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- 9q21.13q21.31 deletion in a patient with intellectual disability severe speech delay and and dysmorphic features a newly recognized microdeletion syndromePublication . Marques, Barbara; Serafim, Silvia; Pedro, Sonia; Tarelho, Ana Rita; Ferreira, Cristina; Gonçalves, Rui; Correia, HildebertoThe increased use of chromosomal microarray analysis has led to the identification of new microdeletion/microduplication syndromes, enabling better genotype-phenotype correlations. Interstitial deletions involving the long arm of chromosome 9 are rare but recently a microdeletion syndrome at 9q21.13 was suggested, with mental retardation, speech delay, epilepsy, autistic behaviour and moderate facial dysmorphism as the main characteristics. Here we present a male child with intellectual disability, severe speech delay, microcephaly and dysmorphic features carrying an interstitial deletion, detected by the Affymetrix Cytoscan HD microarray, of 6.56 Mb at 9q21.13q21.31 region encompassing 16 OMIM genes (arr[GRCh37] 9q21.13q21.31(76551542_83116342)x1). Among the genes in the deleted region the PRUNE2, PCSK5, RORB and TRPM6 genes are expressed in the nervous system and have been describe as being candidate genes to play a role in mental retardation or neurological disorders. Although the cohort of patients identified with deletions in this region is still small our patient phenotype partially overlaps the others described in the literature. The collection of more cases with deletion of the 9q21.13 region will help establishing a clear classification for this CNV, finding the real weight in the patient’s phenotype, delineating the genetic counseling for their families, and clearly establishing this microdeletion as a syndrome.
- Age Dependency of the Prognostic Impact of Tumor Genomics in Localized Resectable MYCN-Nonamplified Neuroblastomas. Report From the SIOPEN Biology Group on the LNESG Trials and a COG Validation GroupPublication . Ambros, Inge M.; Tonini, Gian-Paolo; Pötschger, Ulrike; Gross, Nicole; Mosseri, Véronique; Beiske, Klaus; Berbegall, Ana P.; Bénard, Jean; Bown, Nick; Caron, Huib; Combaret, Valérie; Couturier, Jerome; Defferrari, Raffaella; Delattre, Olivier; Jeison, Marta; Kogner, Per; Lunec, John; Marques, Barbara; Martinsson, Tommy; Mazzocco, Katia; Noguera, Rosa; Schleiermacher, Gudrun; Valent, Alexander; Van Roy, Nadine; Villamon, Eva; Janousek, Dasa; Pribill, Ingrid; Glogova, Evgenia; Attiyeh, Edward F.; Hogarty, Michael D.; Monclair, Tom F.; Holmes, Keith; Valteau-Couanet, Dominique; Castel, Victoria; Tweddle, Deborah A.; Park, Julie R.; Cohn, Sue; Ladenstein, Ruth; Beck-Popovic, Maja; De Bernardi, Bruno; Michon, Jean; Pearson, Andrew D.J.; Ambros, Peter F.Purpose: For localized, resectable neuroblastoma without MYCN amplification, surgery only is recommended even if incomplete. However, it is not known whether the genomic background of these tumors may influence outcome. Patients and methods: Diagnostic samples were obtained from 317 tumors, International Neuroblastoma Staging System stages 1/2A/2B, from 3 cohorts: Localized Neuroblastoma European Study Group I/II and Children's Oncology Group. Genomic data were analyzed using multi- and pangenomic techniques and fluorescence in-situ hybridization in 2 age groups (cutoff age, 18 months) and were quality controlled by the International Society of Pediatric Oncology European Neuroblastoma (SIOPEN) Biology Group. Results: Patients with stage 1 tumors had an excellent outcome (5-year event-free survival [EFS] ± standard deviation [SD], 95% ± 2%; 5-year overall survival [OS], 99% ± 1%). In contrast, patients with stage 2 tumors had a reduced EFS in both age groups (5-year EFS ± SD, 84% ± 3% in patients < 18 months of age and 