congenital abnormalities who had in 13% of his cells a unique supernumerary ring chromosome 18 that was composed of 6 copies of the 5 Mb gene rich region of 18q11.

Chemokine receptor CCR7 expression predicts poor outcome in uveal melanoma and relates to liver metastasis whereas expression of CXCR4 is not of clinical relevance.

PURPOSEnTo examine the prognostic relevance of expression of the chemokine receptors CCR7 and CXCR4 and its ligand CXCL12 in uveal melanoma in nonmetastatic and metastatic patients with correlation to liver metastasis and overall survival.nnnMETHODSnPrimary uveal melanoma specimens from 19 patients with correlating liver metastasis specimens and 30 primary uveal melanoma specimens of patients without metastasis were collected between the years 1988 and 2008. Expression of CCR7, CXCR4, and CXCL12 were studied using immunohistochemistry. Single nucleotide polymorphism (SNP) arrays were used to examine gains or losses of chromosomes 1, 3, 6, and 8 and the regions of CCR7 (17q12-q21.2), CXCR4 (2q21), and CXCL12 (10q11.1) genes.nnnRESULTSnStrong cytoplasmic staining for CCR7 correlated with the presence of epithelioid cells (P = 0.037), tumor thickness (P = 0.011), lymphocytic infiltration (P = 0.041), and necrosis (P = 0.045). Nuclear staining for CXCR4 correlated with lymphocytic infiltration (P = 0.017). CXCL12 showed no correlation to histologic parameters. Single nucleotide polymorphism analyses showed no copy number variations in the regions of CCR7, CXCR4, or CXCL12. Strong expression of CCR7 was observed in 76% of the metastatic patients and 0% of nonmetastasis patients. In multivariate analysis, CCR7 staining was inversely correlated to overall survival and disease-free survival, whereas CXCR4 nuclear staining was not.nnnCONCLUSIONSnOur data suggest that CCR7 plays a role in uveal melanoma metastasis and is associated with poor survival. CCR7 and its involved related pathways are of prognostic value in uveal melanoma and may prove to be a target for therapeutic intervention.

The Prognostic Value of Extraocular Extension in Relation to Monosomy 3 and Gain of Chromosome 8q in Uveal Melanoma

PURPOSEnTo identify the prognostic value of extraocular extension in enucleated uveal melanoma (UM) patients and to correlate extraocular extension to chromosomal aberrations, metastasis-free survival (MFS), and clinico-histopathological risk factors.nnnMETHODSnRetrospective study of patients with UM treated with enucleation between 1987 and 2011. Melanoma-related metastasis and death were recorded. Statistical analysis (log-rank test or Cox regression analysis) was performed to correlate MFS with tumor characteristics, extraocular extension, episcleral diameter of the extraocular extension, cell type, extracellular matrix patterns, inflammation, loss of chromosome 3, and gain of chromosome 8q.nnnRESULTSnIn 43 (12%) of 357 patients, extraocular extension was observed. In this subset of patients, we noted a reduced survival of 70 months (105.5 months, P = 0.010) compared with patients without extraocular extension (175.8 months). Patients with gain of chromosomal region 8q in UM with extraocular extension had an increased risk of metastatic disease (P < 0.001). In multivariate Cox proportional hazard analysis, largest basal tumor diameter (P = 0.001), extracellular matrix patterns (P = 0.009), episcleral diameter of the extraocular extension (P = 0.016), loss of chromosome 3 (P < 0.001), and gain of 8q (P < 0.001) were independent predictors for MFS.nnnCONCLUSIONSnLarger episcleral diameter of the extraocular extension and additional gain of chromosome 8q in extraocular extension UM correlates to a worse prognosis. MFS is significantly reduced in UM with a large basal tumor diameter, extracellular matrix patterns, loss of chromosome 3, and gain of chromosome 8q.

Complex MAX Rearrangement in a Family With Malignant Pheochromocytoma, Renal Oncocytoma, and Erythrocytosis.

CONTEXTnFamilial pheochromocytoma (PCC) has been associated with germline mutations in 16 genes. Here we investigated three siblings presenting with bilateral pheochromocytomas. In addition, the index patient also exhibited renal oncocytoma and erythrocytosis, whereas the second sibling presented with a lymph node metastasis.nnnDESIGNnFirst, single-nucleotide polymorphism array and exome sequencing were performed on germline and PCC-derived DNA to identify genomic alterations in the index patient. Second, alterations were confirmed and validated by Sanger sequencing, analyzed by (multiplexed) PCR to determine the loss of the wild-type allele, and investigated by immunohistochemistry in the tumors of the three siblings.nnnRESULTSnThe index patients germline DNA revealed a large complex genomic alteration encompassing the intragenic and promoter regions of Myc-associated factor X (MAX) and alpha-(1,6)-fucosyltransferase (FUT8). In all three siblings the MAX alteration was confirmed, and the loss of the wild-type MAX and FUT8 alleles was demonstrated in all tumors. Uniparental disomy of chromosome 14q, previously demonstrated as a hallmark for MAX-related PCC, was shown in the index patients PCC by single-nucleotide polymorphism array. Loss of MAX and FUT8 protein expression was demonstrated by immunohistochemistry in the tumors from the three siblings.nnnCONCLUSIONSnOur results indicate that large genomic deletions of MAX should be considered in familial and bilateral PCC with prior negative testing for gene mutations. In addition, our results confirm that MAX is a tumor suppressor gene for renal oncocytomas.

