Radiomic features define risk and are linked to DNA methylation attributes in primary CNS lymphoma.

Background: The prognostic roles of clinical and laboratory markers have been exploited to model risk in patients with primary CNS lymphoma, but these approaches do not fully explain the observed variation in outcome. To date, neuroimaging or molecular information is not used. The aim of this study was to determine the utility of radiomic features to capture clinically relevant phenotypes, and to link those to molecular profiles for enhanced risk stratification. Methods: In this retrospective study, we investigated 133 patients across 9 sites in Austria (2005-2018) and an external validation site in South Korea (44 patients, 2013-2016). We used T1-weighted contrast-enhanced MRI and an L1-norm regularized Cox proportional hazard model to derive a radiomic risk score. We integrated radiomic features with DNA methylation profiles using machine learning-based prediction, and validated the most relevant biological associations in tissues and cell lines. Results: The radiomic risk score, consisting of 20 mostly textural features, was a strong and independent predictor of survival (multivariate hazard ratio = 6.56 [3.64-11.81]) that remained valid in the external validation cohort. Radiomic features captured gene regulatory differences such as in BCL6 binding activity, which was put forth as testable treatment target for a subset of patients. Conclusions: The radiomic risk score was a robust and complementary predictor of survival and reflected characteristics in underlying DNA methylation patterns. Leveraging imaging phenotypes to assess risk and inform epigenetic treatment targets provides a concept on which to advance prognostic modeling and precision therapy for this aggressive cancer.

Buffy coat signatures of breast cancer risk in a prospective cohort study.

BACKGROUND: Epigenetic alterations are a near-universal feature of human malignancy and have been detected in malignant cells as well as in easily accessible specimens such as blood and urine. These findings offer promising applications in cancer detection, subtyping, and treatment monitoring. However, much of the current evidence is based on findings in retrospective studies and may reflect epigenetic patterns that have already been influenced by the onset of the disease. METHODS: Studying breast cancer, we established genome-scale DNA methylation profiles of prospectively collected buffy coat samples (n = 702) from a case-control study nested within the EPIC-Heidelberg cohort using reduced representation bisulphite sequencing (RRBS). RESULTS: We observed cancer-specific DNA methylation events in buffy coat samples. Increased DNA methylation in genomic regions associated with SURF6 and REXO1/CTB31O20.3 was linked to the length of time to diagnosis in the prospectively collected buffy coat DNA from individuals who subsequently developed breast cancer. Using machine learning methods, we piloted a DNA methylation-based classifier that predicted case-control status in a held-out validation set with 76.5% accuracy, in some cases up to 15 years before clinical diagnosis of the disease. CONCLUSIONS: Taken together, our findings suggest a model of gradual accumulation of cancer-associated DNA methylation patterns in peripheral blood, which may be detected long before clinical manifestation of cancer. Such changes may provide useful markers for risk stratification and, ultimately, personalized cancer prevention.

Comparative analysis of genome-scale, base-resolution DNA methylation profiles across 580 animal species.

Methylation of cytosines is a prototypic epigenetic modification of the DNA. It has been implicated in various regulatory mechanisms across the animal kingdom and particularly in vertebrates. We mapped DNA methylation in 580 animal species (535 vertebrates, 45 invertebrates), resulting in 2443 genome-scale DNA methylation profiles of multiple organs. Bioinformatic analysis of this large dataset quantified the association of DNA methylation with the underlying genomic DNA sequence throughout vertebrate evolution. We observed a broadly conserved link with two major transitions-once in the first vertebrates and again with the emergence of reptiles. Cross-species comparisons focusing on individual organs supported a deeply conserved association of DNA methylation with tissue type, and cross-mapping analysis of DNA methylation at gene promoters revealed evolutionary changes for orthologous genes. In summary, this study establishes a large resource of vertebrate and invertebrate DNA methylomes, it showcases the power of reference-free epigenome analysis in species for which no reference genomes are available, and it contributes an epigenetic perspective to the study of vertebrate evolution.