75% ± 7% in patients ≥ 18 months of age). However, OS was significantly decreased only in the latter group (5-year OS ± SD in < 18months and ≥ 18months, 96% ± 2% and 81% ± 7%, respectively; P = .001). In < 18months, relapses occurred independent of segmental chromosome aberrations (SCAs); only 1p loss decreased EFS (5-year EFS ± SD in patients 1p loss and no 1p loss, 62% ± 13% and 87% ± 3%, respectively; P = .019) but not OS (5-year OS ± SD, 92% ± 8% and 97% ± 2%, respectively). In patients ≥ 18 months, only SCAs led to relapse and death, with 11q loss as the strongest marker (11q loss and no 11q loss: 5-year EFS ± SD, 48% ± 16% and 85% ± 7%, P = .033; 5-year OS ± SD, 46% ± 22% and 92% ± 6%, P = .038). Conclusion: Genomic aberrations of resectable non-MYCN-amplified stage 2 neuroblastomas have a distinct age-dependent prognostic impact. Chromosome 1p loss is a risk factor for relapse but not for diminished OS in patients < 18 months, SCAs (especially 11q loss) are risk factors for reduced EFS and OS in those > 18months. In older patients with SCA, a randomized trial of postoperative chemotherapy compared with observation alone may be indicated.
- Detection of copy number variants in the human genome: Is long-read sequencing an alternative to genomic microarrays?Publication . Silva, Catarina; Ferrão, José; Marques, Barbara; Pedro, Sónia; Correia, Hildeberto; Rodrigues, António Sebastião; Vieira, LuísIntroduction: Copy number variations (CNVs) represent ~13% of the human genome and can harbour important genes and regulatory elements. High-resolution whole genome microarray (MA) analysis is the gold standard tool for detection of CNVs associated with genetic disorders. While short-read sequencing (SRS) can address SV detection, the use of long-read sequencing as proven to overcome SRS mapping inaccuracy in highly repetitive DNA regions and improve genome contiguity. We applied whole genome nanopore sequencing (NS) to call CNVs and compared the results with those obtained by microarray. Methodology: Genomic DNA from 2 cell lines (EOL-1 and 697) were processed using the CytoSan HD Array (Affymetrix) and ChAS software (ThermoFisher). A minimum CNV calling size threshold of 35 Kb was used. DNA was also sequenced on the MinION device (Oxford Nanopore Technologies) following a rapid library preparation method. Sequencing data were basecalled using Guppy, mapped with LRA, and SVs called using both CuteSV and Sniffles2. Sanger sequencing was performed to demonstrate breakpoint positions for 3 CNVs. R packages were used to perform comparisons between MA and NS data. Results: A total of 49 CNVs were confirmed after curated MA analysis in both cell lines, ranging in size from 35 Kb to 79 Mb. From those, 43 CNVs (87.7%) were called in nanopore data by either one (4 CNVs) or both (39 CNVs) callers with a mean whole genome coverage of ~12X. Six of 43 CNVs were called as inversions instead. In 3 CNVs the size of the variant was found to be smaller (ranging from ~5 to 22 Kb) than the threshold of MA analysis. The correlation between CNV sizes obtained with MA and NS was of 0.71 with Sniffles2 and 0.74 with CuteSV, whereas the correlation between callers was of 0.99. The breakpoint precision obtained for NS was much higher (ranging for CuteSV from 2 to 42 bp; and for Sniffles2 from 0 to 87 bp) than the one obtained for MA (ranging from 774 to 7618 bp). Conclusions: NS technology proved to be technically effective in the detection of CNVs of different types and sizes and thus posing itself as an alternative to MA in the detection of pathogenic SVs associated with genetic diseases. However, NS data analysis requires fine-tuning of the analysis conditions as well as the use of different methods, for greater reliability of results in a clinical context.
