Effect of different mutations in the ATM gene on the cellular response to ionizing radiation

Ataxia Telangiectasia - an autosomal recessive, multisystem disorder - is characterized by progressive cerebellar ataxia, telangiectasia, immune defects and a predisposition to malignancy and was first described in 1926 by Syllaba and Henner (Syllaba, L., Henner, K. 1926). The gene – Ataxia Telangiectasia Mutated (ATM) - was identified on chromosome 11q22-23 and up to now more than 350 different mutations have been described. The ATM protein is a 350 kDa serine-threonine protein kinase that localizes mainly to the nucleus and plays a key role in the repair of DNA double strand breaks that are typically induced by ionizing radiation (IR). ATM is activated after IR-induced double strand breaks occur and subsequently phosphorylates many downstream targets that are involved in DNA damage repair mechanisms and cell cycle control. This activation cascade leads either to cell cycle arrest, repair of the DNA damage or induction of apoptosis. Disturbances in these mechanisms can result in an accumulation of DNA alterations that predispose the damaged cells to malignant transformation. Since 1991, ATM has been discussed as a susceptibility gene for breast cancer (Swift et al. 1991), in particular the missense mutation c.7271T>G, p.Val2424Gly. For this missense variation a dominant-negative mechanism is described in the literature and in 2006 Waddell used expression profiles to discriminate c.7271T>G carriers from healthy controls (Chenevix-Trench et al. 2002; Waddell et al. 2006). In the present study proteomics-based approaches in addition to different cell biological and biochemical investigations were used to elucidate the cellular reactions upon ionizing radiation of lymphoblastoid cell lines from A-T patients carrying different ATM-mutations with one AT patient homozygous for c.7271T>G, breast cancer patients heterozygous for c.7271T>G, and healthy controls. FACS analysis could confirm already published data for a higher susceptibility to cell death and G1 arrest (f.e. (Bakkenist und Kastan 2003) for the cell lines derived from breast cancer patients harboring the heterozygous c.7271T>G variant upon IR. In contrast the Western Blot analysis could not confirm the dominant negative effect that was postulated by Chenevix-Trench and Waddell for the cell lines harbouring the heterozygous c.7271T>G substitution. In contrast to the published data the present study could show that downstream targets of ATM are still phosphorylated upon IR. The pedigree of the family A published 2002 by Chenevix-Trench shows that the breast cancer is inherited within the same haplotype, so there could be other factors accounting for this phenotype. Two proteomics-based approaches, DIGE analysis and SWATH acquisition, were used to investigate the cellular response upon ionizing radiation for all cell lines. Two members of the ATM pathway –SMC1A and MRE11 – were significantly downregulation in the healthy control cell lines, with MRE11 also downregulated in the AT cell lines. A shift in the 2D gel of the phosphorylated proteins SMC1A and MRE11 would explain that downregulation upon irradiation. Additionally, the AT patients and control cell lines showed an upregulation of RAD23B – a protein involved in Nucleotide Excision Repair (NER) – upon IR in the SWATH approach, which was not present in the cell lines derived from breast cancer patients. The upregulation of RAD23B was also found in the AT patient cell line in the DIGE analysis. This regulation of RAD23B gives a first hint to a restricted function of NER and maybe DNA mismatch repair (MMR) in the cell lines derived from the breast cancer patients harbouring the c.7271T>G substitution. Additionally, the SWATH-MS approach showed a reduced regulation of proteins involved in NER and MMR in the c.7271T>G heterozygous cell lines compared to the controls. Therefore, these proteomics data show for the first time the postulated link between ATM and the MMR pathway ( (Romeo et al. 2011), which needs further investigation

Early Embryonic Chromosome Instability Results in Stable Mosaic Pattern in Human Tissues

The discovery of copy number variations (CNV) in the human genome opened new perspectives on the study of the genetic causes of inherited disorders and the aetiology of common diseases. Here, a single-cell-level investigation of CNV in different human tissues led us to uncover the phenomenon of mitotically derived genomic mosaicism, which is stable in different cell types of one individual. The CNV mosaic ratios were different between the 10 individuals studied. However, they were stable in the T lymphocytes, immortalized B lymphoblastoid cells, and skin fibroblasts analyzed in each individual. Because these cell types have a common origin in the connective tissues, we suggest that mitotic changes in CNV regions may happen early during embryonic development and occur only once, after which the stable mosaic ratio is maintained throughout the differentiated tissues. This concept is further supported by a unique study of immortalized B lymphoblastoid cell lines obtained with 20 year difference from two subjects. We provide the first evidence of somatic mosaicism for CNV, with stable variation ratios in different cell types of one individual leading to the hypothesis of early embryonic chromosome instability resulting in stable mosaic pattern in human tissues. This concept has the potential to open new perspectives in personalized genetic diagnostics and can explain genetic phenomena like diminished penetrance in autosomal dominant diseases. We propose that further genomic studies should focus on the single-cell level, to better understand the aetiology of aging and diseases mediated by somatic mutations