Multimodal analysis of cell-free DNA whole-genome sequencing for pediatric cancers with low mutational burden.

Sequencing of cell-free DNA in the blood of cancer patients (liquid biopsy) provides attractive opportunities for early diagnosis, assessment of treatment response, and minimally invasive disease monitoring. To unlock liquid biopsy analysis for pediatric tumors with few genetic aberrations, we introduce an integrated genetic/epigenetic analysis method and demonstrate its utility on 241 deep whole-genome sequencing profiles of 95 patients with Ewing sarcoma and 31 patients with other pediatric sarcomas. Our method achieves sensitive detection and classification of circulating tumor DNA in peripheral blood independent of any genetic alterations. Moreover, we benchmark different metrics for cell-free DNA fragmentation analysis, and we introduce the LIQUORICE algorithm for detecting circulating tumor DNA based on cancer-specific chromatin signatures. Finally, we combine several fragmentation-based metrics into an integrated machine learning classifier for liquid biopsy analysis that exploits widespread epigenetic deregulation and is tailored to cancers with low mutation rates. Clinical associations highlight the potential value of cfDNA fragmentation patterns as prognostic biomarkers in Ewing sarcoma. In summary, our study provides a comprehensive analysis of circulating tumor DNA beyond recurrent genetic aberrations, and it renders the benefits of liquid biopsy more readily accessible for childhood cancers.

Finding underlying genetic mechanisms of two patients with autism spectrum disorder carrying familial apparently balanced chromosomal translocations.

BACKGROUND: Genetic etiologies of autism spectrum disorders (ASD) are complex, and the genetic factors identified so far are very diverse. In complex genetic diseases such as ASD, de novo or inherited chromosomal abnormalities are valuable findings for researchers with respect to identifying the underlying genetic risk factors. With gene mapping studies on these chromosomal abnormalities, dozens of genes have been associated with ASD and other neurodevelopmental genetic diseases. In the present study, we aimed to idenitfy the causative genetic factors in patients with ASD who have an apparently balanced chromosomal translocation in their karyotypes. METHODS: For mapping the broken genes as a result of chromosomal translocations, we performed whole genome DNA sequencing. Chromosomal breakpoints and large DNA copy number variations (CNV) were determined after genome alignment. Identified CNVs and single nucleotide variations (SNV) were evaluated with VCF-BED intersect and Gemini tools, respectively. A targeted resequencing approach was performed on the JMJD1C gene in all of the ASD cohorts (220 patients). For molecular modeling, we used a homology modeling approach via the SWISS-MODEL. RESULTS: We found that there was no contribution of the broken genes or regulator DNA sequences to ASD, whereas the SNVs on the JMJD1C, CNKSR2 and DDX11 genes were the most convincing genetic risk factors for underlying ASD phenotypes. CONCLUSIONS: Genetic etiologies of ASD should be analyzed comprehensively by taking into account of the all chromosomal structural abnormalities and de novo or inherited CNV/SNVs with all possible inheritance patterns.

Identifying disease-causing mutations with privacy protection.

MOTIVATION: The use of genome data for diagnosis and treatment is becoming increasingly common. Researchers need access to as many genomes as possible to interpret the patient genome, to obtain some statistical patterns and to reveal disease-gene relationships. The sensitive information contained in the genome data and the high risk of re-identification increase the privacy and security concerns associated with sharing such data. In this article, we present an approach to identify disease-associated variants and genes while ensuring patient privacy. The proposed method uses secure multi-party computation to find disease-causing mutations under specific inheritance models without sacrificing the privacy of individuals. It discloses only variants or genes obtained as a result of the analysis. Thus, the vast majority of patient data can be kept private. RESULTS: Our prototype implementation performs analyses on thousands of genomic data in milliseconds, and the runtime scales logarithmically with the number of patients. We present the first inheritance model (recessive, dominant and compound heterozygous) based privacy-preserving analyses of genomic data to find disease-causing mutations. Furthermore, we re-implement the privacy-preserving methods (MAX, SETDIFF and INTERSECTION) proposed in a previous study. Our MAX, SETDIFF and INTERSECTION implementations are 2.5, 1122 and 341 times faster than the corresponding operations of the state-of-the-art protocol, respectively. AVAILABILITY AND IMPLEMENTATION: https://gitlab.com/DIFUTURE/privacy-preserving-genomic-diagnosis. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Detection of allele frequencies of common c. 511C>T and c.625G>A variants in the ACADS gene in the Turkish population.