- Disruption of NUBPL due to balanced translocation [t(3;14) (q26.33;q14)] increases severity of a family-specific PGK1 mutationPublication . David, Dezső; Haltrich, Iren; Marques, Barbara; Fernandes, Cristina; Malveiro, Sara; Fekete, GyörgyAn intriguing group of familiar translocations are those which not always segregate with the “associated” disorder. Here we report the genetic alterations underlying a clinical phenotype characterized by haemolytic anemia and neuro-myopathy, seemingly associated with the familial translocation [t(3;14)(3q26.33;q14)]. Two affected probands and two unaffected relatives have been identified as carriers of this translocation. The 3q26.33 breakpoint was mapped about 40 kb from the TTC14 5’ end, at position 180.28 Mb and the 14q14 breakpoint within IVS 6 of NUBPL. The latter has been implicated in the aetiology of mitochondrial complex I deficiency (OMIM 252010). The most important additional possible candidate gene identified in this region is DNAJC19 causing an autosomal recessive disorder (OMIM 610198) that partially overlaps the reported phenotype. The recognition that a deceased relative most likely suffered from a similar disorder suggested the possibility of an X-linked disorder. Exclusion of additional genomic alterations within the breakpoint regions or elsewhere in the genome, familial X-chromosome segregation analysis and whole exome sequencing identified a novel missense mutation, c.358G>A, p.Glu120Lys, in exon 4 of phosphoglycerate kinase 1 (PGK1). Segregation analysis confirmed the association of this mutation with the disease phenotype. Re-evaluation of clinical data indicates that myopathy is considerably more severe in PGK1 deficient patients carriers of the translocation. The confirmation of this observation is currently underway. In conclusion, we have identified a novel PGK1 mutation whose clinical phenotype is exacerbated by co-inheritance of the disrupted NUBPL and/or by alterations affecting the genes in the breakpoint regions.
- Disruption of NUBPL due to balanced translocation [t(3;14)(q26.33;q14)] increases severity of a family-specific PGK1 mutationPublication . David, Dezső; Haltrich, Iren; Marques, Barbara; Fernandes, Catarina; Malveiro, Sara; Fekete, GyörgyAn intriguing group of familiar translocations are those which not always segregate with the “associated” disorder. Here we report the genetic alterations underlying a clinical phenotype characterized by haemolytic anemia and neuro-myopathy, seemingly associated with the familial translocation [t(3;14)(3q26.33;q14)]. Two affected probands and two unaffected relatives have been identified as carriers of this translocation. The 3q26.33 breakpoint was mapped about 40 kb from the TTC14 5’ end, at position 180.28 Mb and the 14q14 breakpoint within IVS 6 of NUBPL. The latter has been implicated in the aetiology of mitochondrial complex I deficiency (OMIM 252010). The most important additional possible candidate gene identified in this region is DNAJC19 causing an autosomal recessive disorder (OMIM 610198) that partially overlaps the reported phenotype. The recognition that a deceased relative most likely suffered from a similar disorder suggested the possibility of an X-linked disorder. Exclusion of additional genomic alterations within the breakpoint regions or elsewhere in the genome, familial X-chromosome segregation analysis and whole exome sequencing identified a novel missense mutation, c.358G>A, p.Glu120Lys, in exon 4 of phosphoglycerate kinase 1 (PGK1). Segregation analysis confirmed the association of this mutation with the disease phenotype. Re-evaluation of clinical data indicates that myopathy is considerably more severe in PGK1 deficient patients carriers of the translocation. The confirmation of this observation is currently underway. In conclusion, we have identified a novel PGK1 mutation whose clinical phenotype is exacerbated by co-inheritance of the disrupted NUBPL and/or by alterations affecting the genes in the breakpoint regions.
