SVA retrotransposon insertion-associated deletion represents a novel mutational mechanism underlying large genomic copy number changes with non-recurrent breakpoints

Background: Genomic disorders are caused by copy number changes that may exhibit recurrent breakpoints processed by nonallelic homologous recombination. However, region-specific disease-associated copy number changes have also been observed which exhibit non-recurrent breakpoints. The mechanisms underlying these non-recurrent copy number changes have not yet been fully elucidated. Results: We analyze large NF1 deletions with non-recurrent breakpoints as a model to investigate the full spectrum of causative mechanisms, and observe that they are mediated by various DNA double strand break repair mechanisms, as well as aberrant replication. Further, two of the 17 NF1 deletions with non-recurrent breakpoints, identified in unrelated patients, occur in association with the concomitant insertion of SINE/variable number of tandem repeats/Alu (SVA) retrotransposons at the deletion breakpoints. The respective breakpoints are refractory to analysis by standard breakpoint-spanning PCRs and are only identified by means of optimized PCR protocols designed to amplify across GC-rich sequences. The SVA elements are integrated within SUZ12P intron 8 in both patients, and were mediated by target-primed reverse transcription of SVA mRNA intermediates derived from retrotranspositionally active source elements. Both SVA insertions occurred during early postzygotic development and are uniquely associated with large deletions of 1 Mb and 867 kb, respectively, at the insertion sites. Conclusions: Since active SVA elements are abundant in the human genome and the retrotranspositional activity of many SVA source elements is high, SVA insertion-associated large genomic deletions encompassing many hundreds of kilobases could constitute a novel and as yet under-appreciated mechanism underlying large-scale copy number changes in the human genome

Mutational spectrum by phenotype: panel-based NGS testing of patients with clinical suspition of RASopathy and children with multiple café-au-lait macules

Children with neurofibromatosis type 1 (NF1) may exhibit an incomplete clinical presentation, making difficult to reach a clinical diagnosis. A phenotypic overlap may exist in children with other RASopathies or with other genetic conditions if only multiple café‐au‐lait macules (CALMs) are present. The syndromes that can converge in these inconclusive phenotypes have different clinical courses. In this context, an early genetic testing has been proposed to be clinically useful to manage these patients. We present the validation and implementation into diagnostics of a custom NGS panel (I2HCP, ICO‐IMPPC Hereditary Cancer Panel) for testing patients with a clinical suspicion of a RASopathy (n = 48) and children presenting multiple CALMs (n = 102). We describe the mutational spectrum and the detection rates identified in these two groups of individuals. We identified pathogenic variants in 21 out of 48 patients with clinical suspicion of RASopathy, with mutations in NF1 accounting for 10% of cases. Furthermore, we identified pathogenic mutations mainly in the NF1 gene, but also in SPRED1, in more than 50% of children with multiple CALMs, exhibiting an NF1 mutational spectrum different from a group of clinically diagnosed NF1 patients (n = 80). An NGS panel strategy for the genetic testing of these two phenotype‐defined groups outperforms previous strategie

SVA retrotransposon insertion-associated deletion represents a novel mutational mechanism underlying large genomic copy number changes with non-recurrent breakpoints

Background: Genomic disorders are caused by copy number changes that may exhibit recurrent breakpoints processed by nonallelic homologous recombination. However, region-specific disease-associated copy number changes have also been observed which exhibit non-recurrent breakpoints. The mechanisms underlying these non-recurrent copy number changes have not yet been fully elucidated. Results: We analyze large NF1 deletions with non-recurrent breakpoints as a model to investigate the full spectrum of causative mechanisms, and observe that the

Examination of the genetic factors underlying the cognitive variability associated with neurofibromatosis type 1

Purpose: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder associated with cognitive deficits. The NF1 cognitive phenotype is generally considered to be highly variable, possibly due to the observed T2-weighted hyperintensities, loss of heterozygosity, NF1-specific genetic modifiers, or allelic imbalance. Methods: We investigated cognitive variability and assessed the contribution of genetic factors by performing a retrospective cohort study and a monozygotic twin case series. We included data of 497 children with genetically confirmed NF1 and an IQ assessment, including 12 monozygotic twin and 17 sibling sets. Results: Individuals carrying an NF1 chromosomal microdeletion showed significant lower full-scale IQ (FSIQ) scores than individuals carrying intragenic pathogenic NF1 variants. For the intragenic subgroup, the variability in cognitive ability and the correlation of IQ between monozygotic NF1 twin pairs or between NF1 siblings is similar to the general population. Conclusions: The variance and heritability of IQ in individuals with NF1 are similar to that of the general population, and hence mostly driven by genetic background differences. The only factor that significantly attenuates IQ in NF1 individuals is the NF1 chromosomal microdeletion genotype. Implications for clinical management are that individuals with intragenic NF1 variants that score <1.5–2 SD below the mean of the NF1 population should be screened for additional causes of cognitive disability