PAIN MANAGEMENT IN INTELLECTUALLY DISABLED CHILDREN: ASSESSMENT, TREATMENT, AND TRANSLATIONAL RESEARCH

The primary focus of pain research in intellectually disabled individuals is still on pain assessment. Several observational pain assessment scales are available, each with its own characteristics, its own target group and its own validated use. Observational studies report differences in the treatment of intra- and postoperative pain of intellectually disabled children and almost all children with intellectual disability have comorbidities that need to be addressed. The scope of research has started to broaden. In this review we aim to answer the question: Can we integrate validated ways of pain assessment and postoperative pain treatment in intellectually disabled children to develop specific analgesic algorithms? Regrettably there is little knowledge on possible interaction effects and other relevant pharmacological issues. Possible genotype-phenotype associations related to pain in children with Down syndrome have several promises as six possible candidate genes are located on chromosome 21. In conclusion, the pain assessment tools for intellectually disabled children are there. We should now focus on tailoring the pain treatment. To this aim we need to perform pharmacokinetic and pharmacodynamic studies of analgesics and obtain information about the genotype-phenotype relationships for pain. This can lead to the development of specific analgesic algorithms.

Risk factors associated with secondary enucleation after fractionated stereotactic radiotherapy in uveal melanoma

To evaluate risk factors for secondary enucleation after fractionated stereotactic radiotherapy (fSRT) in uveal melanoma.

Metastatic disease in polyploid uveal melanoma patients is associated with BAP1 mutations

PURPOSEnMost of the uvea melanoma (UM) display a near-diploid (normal, -2N) karyotype with only a few chromosomal changes. In contrast to these simple aberrations 18% of the UM samples show a polyploid character (>2N) and this was associated with an unfavorable prognosis. This study attempts to gain insight in the prognostic value of polyploidy in UM.nnnMETHODSnIn 202 patients the ploidy status of the UM was determined using cytogenetic analysis, fluorescence-in-situ-hybridization (FISH), multiplex ligation dependent probe amplification (MLPA), and/or single nucleotide polymorphism (SNP) array analysis. Immunohistochemistry was used to determine the BAP1 expression and mutation analyses of BAP1 (coding regions) and the mutation hotspots for the SF3B1, EIF1AX, GNAQ, and GNA11 genes was carried out using Sanger sequencing or whole-exome sequencing.nnnRESULTSnTwenty-three patients had a polyploid UM karyotype (11.4%). Patients with a polyploid tumor had larger tumors (15.61 vs. 13.13 mm, P = 0.004), and more often loss of heterozygosity of chromosome 3 (P = 0.003). No difference in occurrence of mutations between polyploid and diploid tumors was observed for BAP1, SF3B1, EIF1AX, GNAQ, and GNA11. Polyploidy did not affect survival (P = 0.143). BAP1 deficiency was the only significant independent prognostic predictor for patients with polyploid tumors, with a 16-fold increased hazard ratio (HR 15.90, P = 0.009).nnnCONCLUSIONSnThe prevalence of mutations in the UM related genes is not different in polyploid UM compared with diploid UM. Moreover, similar to patients with diploid UM, BAP1 mutation is the most significant prognostic predictor of metastasis in patients with polyploid UM.