- Frequency and Prognostic Impact of ALK Amplifications and Mutations in the European Neuroblastoma Study Group (SIOPEN) High-Risk Neuroblastoma Trial (HR-NBL1)Publication . Bellini, Angela; Pötschger, Ulrike; Bernard, Virginie; Lapouble, Eve; Baulande, Sylvain; Ambros, Peter F.; Auger, Nathalie; Beiske, Klaus; Bernkopf, Marie; Betts, David R.; Bhalshankar, Jaydutt; Bown, Nick; de Preter, Katleen; Clément, Nathalie; Combaret, Valérie; Font de Mora, Jaime; George, Sally L.; Jiménez, Irene; Jeison, Marta; Marques, Barbara; Martinsson, Tommy; Mazzocco, Katia; Morini, Martina; Mühlethaler-Mottet, Annick; Noguera, Rosa; Pierron, Gaelle; Rossing, Maria; Taschner-Mandl, Sabine; Van Roy, Nadine; Vicha, Ales; Chesler, Louis; Balwierz, Walentyna; Castel, Victoria; Elliott, Martin; Kogner, Per; Laureys, Geneviève; Luksch, Roberto; Malis, Josef; Popovic-Beck, Maja; Ash, Shifra; Delattre, Olivier; Valteau-Couanet, Dominique; Tweddle, Deborah A.; Ladenstein, Ruth; Schleiermacher, GudrunPurpose: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through activating point mutations or genomic amplification. We studied ALK genetic alterations in high-risk (HR) patients on the HR-NBL1/SIOPEN trial to determine their frequency, correlation with clinical parameters, and prognostic impact. Materials and methods: Diagnostic tumor samples were available from 1,092 HR-NBL1/SIOPEN patients to determine ALK amplification status (n = 330), ALK mutational profile (n = 191), or both (n = 571). Results: Genomic ALK amplification (ALKa) was detected in 4.5% of cases (41 out of 901), all except one with MYCN amplification (MNA). ALKa was associated with a significantly poorer overall survival (OS) (5-year OS: ALKa [n = 41] 28% [95% CI, 15 to 42]; no-ALKa [n = 860] 51% [95% CI, 47 to 54], [P < .001]), particularly in cases with metastatic disease. ALK mutations (ALKm) were detected at a clonal level (> 20% mutated allele fraction) in 10% of cases (76 out of 762) and at a subclonal level (mutated allele fraction 0.1%-20%) in 3.9% of patients (30 out of 762), with a strong correlation between the presence of ALKm and MNA (P < .001). Among 571 cases with known ALKa and ALKm status, a statistically significant difference in OS was observed between cases with ALKa or clonal ALKm versus subclonal ALKm or no ALK alterations (5-year OS: ALKa [n = 19], 26% [95% CI, 10 to 47], clonal ALKm [n = 65] 33% [95% CI, 21 to 44], subclonal ALKm (n = 22) 48% [95% CI, 26 to 67], and no alteration [n = 465], 51% [95% CI, 46 to 55], respectively; P = .001). Importantly, in a multivariate model, involvement of more than one metastatic compartment (hazard ratio [HR], 2.87; P < .001), ALKa (HR, 2.38; P = .004), and clonal ALKm (HR, 1.77; P = .001) were independent predictors of poor outcome. Conclusion: Genetic alterations of ALK (clonal mutations and amplifications) in HR-NB are independent predictors of poorer survival. These data provide a rationale for integration of ALK inhibitors in upfront treatment of HR-NB with ALK alterations.