The Human Genome Puzzle – the Role of Copy Number Variation in Somatic Mosaicism

The discovery of copy number variations (CNV) in the human genome opened new perspectives in the study of the genetic causes of inherited disorders and the etiology of common diseases. Differently patterned instances of somatic mosaicism in CNV regions have been shown to be present in monozygotic twins and throughout different tissues within an individual. A single-cell-level investigation of CNV in different human cell types led us to uncover mitotically derived genomic mosaicism, which is stable in different cell types of one individual. A unique study of immortalized B-lymphoblastoid cell lines obtained with 20 year interval from the same two subjects shows that mitotic changes in CNV regions may happen early during embryonic development and seem to occur only once, as levels of mosaicism remained stable. This finding has the potential to change our concept of dynamic human genome variation. We propose that further genomic studies should focus on the single-cell level, to understand better the etiology and physiology of aging and diseases mediated by somatic variations

Doublet-Mediated DNA Rearrangement-A Novel and Potentially Underestimated Mechanism for the Formation of Recurrent Pathogenic Deletions

Deletions and duplications of genomic DNA contribute to evolution, phenotypic diversity, and human disease. The underlying mechanisms are incompletely understood. We identified deletions of exon 10 of the SPAST gene in two unrelated families with hereditary spastic paraplegia. We excluded a founder event, but observed that the breakpoints map to identical repeat regions. These regions likely represent an intragenic doublet, that is, an enigmatic class of local duplications. The fusion sequences for both deletions are compatible with recombination-based as well as with replication-based mechanisms. Searching the literature, we identified a partial SLC24A4 deletion that involved two copies of another doublet, and was likely formed in an analogous way. Comparing the SPAST and the SLC24A4 doublets with doublets identified previously suggested that many additional doublets have a high potential for triggering rearrangements. Considering that doublets are still being formed in the human genome, and that they likely create high local instability, we suggest that a two-step mechanism consisting of doublet generation and subsequent doublet-mediated deletion/duplication may underlie certain copy-number changes for which other mechanisms are currently assumed. Further studies are necessary to delineate the significance of the thus-far understudied doublets for the formation of copy-number variation. (c) 2016 Wiley Periodicals, Inc

Further evidence for complex inheritance of holoprosencephaly: Lessons learned from pre‐ and postnatal diagnostic testing in Germany

Holoprosencephaly (HPE) has been defined as a distinct clinical entity with characteristic facial gestalt, which may-or may not-be associated with the true brain malformation observed post-mortem in autopsy or in pre- or postnatal imaging. Affected families mainly show autosomal dominant inheritance with markedly reduced penetrance and extremely broad clinical variability even between mutation carriers within the same families. We here present advances in prenatal imaging over the last years, increasing the proportion of individuals with HPE identified prenatally including milder HPE forms and more frequently allowing to detect more severe forms already in early gestation. We report the results of diagnostic genetic testing of 344 unrelated patients for HPE at our lab in Germany since the year 2000, which currently with the application of next generation sequencing (NGS) panel sequencing identifies causal mutations for about 31% (12/38) of unrelated individuals with normal chromosomes when compared to about 15% (46/306) using conventional Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). More comprehensive genetic testing by our in house NGS panel sequencing of 10 HPE associated genes (MiSeq (TM) and NextSeq (TM) 500, Illumina, Inc., San Diego, CA) not only allowed to include genes with smaller contribution to the phenotype, but may also unravel additional low frequency or more common genetic variants potentially contributing to the observed large intrafamiliar variability and may ultimately guide our understanding of the individual clinical manifestation of this complex developmental disorder

An unexpected finding in a child with neurological problems: mosaic ring chromosome 18

