Genome-wide screening methods in tumors of the central nervous system and cancer predisposition

Since the development of screening methods that can be used on an exome- or genome-wide scale such as array-based comparative genomic hybridization (array-CGH) and next generation sequencing (NGS), these techniques have been employed to analyze large patient and tumor cohorts and are also frequently used for diagnostic purposes. In this study, array-CGH was used to analyze DNA from tumors of the central nervous system to identify somatic copy number changes. Approximately 300 tumor samples from the German Glioma Network and an intracranial malignant peripheral nerve sheath tumor were analyzed using array-CGH to identify specific patterns of copy number alterations in the different tumor entities. Furthermore, array-CGH and whole exome sequencing (WES) were performed on DNA from peripheral blood from a patient presenting with a complex phenotype including cancer predisposition to identify causative germline aberrations. The first project addressed molecular aberrations in gliomas classified as grade II and grade III by the World Health Organization (WHO) including astrocytomas, oligoastrocytomas and oligodendrogliomas as well as anaplastic astrocytomas and anaplastic oligoastrocytomas. Tumor samples of the different glioma entities were analyzed using array-CGH, in order to detect common genetic imbalances in the gliomas of WHO grade II and III. Together with the German Glioma Network the mutation status in the IDH1 (isocitrate-dehydrogenase 1) and IDH2 genes was determined. Most of the WHO grade II and grade III gliomas harbored an IDH1 or IDH2 mutation. It could be shown that WHO grade II gliomas displayed DNA copy number changes less frequently than WHO grade III gliomas. Interestingly, a small group of IDH1/2 wild-type WHO grade II astrocytomas were detected which displayed glioblastoma-like genomic imbalances. Patients with an IDH1/2 wild-type astrocytoma of WHO grade II displaying a glioblastoma-like genomic profile, i.e. gains on chromosomes 7, 19 and 20 as well as losses of chromosomes 9 and 10, possibly would benefit from a more intensive therapy strategy. Therefore these analyses have future implications. Furthermore, frequent deletions of chromosomal arms 1p and 19q were found in oligodendroglial tumors or in mixed astrocytic tumors displaying also an oligodendroglial component. Both alterations were significantly less frequent in astrocytic tumors. The molecular analysis of primary WHO grade IV glioblastomas was subdivided into two parts. As the prognosis of primary glioblastoma is still poor, long-term survival of more than three years after diagnosis is rare in these patients. Thus, the first part of the analysis (project 2 of this work) focused on tumors from patients who exhibited long-term survival. Genomic profiles of glioblastomas from long-term survivors were compared to those from short-term and intermediate-term survivors. The IDH1/2 mutation and the MGMT promoter methylation status were also determined in these tumors. This analysis showed that patients with long-term survival were younger and corresponding tumors more often had an IDH1/2 mutation and a MGMT promoter methylation. Genomic imbalances were prominently different between IDH1/2 mutant and IDH1/2 wild-type tumors, but not between survival groups of IDH1/2 wild-type glioblastoma patients, suggesting that long-term survival is due to other, e.g. host-related factors. The second part of the analysis (project 3 of this work) focused on tumor recurrence of primary WHO grade IV glioblastomas. Glioblastomas have a tendency to recur despite combined surgical resection, radiotherapy and temozolomide chemotherapy. When the tumor recurs the WHO grade remains the same, therefore, the recurrent tumor is treated similar to the primary tumor. Genomic profiles of 27 primary and recurrent IDH1/2 wild-type glioblastoma from the same patient were compared to determine genetic patterns of glioblastoma progression. After comparing the array-CGH profiles of the primary and recurrent tumors, taking the tumor cell content into account, a difference profile for each tumor pair was generated. Subsequently, three molecular relapse groups were defined (Equal, Sequential and Discrepant). Seven of the 27 (26%) tumor pairs were identified to be Equal pairs, showing no DNA copy number differences between primary and recurrent tumor, suggesting a monoclonal cell composition of both tumors. In nine of 27 (33%) tumor pairs, the same and additional chromosomal imbalances were found in the recurrent tumor as compared to the primary tumor (Sequential pairs). These findings suggest a sequential acquisition or selection for aberrations during tumor progression. In eleven of 27 (41%) pairs, the difference profiles of primary and recurrent tumors were divergent, i.e. the recurrent tumors contained additional chromosomal aberrations but had also lost others (Discrepant pairs). These findings suggest a polyclonal composition of the primary tumors and considerable clonal evolution. Interestingly, losses on chromosomal band 9p21.3, harboring the CDKN2A/B locus, were significantly more common in primary tumors of the Sequential and Discrepant tumor pairs, also called non-Equal pairs. Analyzing regions of chromosomal differences between primary and recurrent tumors 46 candidate genes associated with tumor recurrence were identified. Frequently, the identified genes for apoptosis regulators, possibly explaining