- Genetic alterations of ALK in high-risk neuroblastoma patients. A SIOPEN studyPublication . Bellini, Angela; Bernard, Virginie; Ambros, Inge; F. Ambros, Peter; de Preter, Katleen; Combaret, Valérie; Beiske, Klaus; Jeison, Marta; Marques, Barbara; Morini, Martina; Mazzocco, Katia; Defferrari, Raffaella; Betts, David; Martinsson, Tommy; Mühlethaler-Mottet, Annick; Noguera, Rosa; Font de Mora, Jaime; Vicha, Ales; Ladenstein, Ruth; Valteau-Couanet, Dominique; Rossing, Caroline Maria; Bown, Nick; Tweddle, Deborah; Avigad, Smadar; Lapouble, Eve; Chicard, Mathieu; Leprovost, Nada; Clement, Nathalie; Baulande, Sylvain; Pierron, Gaelle; Irene, Jimenez; Jaydutt, Bhalshankar; Delattre, Olivier; Michon, Jean; Schleiermacher, GudrunBackground: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through genomic amplification or activating point mutations. We studied ALK genetic alterations in high-risk NB patients to determine their frequency and prognostic impact. Methods: Diagnostic NB samples from 1039 patients enrolled in the SIOPEN-HR-NBL1 trial were studied to determine the ALK amplification status (copy number analysis; n=337), the ALK mutational profile (Sanger and/or NGS including deep sequencing, n=203) or both (n=499). The sensitivity of ALK mutated/ALK amplified or ALK wildtype NB cell lines ((CLB-GA (R1275Q), CLB-GE (F1174V; ALK-A), SKNBE-2C (ALK wt)) to simultaneous or consecutive combinations of ALK TKIs (crizotinib/lorlatinib) and/or chemotherapy (Etoposide and Doxorubicin) was then tested. Results: Genomic ALK amplifications were detected in 4.4% of cases (37/836); all but 2 showed MYCN amplification. ALK mutations were detected at a clonal level (>20% mutated allele fraction, MAF) in 9.8% of cases (69/702) (F1174 n=25, R1275 n=32, both F1174 and R1275 n=1, F1245 n=6, others n=5) and at a subclonal level (MAF 0.5-20%) in 3.7% of patients (22/586) (F1174 n=11, R1275 n=6, both F1174 and R1275 or F1174 and F1245 n=3, other n=2). A significantly poorer OS and EFS was observed in cases with clonal ALK mutations, versus all others (3-years OS 47% +/-6.4% versus 65% +/-2%, logrank, p< 0.0001) and in those with ALK amplifications, versus all others (3-years OS 31% +/-8.5% versus 66% +/- 1.9%; logrank, p<0.0001). A Cox proportional hazards procedure (450 patients with complete clinical/biological datasets) retained stage 4 disease (as opposed to non-stage 4) and ALK amplification as factors with a higher hazard of relapse/progression (hazard 2.3 and 2.2, respectively), whereas ALK mutation, MYCN amplification and age>18 months were not retained. The consecutive treatment of Doxorubicin followed by Lorlatinib had a synergistic effect in ALK mutated/amplified NB cell lines. Conclusion: Genetic alterations of ALK (clonal mutations, amplifications) in high-risk NB patients are associated with poorer survival. Further preclinical data are required to determine optimal treatment modalities for integration of TKI in upfront treatment strategies of HR NB patients with ALK alterations.
- Genetic alterations of ALK in high-risk neuroblastoma patients: a SIOPEN studyPublication . Bellini, Angela; Bernard, Virginie; Ambros, Inge; Ambros, Peter F.; de Preter, Katleen; Combaret, Valérie; Beiske, Klaus; Jeison, Marta; Marques, Barbara; Morini, Martina; Mazzocco, Katia; Defferrari, Raffaella; Betts, David; Martinsson, Tommy; Mühlethaler-Mottet, Annick; Noguera, Rosa; Font de Mora, Jaime; Vicha, Ales; Ladenstein, Ruth; Valteau-Couanet, Dominique; Rossing, Caroline Maria; Bown, Nick; Tweddle, Deborah; Avigad, Smadar; Lapouble, Eve; Chicard, Mathieu; Leprovost, Nada; Clement, Nathalie; Baulande, Sylvain; Pierron, Gaelle; Irene, Jimenez; Jaydutt, Bhalshankar; Delattre, Olivier; Michon, Jean; Schleiermacher, GudrunBackground: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated either through genomic amplification or activating point mutations. We studied ALK genetic alterations in high-risk NB patients to determine their frequency and prognostic impact.