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare inborn error of mitochondrial fatty acid oxidation and protein misfolding disorder. Our aim was to detect the number of Turkish patients diagnosed with SCADD in the literature and to determine the allele frequencies of two common variants (c.511C > T and c.625G > A) in the Turkish population. Five Turkish patients with SCADD were reported in the literature from four unrelated families. We also investigated allele frequencies of common variants of c.511C > T and c.625G > A, which confer susceptibility to SCADD, which were found to be 1.7% and 20.2%, respectively. Both of these susceptibility variants were found to be high in the Turkish population as they are worldwide.

GPR56 homozygous nonsense mutation p.R271* associated with phenotypic variability in bilateral frontoparietal polymicrogyria.

Öncü-Öner T, Ünalp A, Porsuk-Doru I, Agilkaya S, Güleryüz H, Saraç A, Ergüner B, Yüksel B, Hiz-Kurul S, Cingöz S. GPR56 homozygous nonsense mutation p.R271* associated with phenotypic variability in bilateral frontoparietal polymicrogyria. Turk J Pediatr 2018; 60: 229-237. Polymicrogyria is a disorder of neuronal migration characterized by excessive cortical folding and partially fused gyri separated by shallow sulci. Homozygous mutations in the GPR56 gene, which regulates migration of neural precursor cells, are associated with bilateral frontoparietal polymicrogyria (BFPP) syndrome including white matter changes, brainstem and cerebellar involvement. Herein, we describe three siblings of consanguineous parents with a homozygous germline mutation (p.R271*) located in the seventh exon of the GPR56 gene that was previously detected in only one Portuguese patient. Phenotypic/genotypic relationships were analysed according to the clinical characteristics in only index patient. While earlier reported patient was exhibiting seizures provoked by hot water, macrocephaly, cerebellar/brainstem hypoplasia and corpus callosum abnormalities, the index patient showed only hypoplasia of brainstem, focal onset bilateral tonic clonic seizure. Despite the phenotypic similarities in two patients, the potential causes of the variation in the expression of the p.R271* variant between the two affected families might be genetic or epigenetic factors beyond the GPR56 gene. Consequently, the present findings show that the same mutation in GPR56 gene can have different phenotypic effects. Therefore, additional functional studies are needed to detect the phenotypic spectrum of the p.R271* mutation in GPR56, and provide insight into the mechanism of normal cortical development and regional patterning of the cerebral cortex.

The DNA methylation landscape of glioblastoma disease progression shows extensive heterogeneity in time and space.

Glioblastoma is characterized by widespread genetic and transcriptional heterogeneity, yet little is known about the role of the epigenome in glioblastoma disease progression. Here, we present genome-scale maps of DNA methylation in matched primary and recurring glioblastoma tumors, using data from a highly annotated clinical cohort that was selected through a national patient registry. We demonstrate the feasibility of DNA methylation mapping in a large set of routinely collected FFPE samples, and we validate bisulfite sequencing as a multipurpose assay that allowed us to infer a range of different genetic, epigenetic, and transcriptional characteristics of the profiled tumor samples. On the basis of these data, we identified subtle differences between primary and recurring tumors, links between DNA methylation and the tumor microenvironment, and an association of epigenetic tumor heterogeneity with patient survival. In summary, this study establishes an open resource for dissecting DNA methylation heterogeneity in a genetically diverse and heterogeneous cancer, and it demonstrates the feasibility of integrating epigenomics, radiology, and digital pathology for a national cohort, thereby leveraging existing samples and data collected as part of routine clinical practice.

Myophosphorylase (PYGM) mutations determined by next generation sequencing in a cohort from Turkey with McArdle disease.