why these cells escape therapy induced apoptosis. Taken together about 75% of IDH1/2 wild-type recurrent glioblastomas acquire additional genomic alterations during progression. This process is possibly facilitated by the loss of genetic material from chromosomal band 9p21.3 in the primary glioblastomas. These tumor recurrence-associated chromosomal changes may contribute to therapy resistance, e.g. by copy number alterations of apoptosis regulatory genes. The analysis of the genomic differences between primary and recurrent glioblastomas may identify newly acquired genetic properties targetable by salvage therapies for a more effective treatment of patients with recurrent glioblastoma. In the fourth project of this work, two intracranial tumor samples from a 47-year old female patient were retrospectively analyzed using array-CGH in order to determine their clonal relationship. The histological diagnosis of the first tumor was an unusual pituitary adenoma, but the second tumor that had developed 3 months later was diagnosed as a rare malignant peripheral nerve sheath tumor. Though the two tumor samples were different in their histopathology, the question arose if the first and the second tumor contained the same copy number changes and thus most likely developed from the same origin. Array-CGH of the first tumor revealed a complex pattern of chromosomal imbalances affecting all chromosomes but one (chromosome 16). Array-CGH of the second tumor revealed a similarly complex profile. About 80% of the 29 copy number changes detected in the second tumor were already present in the first tumor. These findings provide strong evidence for a clonal relationship between the two tumor samples and suggest that the second tumor was a recurrent tumor of the first lesion. It also could be shown that the genomic profiles of both tumors were highly similar to those of already published MPNST cases, indicating that the analyzed tumor indeed is a MPNST. Taken together, it could be shown that array-CGH can be successfully used to identify the clonal relationship between two histologically distinct tumors. The fifth project of this work aimed at identifying the germline aberrations underlying a complex phenotype including cancer predisposition. Symptoms of the patient included cognitive impairment, two neoplastic diseases (a both-sided mixed malignant germ cell tumor of the ovaries and an acute pre-B-lymphoblastic leukemia) prior to the age of 20 years, anomalies of skin pigmentation and short stature. Using array-CGH on DNA from peripheral blood from the patient and her mother, two maternally inherited microduplications in the chromosomal bands 6q27 and 22q11.21 were detected. The microduplication with the size of 0.26 Mb in 6q27 encompassed parts of the MLLT4 gene, a known fusion partner of MLL in leukemic cells. The microduplication in chromosomal band 22q11.21 had a size of 2.5 Mb, harboring approximately 50 genes. This region has been reported to be susceptible to chromosomal rearrangements and to cause the 22q11.21 microduplication syndrome when duplicated and is associated with a high variability, explaining in part the patient’s intellectual disability but not its severity. Particularly the two malignancies of the patient were not explained by the detected microduplications. Therefore, DNA from peripheral blood from the patient as well as from her patents was screened by WES in order to find further causative germline aberrations. A trio-based de novo analysis, subtracting the parental variants from variants detected in the patient, revealed a de novo CHEK2 variant (CHEK2,c.1427C>T;p.Thr476Met). This rare missense variant is a “HGMD disease mutation” contributing to breast cancer susceptibility. Using a different filter strategy for the WES data set of the patient, two known BLM founder mutations (BLM,c.1642C>T;p.Gln548X; BLM,c.2695C>T;p.Arg899X) were also detected. Sanger sequencing revealed that the patient was compound heterozygous for these two stop mutations in the BLM gene, i.e. that one of the mutations was inherited from the mother and the other from the father. Bloom syndrome, caused by mutations in the BLM gene is a rare autosomal recessive disorder associated with most of the symptoms found in the patient, including short stature, mild craniofacial dysmorphia, hypo- and hyper-pigmented skin lesions and cancer predisposition. Taken together, two genome-wide screening methods have been used to unravel the highly complex phenotype of the patient. Employing array-CGH and WES, two inherited microduplications as well as two inherited recessive founder mutations (BLM) and one de novo missense variant (CHEK2) were identified. The combination of Bloom syndrome with the 22q11.2 microduplication syndrome and the CHEK2 associated multi-cancer susceptibility syndrome, are presumed to cause the severe intellectual disability, and explain the cancer predisposition in the patient. This case demonstrated that complex phenotypes may be caused by more than one genetic alteration, rather a combination of copy number variants and point mutations may be the cause. In summary, array-CGH was used to detect somatic tumor aberrations in WHO grade II to WHO grade IV glioma entities as well as a case of a MPNST. Further, employing two genome-wide screening methods, array-CGH and WES, the complex genotype of a patient with syndromic cancer predisposition was unraveled, indicating that complex phenotypes may be caused by a number of different genetic alterations