Major neurological disorders may accompany rare chromosomal abnormalities. As an example of this rare condition, we present a case with microcephaly, mental retardation, developmental delay, hyperactivity, stereotypic movements, seizures and dysmorphic facial appearance in whom a mosaic ring chromosome 18 was found [45,XX,-18/46,XX,r(18)/46,XX,dicr(18)]. Although ring chromosome 18 phenotype has been known for a long time, this is the third reported patient with a dicentric ring chromosome 18 mosaicism. The presented case will contribute to the identification of the genotype-phenotype correlation in chromosome 18 anomalies

High Frequency of Pathogenic Rearrangements in SPG11 and Extensive Contribution of Mutational Hotspots and Founder Alleles

Biallelic loss-of-function mutations in SPG 11 cause a wide spectrum of recessively inherited, neuro-degenerative disorders including hereditary spastic paraplegia (HSP), amyotrophic lateral sclerosis, and Charcot-Marie-Tooth disease. By comprehensive screening of three large cohorts of HSP index patients, we identified 83 alleles with "small" mutations and 13 alleles that carry large genomic rearrangements. Including relevant data from previous studies, we estimate that copy number variants (CNVs) account for similar to 19% of pathogenic SPG11 alleles. The breakpoints for all novel and some previously reported CNVs were determined by long-range PCR and sequencing. This revealed several Alu-associated recombination hotspots. We also found evidence for additional mutational mechanisms, including for a two-step event in which an Alu retrotransposition preceded the actual rearrangement. Apparently independent samples with identical breakpoints were analyzed by microsatellite PCRs. The resulting haplotypes suggested the existence of two rearrangement founder alleles. Our findings widen the spectra of mutations and mutational mechanisms in SPG11, underscore the pivotal role played by Alus, and are of high diagnostic relevance for a wide spectrum of clinical phenotypes including the most frequent form of recessive HSP. (C) 2016 Wiley Periodicals, Inc

Clinical Long-Time Course, Novel Mutations And Genotype-Phenotype Correlation In A Cohort Of 27 Families With Pomt1-Related Disorders

Background The protein O-mannosyltransferase 1, encoded by the POMT1 gene, is a key enzyme in the glycosylation of α-dystroglycan. POMT1–related disorders belong to the group of dystroglycanopathies characterized by a proximally pronounced muscular dystrophy with structural or functional involvement of the brain and/or the eyes. The phenotypic spectrum ranges from the severe Walker-Warburg syndrome (WWS) to milder forms of limb girdle muscular dystrophy (LGMD). The phenotypic severity of POMT1-related dystroglycanopathies depends on the residual enzyme activity. A genotype-phenotype correlation can be assumed. Results The clinical, neuroradiological, and genetic findings of 35 patients with biallelic POMT1 mutations (15 WWS, 1 MEB (muscle-eye-brain disease), 19 LGMD) from 27 independent families are reported. The representative clinical course of an infant with WWS and the long-term course of a 32 years old patient with LGMD are described in more detail. Specific features of 15 patients with the homozygous founder mutation p.Ala200Pro are defined as a distinct and mildly affected LGMD subgroup. Ten previously reported and 8 novel POMT1 mutations were identified. Type and location of each of the POMT1 mutations are evaluated in detail and a list of all POMT1 mutations reported by now is provided. Patients with two mutations leading to premature protein termination had a WWS phenotype, while the presence of at least one missense mutation was associated with milder phenotypes. In the patient with MEB-like phenotype two missense mutations were observed within the catalytic active domain of the enzyme. Conclusions Our large cohort confirms the importance of type and location of each POMT1 mutation for the individual clinical manifestation and thereby expands the knowledge on the genotype-phenotype correlation in POMT1-related dystroglycanopathies. This genotype-phenotype correlation is further supported by the observation of an intrafamiliar analogous clinical manifestation observed in all affected 13 siblings from 5 independent families. Our data confirm the progressive nature of the disease also in milder LGMD phenotypes, ultimately resulting in loss of ambulation at a variable age. Our data define two major clinical POMT1 phenotypes, which should prompt genetic testing including the POMT1 gene: patients with a severe WWS manifestation predominantly present with profound neonatal muscular hypotonia and a severe and progressive hydrocephalus with involvement of brainstem and/or cerebellum. The presence of an occipital encephalocele in a WWS patient might point to POMT1 as causative gene within the different genes associated with WWS. The milder LGMD phenotypes constantly show markedly elevated creatine kinase values in combination with microcephaly and cognitive impairment. Electronic supplementary material The online version of this article (10.1186/s13023-019-1119-0) contains supplementary material, which is available to authorized users.PubMedWoSScopu