- A multilocus technique for risk evaluation of patients with neuroblastomaPublication . Ambros, Inge M.; Brunner, Bettina; Aigner, Gerhard; Bedwell, Clare; Beiske, Klaus; Bénard, Jean; Bown, Nick; Combaret, Valerie; Couturier, Jerome; Defferrari, Raffaella; Gross, Nicole; Jeison, Marta; Lunec, John; Marques, Barbara; Martinsson, Tommy; Mazzocco, Katia; Noguera, Rosa; Schleiermacher, Gudrun; Speleman, Frank; Stallings, Ray; Tonini, Gian Paolo; Tweddle, Deborah A.; Valent, Alexander; Vicha, Ales; Van Roy, Nadine; Villamon, Eva; Ziegler, Andrea; Preuner, Sandra; Drobics, Mario; Ladenstein, Ruth; Amann, Gabriele; Schuit, Robert J.L.; Pötschger, Ulrike; Ambros, Peter F.Purpose: Precise and comprehensive analysis of neuroblastoma genetics is essential for accurate risk evaluation and only pangenomic/multilocus approaches fulfill the present-day requirements. We present the establishment and validation of the PCR-based multiplex ligation-dependent probe amplification (MLPA) technique for neuroblastoma. Experimental Design: A neuroblastoma-specific MLPA kit was designed by the SIOP Europe Neuroblastoma Biology Committee in cooperation with MRC-Holland. The contained target sequences cover 19 chromosomal arms and reference loci. Validation was performed by single locus and pangenomic techniques (n ¼ 174). Dilution experiments for determination of minimal tumor cell percentage were performed and testing of reproducibility was checked by interlaboratory testing (n ¼ 15). Further 156 neuroblastomas were used for establishing the amplification cutoff level. Results: The MLPA technique was tested in 310 neuroblastomas and 8 neuroblastoma cell lines (including validation and amplification cutoff level testing). Intertechnique validation showed a high concordance rate (99.5%). Interlaboratory MLPA testing (k ¼ 0.95, P < 0.01) revealed 7 discrepant of 1,490 results (0.5%). Validation by pangenomic techniques showed a single discordance of 190 consensus results (0.5%). The test results led to formulation of interpretation standards and to a kit revision. The minimal tumor cell percentage was fixed at 60%. Conclusions: The recently designed neuroblastoma-specific MLPA kit covers all chromosomal regions demanded by the International Neuroblastoma Risk Group for therapy stratification and includes all hitherto described genetic loci of prognostic interest for future studies and can be modified or extended at any time. Moreover, the technique is cost effective, reliable, and robust with a high interlaboratory and intertechnique concordance.
- Small Deletion of 143 Kb Encompassing Exon 2 of the AUTS2: Rise of a NewMicrodeletion Syndrome?Publication . Serafim, Silvia; Marques, Barbara; Filomena, Brito; Pedro, Sónia; Ferreira, Cristina; Ventura, Catarina; Gaspar, Isabel; Correia, HildebertoChromosome microarray analysis is a powerful diagnostic tool and is being used as a first-line approach to detect chromosome imbalances associated with intellectual disability, dysmorphic features and congenital anomalies. This test enables the identification of new copy number variants (CNVs) and their association with new microdeletion/microduplication syndromes in patients previously without diagnosis. Here we report the case of a 17 year-old female with severe intellectual disability, absence of speech, microcephaly and congenital abnormalities with a previous normal karyotype performed at a younger age. Affymetrix CytoScan HD chromosome microarray analysis was performed detecting a 143 Kb deletion at the 7q11.22 breakpoint, encompassing exon 2 of AUTS2 gene: arr[hg19] 7q11.22(69238957- 69381975)×1. The AUTS2 gene has been recently implicated in neurodevelopment and is a candidate gene for numerous neurological disorders. Common clinical features described in patients with deletions in AUTS2 gene include intellectual disability, speech delay and microcephaly, among others. Thus, the CNV identified in our patient explains the phenotype observed. We compare our patient with other similar reported cases, adding additional value to the phenotypegenotype correlation of deletions in this region. The growing collection of new cases with similar phenotypes, and the observation of this deletion occurring frequently de novo, indicates this CNV as a possible new single gene microdeletion syndrome.