De novo variants in neurodevelopmental disorders-experiences from a tertiary care center.

Up to 40% of neurodevelopmental disorders (NDDs) such as intellectual disability, developmental delay, autism spectrum disorder, and developmental motor abnormalities have a documented underlying monogenic defect, primarily due to de novo variants. Still, the overall burden of de novo variants as well as novel disease genes in NDDs await discovery. We performed parent-offspring trio exome sequencing in 231 individuals with NDDs. Phenotypes were compiled using human phenotype ontology terms. The overall diagnostic yield was 49.8% (n = 115/231) with de novo variants contributing to more than 80% (n = 93/115) of all solved cases. De novo variants affected 72 different-mostly constrained-genes. In addition, we identified putative pathogenic variants in 16 genes not linked to NDDs to date. Reanalysis performed in 80 initially unsolved cases revealed a definitive diagnosis in two additional cases. Our study consolidates the contribution and genetic heterogeneity of de novo variants in NDDs highlighting trio exome sequencing as effective diagnostic tool for NDDs. Besides, we illustrate the potential of a trio-approach for candidate gene discovery and the power of systematic reanalysis of unsolved cases.

MYSM1 maintains ribosomal protein gene expression in hematopoietic stem cells to prevent hematopoietic dysfunction.

Ribosomopathies are congenital disorders caused by mutations in the genes encoding ribosomal and other functionally related proteins. They are characterized by anemia, other hematopoietic and developmental abnormalities, and p53 activation. Ribosome assembly requires coordinated expression of many ribosomal protein (RP) genes; however, the regulation of RP gene expression, especially in hematopoietic stem cells (HSCs), remains poorly understood. MYSM1 is a transcriptional regulator essential for HSC function and hematopoiesis. We established that HSC dysfunction in Mysm1 deficiency is driven by p53; however, the mechanisms of p53 activation remained unclear. Here, we describe the transcriptome of Mysm1-deficient mouse HSCs and identify MYSM1 genome-wide DNA binding sites. We establish a direct role for MYSM1 in RP gene expression and show a reduction in protein synthesis in Mysm1-/- HSCs. Loss of p53 in mice fully rescues Mysm1-/- anemia phenotype but not RP gene expression, indicating that RP gene dysregulation is a direct outcome of Mysm1 deficiency and an upstream mediator of Mysm1-/- phenotypes through p53 activation. We characterize a patient with a homozygous nonsense MYSM1 gene variant, and we demonstrate reduced protein synthesis and increased p53 levels in patient hematopoietic cells. Our work provides insights into the specialized mechanisms regulating RP gene expression in HSCs and establishes a common etiology of MYSM1 deficiency and ribosomopathy syndromes.

Lessons from exome sequencing in prenatally diagnosed heart defects: A basis for prenatal testing.

Congenital heart defects (CHDs) are the most common birth defect with 30%-40% being explained by genetic aberrations. With next generation sequencing becoming widely available, we sought to evaluate the clinical utility of exome sequencing (ES) in prenatally diagnosed CHD. We retrospectively analyzed the diagnostic yield as well as non-conclusive and incidental findings in 30 cases with prenatally diagnosed CHDs using ES, mostly as parent-child trios. A genetic diagnosis was established in 20% (6/30). Non-conclusive results were found in 13% (4/30) and incidental findings in 10% (3/30). There was a phenotypic discrepancy between reported prenatal and postnatal extracardiac findings in 40% (8/20). However, none of these additional, postnatal findings altered the genetic diagnosis. Herein, ES in prenatally diagnosed CHDs results in a comparably high diagnostic yield. There was a significant proportion of incidental findings and variants of unknown significance as well as potentially pathogenic variants in novel disease genes. Such findings can bedevil genetic counseling and decision making for pregnancy termination. Despite the small cohort size, our data serve as a first basis to evaluate the value of prenatal ES in CHD for further studies emerging in the near future.

Spatially clustering de novo variants in CYFIP2, encoding the cytoplasmic FMRP interacting protein 2, cause intellectual disability and seizures.