Legius Syndrome in Fourteen Families

Legius syndrome presents as an autosomal dominant condition characterized by café-au-lait macules with or without freckling and sometimes a Noonan-like appearance and/or learning difficulties. It is caused by germline loss-of-function SPRED1 mutations and is a member of the RAS-MAPK pathway syndromes. Most mutations result in a truncated protein and only a few inactivating missense mutations have been reported. Since only a limited number of patients has been reported up until now, the full clinical and mutational spectrum is still unknown. We report mutation data and clinical details in fourteen new families with Legius syndrome. Six novel germline mutations are described. The Trp31Cys mutation is a new pathogenic SPRED1 missense mutation. Clinical details in the 14 families confirmed the absence of neurofibromas, and Lisch nodules, and the absence of a high prevalence of central nervous system tumors. We report white matter T2 hyperintensities on brain MRI scans in 2 patients and a potential association between postaxial polydactyly and Legius syndrome. © 2010 Wiley-Liss, Inc

Neurofibromatosis type 1-related pseudarthrosis: Beyond the pseudarthrosis site.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting approximately 1 in 2,000 newborns. Up to 5% of NF1 patients suffer from pseudarthrosis of a long bone (NF1-PA). Current treatments are often unsatisfactory, potentially leading to amputation. To gain more insight into the pathogenesis we cultured cells from PA tissue and normal-appearing periosteum of the affected bone for NF1 mutation analysis. PA cells were available from 13 individuals with NF1. Biallelic NF1 inactivation was identified in all investigated PA cells obtained during the first surgery. Three of five cases sampled during a later intervention showed biallelic NF1 inactivation. Also, in three individuals, we examined periosteum-derived cells from normal-appearing periosteum proximal and distal to the PA. We identified the same biallelic NF1 inactivation in the periosteal cells outside the PA region. These results indicate that NF1 inactivation is required but not sufficient for the development of NF1-PA. We observed that late-onset NF1-PA occurs and is not always preceded by congenital bowing. Furthermore, the failure to identify biallelic inactivation in two of five later interventions and one reintervention with a known somatic mutation indicates that NF1-PA can persist after the removal of most NF1 negative cells

Deep genomic analysis of malignant peripheral nerve sheath tumor cell lines challenges current malignant peripheral nerve sheath tumor diagnosis

Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas of the peripheral nervous system that develop either sporadically or in the context of neurofibromatosis type 1 (NF1). MPNST diagnosis can be challenging and treatment outcomes are poor. We present here a resource consisting of the genomic characterization of 9 widely used human MPNST cell lines for their use in translational research. NF1-related cell lines recapitulated primary MPNST copy number profiles, exhibited NF1 , CDKN2A , and SUZ12/EED tumor suppres-sor gene (TSG) inactivation, and presented no gain-of-function mutations. In contrast, sporadic cell lines collectively displayed different TSG inactivation patterns and presented kinase-activating mutations, fusion genes, altered muta-tional frequencies and COSMIC signatures, and different methylome-based clas-sifications. Cell lines re-classified as melanomas and other sarcomas exhibited a different drug-treatment response. Deep genomic analysis, methylome-based classification, and cell-identity marker expression, challenged the identity of common MPNST cell lines, opening an opportunity to revise MPNST differential diagnosis

Pathogenesis of vestibular schwannoma in ring chromosome 22

<p>Abstract</p> <p>Background</p> <p>Ring chromosome 22 is a rare human constitutional cytogenetic abnormality. Clinical features of neurofibromatosis type 1 and 2 as well as different tumour types have been reported in patients with ring chromosome 22. The pathogenesis of these tumours is not always clear yet.</p> <p>Methods</p> <p>We report on a female patient with a ring chromosome 22 presenting with severe mental retardation, autistic behaviour, café-au-lait macules and facial dysmorphism. Peripheral blood lymphocytes were karyotyped and array CGH was performed on extracted DNA. At the age of 20 years she was diagnosed with a unilateral vestibular schwannoma. Tumour cells were analyzed by karyotyping, array CGH and <it>NF2 </it>mutation analysis.</p> <p>Results</p> <p>Karyotype on peripheral blood lymphocytes revealed a ring chromosome 22 in all analyzed cells. A 1 Mb array CGH experiment on peripheral blood DNA showed a deletion of 5 terminal clones on the long arm of chromosome 22. Genetic analysis of vestibular schwannoma tissue revealed loss of the ring chromosome 22 and a somatic second hit in the <it>NF2 </it>gene on the remaining chromosome 22.</p> <p>Conclusion</p> <p>We conclude that tumours can arise by the combination of loss of the ring chromosome and a pathogenic <it>NF2 </it>mutation on the remaining chromosome 22 in patients with ring chromosome 22. Our findings indicate that patients with a ring 22 should be monitored for NF2-related tumours starting in adolescence.</p

Genotype-phenotype associations in a large PTEN Hamartoma Tumor Syndrome (PHTS) patient cohort