This study aimed to identify PYGM mutations in patients with McArdle disease from Turkey by next generation sequencing (NGS). Genomic DNA was extracted from the blood of the McArdle patients (n = 67) and unrelated healthy volunteers (n = 53). The PYGM gene was sequenced with NGS and the observed mutations were validated by direct Sanger sequencing. A diagnostic algorithm was developed for patients with suspected McArdle disease. A total of 16 deleterious PYGM mutations were identified, of which 5 were novel, including 1 splice-site donor, 1 frame-shift, and 3 non-synonymous variants. The p.Met1Val (27-patients/11-families) was the most common PYGM mutation, followed by p.Arg576* (6/4), c.1827+7A>G (5/4), c.772+2_3delTG (5/3), p.Phe710del (4/2), p.Lys754Asnfs (2/1), and p.Arg50* (1/1). A molecular diagnostic flowchart is proposed for the McArdle patients in Turkey, covering the 6 most common PYGM mutations found in Turkey as well as the most common mutation in Europe. The diagnostic algorithm may alleviate the need for muscle biopsies in 77.6% of future patients. A prevalence of any of the mutations to a geographical region in Turkey was not identified. Furthermore, the NGS approach to sequence the entire PYGM gene was successful in detecting a common missense mutation and discovering novel mutations in this population study.

Exome sequencing identifies a novel homozygous CLN8 mutation in a Turkish family with Northern epilepsy.

Neuronal ceroid lipofuscinosis (NCL), one of the most common neurodegenerative childhood-onset disorders, is characterized by autosomal-recessive inheritance, epileptic seizures, progressive psychomotor deterioration, visual impairment, and premature death. Based on the country of origin of the patients, the clinical features/courses, and the molecular genetics background of the disorder, 14 distinct NCL subtypes have been described to date. CLN8 mutation was first identified in Finnish patients, and the condition was named Northern Epilepsy (NE); however, the severe phenotype of the CLN8 gene was subsequently found outside Finland and named 'variant late-infantile' NCL. In this study, five patients and their six healthy relatives from a large Turkish consanguineous family were enrolled. The study involved detailed clinical, radiological and molecular genetic evaluations. Whole-exome sequencing and homozygosity mapping revealed a novel homozygous CLN8 mutation, c.677T>C (p.Leu226Pro). We defined NE cases in Turkey, caused by a novel mutation in CLN8. WES can be an important diagnostic method in rare cases with atypical courses.

A novel missense mutation, p.(R102W) in WNT7A causes Al-Awadi Raas-Rothschild syndrome in a fetus.

Al-Awadi-Raas-Rothschild syndrome (AARRS) is a rare autosomal recessive disorder which consists of severe malformations of the upper and lower limbs, abnormal genitalia and underdeveloped pelvis. Here, we present a fetus with severe limbs defects, including bilateral humeroradial synostosis, bilateral oligodactyly in hands, underdeveloped pelvis, short femora and tibiae, absence of fibulae, severely small feet, and absence of uterus. An autosomal recessively inherited novel mutation in WNT7A found in the fetus, c.304C > T, affects an evolutionarily well-conserved amino acid, causing the p.(R102W) missense change at protein level. The findings presented in this fetus are compatible with diagnosis of AARRS, expanding the mutational spectrum of limb malformations arising from defects in WNT7A.

Draft Genome Sequences of Two Heat-Resistant Mutant Strains (A52 and B41) of the Photosynthetic Hydrogen-Producing Bacterium Rhodobacter capsulatus.

The draft genome sequences of two heat-resistant mutant strains, A52 and B41, derived from Rhodobacter capsulatus DSM 1710, and with different hydrogen production levels, are reported here. These sequences may help understand the molecular basis of heat resistance and hydrogen production in R. capsulatus.

Hereditary spastic paraplegia with recessive trait caused by mutation in KLC4 gene.