The ARID1B spectrum in 143 patients: from nonsyndromic intellectual disability to Coffin–Siris syndrome

Purpose: Pathogenic variants in ARID1B are one of the most frequent causes of intellectual disability (ID) as determined by large-scale exome sequencing studies. Most studies published thus far describe clinically diagnosed Coffin–Siris patients (ARID1B-CSS) and it is unclear whether these data are representative for patients identified through sequencing of unbiased ID cohorts (ARID1B-ID). We therefore sought to determine genotypic and phenotypic differences between ARID1B-ID and ARID1B-CSS. In parallel, we investigated the effect of different methods of phenotype reporting. Methods: Clinicians entered clinical data in an extensive web-based survey. Results: 79 ARID1B-CSS and 64 ARID1B-ID patients were included. CSS-associated dysmorphic features, such as thick eyebrows, long eyelashes, thick alae nasi, long and/or broad philtrum, small nails and small or absent fifth distal phalanx and hypertrichosis, were observed significantly more often (p < 0.001) in ARID1B-CSS patients. No other significant differences were identified. Conclusion: There are only minor differences between ARID1B-ID and ARID1B-CSS patients. ARID1B-related disorders seem to consist of a spectrum, and patients should be managed similarly. We demonstrated that data collection methods without an explicit option to report the absence of a feature (such as most Human Phenotype Ontology-based methods) tended to underestimate gene-related features

A heritable microduplication encompassing TBL1XR1 causes a genomic sister-disorder for the 3q26.32 microdeletion syndrome