CYFIP2, encoding the evolutionary highly conserved cytoplasmic FMRP interacting protein 2, has previously been proposed as a candidate gene for intellectual disability and autism because of its important role linking FMRP-dependent transcription regulation and actin polymerization via the WAVE regulatory complex (WRC). Recently, de novo variants affecting the amino acid p.Arg87 of CYFIP2 were reported in four individuals with epileptic encephalopathy. We here report 12 independent patients harboring a variety of de novo variants in CYFIP2 broadening the molecular and clinical spectrum of a novel CYFIP2-related neurodevelopmental disorder. Using trio whole-exome or -genome sequencing, we identified 12 independent patients carrying a total of eight distinct de novo variants in CYFIP2 with a shared phenotype of intellectual disability, seizures, and muscular hypotonia. We detected seven different missense variants, of which two occurred recurrently (p.(Arg87Cys) and p.(Ile664Met)), and a splice donor variant in the last intron for which we showed exon skipping in the transcript. The latter is expected to escape nonsense-mediated mRNA decay resulting in a truncated protein. Despite the large spacing in the primary structure, the variants spatially cluster in the tertiary structure and are all predicted to weaken the interaction with WAVE1 or NCKAP1 of the actin polymerization regulating WRC-complex. Preliminary genotype-phenotype correlation indicates a profound phenotype in p.Arg87 substitutions and a more variable phenotype in other alterations. This study evidenced a variety of de novo variants in CYFIP2 as a novel cause of mostly severe intellectual disability with seizures and muscular hypotonia.

A case report and review of the literature indicate that HMGA2 should be added as a disease gene for Silver-Russell syndrome.

Patients with Silver-Russell syndrome (SRS), a syndromic growth retardation syndrome, usually harbor an epimutation at chromosome 11p15 or a maternal uniparental disomy of chromosome 7. However, to date the genetic cause remains unknown in around 40% of SRS cases, suggesting genetic heterogeneity and involvement of other genes. We present a 4-year-old female patient with the clinical diagnosis of SRS and negative results in common genetic SRS diagnostics. Whole exome sequencing identified a de novo heterozygous 7.3 kb deletion on chromosome 12q14.3 including exon 1 and 2 of HMGA2. HMGA2 encodes an architectural transcription factor and has already been linked to body size variations in various genome-wide association studies and mouse models. Reviewing the literature, we found additional four patients with a phenotype of SRS harboring point mutations or structural variants involving HMGA2. We conclude that genetic testing of HMGA2 should be considered in routine diagnostics in patients with the suspicion of SRS.

Increased DNA integrity in colorectal cancer.

BACKGROUND: Blood-based DNA integrity, defined as the relation of long to small fragments of cell-free circulating DNA, is known to be increased in various types of cancers. Since different DNA fragments and formulae are used by different researchers, conflicting results on the relevance of this marker for cancer diagnosis have been reported. PATIENTS AND METHODS: Sera from 24 patients with colorectal cancer, 11 patients with benign gastrointestinal diseases, and 24 healthy individuals were investigated. After DNA isolation, ALU repeats with 115 bp and 247 bp length were measured using quantitative polymerase chain reaction and resulting DNA integrities were calculated by the two formulae of Umetani et al. (DNA Int 1) and Wang et al. (DNA Int 2) RESULTS: DNA integrity by both formulae correlated strongly with each other. DNA integrity was significantly higher in patients with colorectal cancer when compared with healthy controls (p=0.005 and p=0.006, respectively), while there was no significant difference from those with benign colorectal diseases. In receiver operating characteristic curve analysis, areas under the curve of 0.74 and 0.73 and sensitivities of 71% at 75% specificity (DNA Int 1 and 2, respectively) were achieved for the discrimination between patients with colorectal cancer and healthy controls. CONCLUSION: DNA integrity is significantly increased in patients with colorectal cancer and may be useful in prospective trials.

Characterization of H3K9me3- and H4K20me3-associated circulating nucleosomal DNA by high-throughput sequencing in colorectal cancer.

Modified histone tails in nucleosomes circulating in the blood bear the potential as cancer biomarkers. Recently, using chromatin immunopecipitation (ChIP)-related quantitative PCR, we described reduced plasma levels of the two pericentric heterochromatin-specific histone methylation marks H3K9me3 and H4K20me3 in patients with colorectal cancer (CRC). Here, by utilizing ChIP-related high-throughput sequencing, we further characterized these modifications in circulation. Plasma DNA from nucleosomes immunoprecipitated by H3K9me3- and H4K20me3-specific antibodies from patients with CRC (N = 15) and healthy subjects (N = 15) was subjected to the Roche 454 FLX sequencing, and the generated array of ChIP-enriched sequences were compared to the human reference genome. The total number of nucleosomes, of sequence reads and of diverse DNA repetitive elements were statistically compared between the study groups. Total nucleosome amount was not different in both groups. Concerning both histone modifications, lower numbers of sequence reads were detected in CRC patients as compared with healthy controls (medians in H3K9me3: 32 vs. 61; p < 0.01; in H4K20me3: 54 vs. 88; p < 0.01). Size of fragments was not different in both groups. Most abundant sequences were repetitive LINE and SINE elements while simple repeats, LTR, DNA, SAT, and low complexity elements were less frequent. Best discrimination between both groups was achieved by total number of H3K9me3 reads (AUC 0.90) and H3K9me3 LINE elements L1 (AUC 0.93) und L2 (AUC 0.91). The present results confirm earlier findings of lower H3K9me3 levels in CRC and show LINE elements to be the most frequent and best discriminative markers on modified histones.