Correlation of Gene Mutation Status with Copy Number Profile in Uveal Melanoma

Copy number variations (CNV), gene expression profiling, and the recurrent gene mutations in BAP1, SF3B1, or EIF1AX, can be used to stratify uveal melanoma patients. Recently, Decatur et al described an association between specific gene mutations and gene expression profiles. In this report, we describe the relationship between mutational status of BAP1, SF3B1, and EIF1AX and CNV patterns by using conventional karyotyping data to explore the nature of these CNVs. In total, 277 patients from the Rotterdam Ocular Melanoma Study group (ROMS) were included in this study. Details of the patient population and methods are described in the Supplementary Methods (available at www.aaojournals.org). For 207 samples, single nucleotide polymorphism array data and mutational status were available. These patients were divided into 4 subgroups: patients with (1) immunohistochemically BAP1-negative tumors (BAP1), (2) SF3B1-mutated tumors (SF3B1), and (3) EIF1AX-mutated tumors (EIF1AX). Patients in which the tumors were immunohistochemically BAP1 positive and contained no SF3B1 or EIF1AX mutations were classified as (4) no recurrent mutations (NRM) tumors. This group was further split into subgroups with and without the loss of heterozygosity (LOH) of the BAP1 locus (NRMLOHþ and NRM , respectively). We visualized sample distribution of the single nucleotide polymorphism array data across the predefined groups to obtain chromosomal patterns (Fig 1A) and found that BAP1 tumors (n 1⁄4 84) were characterized by loss of chromosome 3 (95% of cases) and gain of the entire long (q) arm of chromosome 8 (80%). Gain of chromosome 8q was often accompanied by loss of chromosome 8p (26%). Loss of chromosomes 1p, 6q, and 16q was also observed frequently (25%, 19%, and 20%, respectively). SF3B1 tumors (n 1⁄4 42) were characterized by gain of chromosomes 6p (85%) and 8q (73%), and loss of chromosomes 6q (52%) and 11q (45%). In addition, loss of chromosome 1p (36%) and gain of chromosome 9q (24%) were present in SF3B1 tumors. BAP1 uveal melanomas (UMs) showed gains or losses of entire chromosomes or chromosome arms, whereas SF3B1 UMs were characterized by structural variants of the distal ends of the chromosomes (e.g., gain of chromosome 8q23qter, 8q13qter, 6p12.3pter or loss of 6q16.1qter and 11q22qter). EIF1AX tumors (n 1⁄4 26) showed gain of chromosome 6p (often 6p21.33pter) in 65% of the cases. No other recurrent CNVs were observed in EIF1AX UMs. NRM tumors (n 1⁄4 28) were characterized by gains of the distal ends of chromosome 6p (53%) and 8q (28%). NRMLOHþ UMs (n 1⁄4 19) were characterized by monosomy 3, because we stratified for LOH of BAP1. These samples showed a very similar CNV profile as the BAP1 UMs because these tumors also contained the typical gain of the entire arm of chromosome 8q, often accompanied by loss of chromosome 8p, and gain of the entire arm of chromosome 6p with loss of chromosome 6q. To substantiate the finding that BAP1 UMs contain mainly entire chromosome or chromosome arm CNVs and SF3B1 UMs smaller, partial chromosome CNVs, we calculated the percentages of aneuploidy and the number of copynumber (CN) events for all groups. These results (Fig 1B) showed significantly greater percentages of aneuploidy in BAP1 tumors compared with SF3B1 tumors (11.8% and 9.3%, respectively; P 1⁄4 0.024). EIF1AX tumors and NRM UMs harbored the least percentage of aneuploidy (1.7% and 4.3%, respectively), which were significantly lower than in BAP1 and SF3B1 tumors (P < 0.001 for all). As for CN events, SF3B1 UMs harbored the most CN events, although this was not significantly higher than BAP1 UMs (P 1⁄4 0.074) in the single nucleotide polymorphism array data when corrected for multiple testing. However, both groups harbored more CN events than EIF1AX and NRM UMs (P 0.01 for all). Furthermore, cytogenetic data and mutational status of 70 patients were used for analysis of the different types of chromosomal anomalies. Translocations or partial chromosome arm CNVs were categorized as chromosomal structural variants (CSVs) and were numerated manually. Although cases were limited, SF3B1 UMs strongly associated with >3 CSVs per tumor compared with BAP1 (P 1⁄4 0.002; Fig 1D). Moreover, 70% (7 of 10) of the UMs with >3 CSVs harbored an SF3B1 mutation, showing that tumors with multiple CSVs are most likely to be SF3B1 mutated. Another type of chromosomal anomaly found in UMs were isochromosomes. These were observed in 74% (25/34) of all BAP1 tumors (Table S1, available at www.aaojournals.org), whereas both SF3B1 and EIF1AX UMs harbored no isochromosomes. Remarkably, beside isochromosomes 8q, isochromosome 6p was recurrent in BAP1 UMs. Other chromosomal changes in BAP UMs were almost exclusively translocations with breakpoints in the centromeric regions (and thus entire chromosome arms), whereas SF3B1 tumors contained more structural aberrations with recurrent distal breakpoints on chromosomes 6 and 8, suggesting that different mechanisms play a role in causing these anomalies. These novel insights in the chromosomal patterns and different types of chromosomal anomalies show that UMs with different mutated genes represent distinct molecular classes. We and others have shown that stratification based on BAP1, SF3B1, and EIF1AX mutations reveal that patients with these mutated genes have a high, intermediate, or low risk of developing metastases, respectively. A limitation in this study was the use of BAP1 immunohistochemistry to replacedin partdBAP1 sequencing. For example, we expect BAP1 mutations in the NRMLOHþ group, because these tumors revealed similar CNVs as the BAP1 UMs. We are currently investigating this using deep sequencing and Western blotting. Furthermore, in this study all except 2 tumors were obtained from enucleated eyes; therefore, the tumor population is skewed toward larger tumors. These tumors tend to have a worse prognosis than eyes that undergo eye-conserving therapy. Nevertheless, this limitation does not affect the clinical significance of our observations that UMs have different molecular classes. In conclusion, in this report we show that patients with UMs harbor mutation-specific chromosomal patterns in the tumor. These