Background: Pathogenic PTEN germline variants cause PTEN Hamartoma Tumor Syndrome (PHTS), a rare disease with a variable genotype and phenotype. Knowledge about these spectra and genotype-phenotype associations could help diagnostics and potentially lead to personalized care. Therefore, we assessed the PHTS genotype and phenotype spectrum in a large cohort study. Methods: Information was collected of 510 index patients with pathogenic or likely pathogenic (LP/P) PTEN variants (n = 467) or variants of uncertain significance. Genotype-phenotype associations were assessed using logistic regression analyses adjusted for sex and age.Results: At time of genetic testing, the majority of children (n = 229) had macrocephaly (81%) or developmental delay (DD, 61%), and about half of the adults (n = 238) had cancer (51%), macrocephaly (61%), or cutaneous pathology (49%). Across PTEN, 268 LP/P variants were identified, with exon 5 as hotspot. Missense variants (n = 161) were mainly located in the phosphatase domain (PD, 90%) and truncating variants (n = 306) across all domains. A trend towards 2 times more often truncating variants was observed in adults (OR = 2.3, 95%CI = 1.5-3.4) and patients with cutaneous pathology (OR = 1.6, 95%CI = 1.1-2.5) or benign thyroid pathology (OR = 2.0, 95%CI = 1.1-3.5), with trends up to 2-4 times more variants in PD. Whereas patients with DD (OR = 0.5, 95%CI = 0.3-0.9) or macrocephaly (OR = 0.6, 95%CI = 0.4-0.9) had about 2 times less often truncating variants compared to missense variants. In DD patients these missense variants were often located in domain C2.Conclusion: The PHTS phenotypic diversity may partly be explained by the PTEN variant coding effect and the combination of coding effect and domain. PHTS patients with early-onset disease often had missense variants, and those with later-onset disease often truncating variants

Molecular and functional characterization of a new neuro-cardio-facial-cutaneous syndrome: Legius syndrome

We beschreven en karakteriseerden een nieuwe autosomaal dominante aandoening, Legius syndroom, die genetisch verschillend is maar klinisch erg gelijkend op neurofibromatose type 1 (NF1). De klinische kenmerken bestaan uit café-au-lait vlekken met of zonder sproeten, macrocefalie, een relatief kleine gestalte en een verhoogde frequentie van leerproblemen bij kinderen. Sommige patiënten vertonen Noonan-gelijkende dysmorfe karakteristieken en sommige individuen vervullen de klinisch diagnostische NIH criteria voor NF1. Sommige typische NF1-geassocieerde kenmerken zijn systematisch afwezig, zoals Lisch nodules, neurofibromen, NF1-geassocieerde botdefecten en tumoren van het centrale zenuwstelsel. Inactiverende mutaties werden geïdentificeerd in SPRED1, dit gen codeert voor een eiwit dat de RAS-MAPK signaaltransductie cascade negatief reguleert en dit ter hoogte van RAS-RAF interactie. Melanocyten van een café-au-lait vlek van een aangetast individu vertoonden biallelische mutaties in SPRED1 (kiemlijn en somatische SPRED1 mutatie). Deze aandoening is een nieuw lid van de groep van de fenotypisch overlappende neuro-cardio-faciale-cutane (NCFC) syndromen of RASopathieën. Een grote groep van NF1-mutatie negatieve patiënten werd onderzocht en in 1.9% van de patiënten werd een pathogene SPRED1 mutatie geïdentificeerd. In een klein percentage van de gevallen werd een SPRED1 missense mutatie gevonden en functioneel gekarakteriseerd naar pathogeniciteit. We toonden aan dat sommige van deze SPRED1 missense mutaties effectief pathogeen zijn. Omdat leerproblemen gerapporteerd werden bij verscheidene kinderen met Legius syndroom, werd een muis Spred1-/- model gebruikt om zowel hippocampaal afhankelijk leren en geheugen als lange termijn potentiatie te onderzoeken. Spred1-/- muizen vertoonden een verlaagde performantie in de gedragstesten wat leren en geheugen betreft en korte en lange termijn hippocampale synaptische plasticiteitsdefecten werden geobserveerd. We startten een verkennende studie omtrent het Spred Drosophila "loss-of-function" model, omdat dit uiterst geschikt is als screen voor interagerende proteïnen, betrokken celtypes en signaaltransductie cascades. We detecteerden een subtiel groeidefect met een kleinere vleugelgrootte maar met een normaal venenpatroon. We identificeerden verminderde opschrik geïnduceerde locomotie en verminderd agressief gedrag in de Spred "loss-of-function" vliegen. Larvale neuromusculaire juncties vertoonden een verminderd aantal synapsen (boutons) in de Spred-/- vliegen, hoewel de lengte en spier innervatie niet veranderd was. Deze succesvolle initiële verkennende studie in Drosophila toonde verschillende fenotypes aan en laat ons toe om in de toekomst Spred functie en proteïne interacties in dit model te bestuderen.status: publishe