Circulating nucleosomes and biomarkers of immunogenic cell death as predictive and prognostic markers in cancer patients undergoing cytotoxic therapy.

INTRODUCTION: Immunogenic cell death markers are released from apoptotic and necrotic cells upon pathologic or therapeutic causes and stimulate the innate and adaptive immune system. Cell death products such as nucleosomes, damage-associated molecular pattern (DAMP) molecules such as the high-mobility group box 1 protein (HMGB1) and its receptor of advanced glycation end products (sRAGE) are supposed to play an essential role in driving this process. However, this immunogenic activation may have dual effects, either by sensitizing the immune system for more efficient tumor cell removal or by creating a favorable tumor microenvironment that facilitates tumor growth, proliferation and invasiveness. AREAS COVERED: Here, we review recent findings on the relevance of serum nucleosomes, DNAse activity, HMGB1 and sRAGE as biomarkers for the diagnosis, prognosis and therapy prediction in cancer disease. EXPERT OPINION: In comparison with healthy controls, cancer patients demonstrated elevated serum levels of nucleosomes and HMGB1 while sRAGE levels were decreased. During locoregional and systemic cytotoxic therapies, a high release of nucleosomes and HMGB1 as well as low release of sRAGE before and during the initial phase of the treatment was found to be associated with poor response to the therapy and patient survival. Therefore, immunogenic cell death markers are promising tools for the prognosis, therapy prediction and monitoring in cancer patients.

Relevance of histone marks H3K9me3 and H4K20me3 in cancer.

BACKGROUND: Circulating nucleosomes are valuable biomarkers for therapy monitoring and estimation of prognosis in cancer disease. While epigenetic and genetic modifications of DNA have been reported in blood of cancer patients, little is known about modifications of histones on circulating nucleosomes. PATIENTS AND METHODS: Sera of 45 cancer patients (21 colorectal, 4 pancreatic, 15 breast, 5 lung cancer), 12 patients with benign gastrointestinal and inflammatory diseases, and 28 healthy individuals were investigated. Histone modifications were detected by chromatin-immunoprecipitation (ChIP) using antibodies for triple histone methylations at sites H3K9me3 and H4K20me3 and subsequent real-time polymerase chain reaction using primers for the centromeric satellites SAT2. Additionally, the amount of circulating nucleosomes, as well as of carcino-embryonic antigen (CEA) and cancer antigen (CA) 19-9 were measured. RESULTS: Levels of SAT2 on H3K9me3 (median 0.507 ng/ml) and on H4K20me3 (0.292 ng/ml) were elevated in sera of patients with breast cancer when compared with healthy controls (0.049 and 0.035 ng/ml), but were lower in patients with colorectal cancer (0.039 and 0.027 ng/ml). Both histone marks were correlated with each other but did not correlate with CEA or CA 19-9 in cancer patients. When H3K9me3 and H4K20me3 were normalized to nucleosome content in sera, ratios were significantly higher in all types of cancer as well as in colorectal and breast subtypes when compared with healthy controls. Best discrimination was achieved by normalized H4K20me3 reaching areas under the curves (AUC) of 79.1%, 90.4% and 81.2% in receiver operating characteristic (ROC) curves of these three comparisons. CONCLUSION: SAT2 levels on H3K9me3 and H4K20me3 are up-regulated in breast cancer and down-regulated in colorectal cancer. Normalization to total nucleosome content enables better discrimination between cancer and control groups.

Methods for isolation of cell-free plasma DNA strongly affect DNA yield.

Extracellular nucleic acids are present in plasma, serum, and other body fluids and their analysis has gained increasing attention during recent years. Because of the small quantity and highly fragmented nature of cell-free DNA in plasma and serum, a fast, efficient, and reliable isolation method is still a problem and so far there is no agreement on a standardized method. We used spin columns from commercial suppliers (QIAamp DNA Blood Midi Kit from Qiagen; NucleoSpin Kit from Macherey-Nagel; MagNA Pure isolation system from Roche Diagnostics) to isolate DNA from 44 plasma samples in parallel at laboratories in Berlin and Munich. DNA in all samples was quantified by real-time PCR on a LightCycler 480 using three different targets (GAPDH, ß-globin, ERV). The quantities of cell-free DNA for the different isolation methods and genes varied between medians of 1.6 ng/mL and 28.1 ng/mL. This considerable variation of absolute DNA values was mainly caused by the use of different isolation methods (p<0.0001). Comparable results were achieved by the use of the genes GAPDH and ERV while higher values were obtained by use of ß-globin. The laboratory site had only minor influence on DNA yield when manual extraction methods were used.