Recently, a new syndrome with intellectual disability (ID) and dysmorphic features due to deletions or point mutations within the TBL1XR1 gene located in the chromosomal band 3q26.32 has been described (MRD41, OMIM 616944). One recurrent point mutation in the TBL1XR1 gene has been identified as the cause of Pierpont syndrome (OMIM 602342), a distinct intellectual disability syndrome with plantar lipomatosis. In addition, different de novo point mutations in the TBL1XR1 gene have been found in patients with autism spectrum disorders (ASD) and intellectual disability. Here, we report four patients from two unrelated families in whom array-CGH analysis and real-time quantitative PCR of genomic DNA revealed a TBL1XR1-microduplication. Adjacent genes were not affected. The microduplication occurred as a de novo event in one patient, whereas the other three cases occurred in two generations of a second, unrelated family. We compare and contrast the clinical findings in TBL1XR1 microdeletion, point mutation, and microduplication cases and expand the TBL1XR1-associated phenotypic spectrum. ID, hearing loss, and ASD are common features of TBL1XR1-associated diseases. Our clinical observations add to the increasing evidence of the role of TBL1XR1 in brain development, and they simultaneously demonstrate that different genetic disease mechanisms affecting TBL1XR1 can lead to similar ID phenotypes. The TBL1XR1-microduplication syndrome is an intellectual disability/learning disability syndrome with associated incomplete penetrance ASD, hearing loss, and delay of puberty. Its phenotypic overlap indicates that it is a genomic sister-disorder to the 3q26.32 microdeletion syndrome

A step towards precision medicine in management of severe transient polyhydramnios: MAGED2 variant

International audienc

NPHP1 gene-associated nephronophthisis is associated with an occult retinopathy

Biallelic deletions in the NPHP1 gene are the most frequent molecular defect of nephronophthisis, a kidney ciliopathy and leading cause of hereditary end-stage kidney disease. Nephrocystin 1, the gene product of NPHP1, is also expressed in photoreceptors where it plays an important role in intra-flagellar transport between the inner and outer segments. However, the human retinal phenotype has never been investigated in detail. Here, we characterized retinal features of 16 patients with homozygous deletions of the entire NPHP1 gene. Retinal assessment included multimodal imaging (optical coherence tomography, fundus autofluorescence) and visual function testing (visual acuity, full -field electroretinography, color vision, visual field). Fifteen patients had a mild retinal phenotype that predominantly affected cones, but with relative sparing of the fovea. Despite a predominant cone dysfunction, night vision problems were an early symptom in some cases. The consistent retinal phenotype on optical coherence tomography images included reduced reflectivity and often a granular appearance of the ellipsoid zone, fading or loss of the interdigitation zone, and mild outer retinal thinning. However, there were usually no obvious structural changes visible upon clinical examination and fundus autofluorescence imaging (occult retinopathy). More advanced retinal degeneration might occur with ageing. An identified additional CEP290 variant in one patient with a more severe retinal degeneration may indicate a potential role for genetic modifiers, although this requires further investigation. Thus, diagnostic awareness about this distinct retinal phenotype has implications for the differential diagnosis of nephronophthisis and for individual prognosis of visual function

Long-term data on two sisters with C3GN due to an identical, homozygous CFH mutation and autoantibodies

C3 glomendonephritis (C3GN) is a rare but severe form of kidney disease caused by fluid-phase dysregulation of the alternative complement pathway. Causative mutations in complement regulating genes as well as auto-immune forms of C3GN have been described. However, therapy and prognosis in individual patients remain a matter of debate and long-term data are scarce. This also applies for the management of transplant patients as disease recurrence post-transplant is frequent. Here, we depict the clinical courses of two sisters with the unique combination of an identical, homozygous mutation in the complement factor H (CFH) gene as well as autoantibodies with a clinical follow-up of more than 20 years. Interestingly, the sisters presented with discordant clinical courses of C3GN with normal kidney function in one (patient A) and end-stage kidney disease in the other sister (patient B). In patient B, eculizumab was administered immediately prior to and in the course after kidney transplantation, with the result of a stable graft function without any signs of disease recurrence. Comprehensive genetic work-up revealed no further disease-causing mutation in both sisters. Intriguingly, the auto-antibody profile substantially differed in both sisters: autoantibodies in patient A reduced the C3b deposition, while the antibodies identified in patient B increased complement activation and deposition of split products. This study underlines the concept of a personalized-medicine approach in complement-associated diseases after thorough evaluation of the individual risk profile in each patient