We report an association between a new causative gene and spastic paraplegia, which is a genetically heterogeneous disorder. Clinical phenotyping of one consanguineous family followed by combined homozygosity mapping and whole-exome sequencing analysis. Three patients from the same family shared common features of progressive complicated spastic paraplegia. They shared a single homozygous stretch area on chromosome 6. Whole-exome sequencing revealed a homozygous mutation (c.853_871del19) in the gene coding the kinesin light chain 4 protein (KLC4). Meanwhile, the unaffected parents and two siblings were heterozygous and one sibling was homozygous wild type. The 19 bp deletion in exon 6 generates a stop codon and thus a truncated messenger RNA and protein. The association of a KLC4 mutation with spastic paraplegia identifies a new locus for the disease.

Performance comparison of Next Generation sequencing platforms.

Next Generation DNA Sequencing technologies offer ultra high sequencing throughput for very low prices. The increase in throughput and diminished costs open up new research areas. Moreover, number of clinicians utilizing DNA sequencing keeps growing. One of the main concern for researchers and clinicians who are adopting these platforms is their sequencing accuracy. We compared three of the most commonly used Next Generation Sequencing platforms; Ion Torrent from Life Technologies, GS FLX+ from Roche and HiSeq 2000 from Illumina.

Early postzygotic mutations contribute to de novo variation in a healthy monozygotic twin pair.

BACKGROUND: Human de novo single-nucleotide variation (SNV) rate is estimated to range between 0.82-1.70×10(-8) mutations per base per generation. However, contribution of early postzygotic mutations to the overall human de novo SNV rate is unknown. METHODS: We performed deep whole-genome sequencing (more than 30-fold coverage per individual) of the whole-blood-derived DNA samples of a healthy monozygotic twin pair and their parents. We examined the genotypes of each individual simultaneously for each of the SNVs and discovered de novo SNVs regarding the timing of mutagenesis. Putative de novo SNVs were validated using Sanger-based capillary sequencing. RESULTS: We conservatively characterised 23 de novo SNVs shared by the twin pair, 8 de novo SNVs specific to twin I and 1 de novo SNV specific to twin II. Based on the number of de novo SNVs validated by Sanger sequencing and the number of callable bases of each twin, we calculated the overall de novo SNV rate of 1.31×10(-8) and 1.01×10(-8) for twin I and twin II, respectively. Of these, rates of the early postzygotic de novo SNVs were estimated to be 0.34×10(-8) for twin I and 0.04×10(-8) for twin II. CONCLUSIONS: Early postzygotic mutations constitute a substantial proportion of de novo mutations in humans. Therefore, genome mosaicism resulting from early mitotic events during embryogenesis is common and could substantially contribute to the development of diseases.

TMCO1 deficiency causes autosomal recessive cerebrofaciothoracic dysplasia.

Cerebrofaciothoracic dysplasia (CFT) (OMIM #213980) is a multiple congenital anomaly and intellectual disability syndrome involving the cranium, face, and thorax. The characteristic features are cranial involvement with macrocrania at birth, brachycephaly, various CT/MRI findings including hypoplasia of corpus callosum, enlargement of septum pellicidum, and diffuse hypodensity of the grey matter, flat face, hypertelorism, cleft lip and cleft palate, low-set, posteriorly rotated ears, short neck, and multiple costal and vertebral anomalies. The underlying genetic defect remains unknown. Using combination of homozygosity mapping and whole-exome sequencing, we identified a homozygous nonsense founder mutation, p.Arg87Ter (c.259 C>T), in the human transmembrane and coiled-coil domains protein 1 (TMCO1) in four out of five families of Turkish origin. The entire critical region on chromosome 1q24 containing TMCO1 was excluded in the fifth family with characteristic findings of CFT providing evidence for genetic heterogeneity of CFT spectrum. Another founder TMCO1 mutation has recently been reported to cause a unique genetic condition, TMCO1-defect syndrome (OMIM #614132). TMCO1-defect syndrome shares many features with CFT. This study supports the fact that "TMCO1-defect syndrome," initially thought to represent a distinct disorder, indeed belongs to the genetically heterogeneous CFT dysplasia spectrum.

Mutation in MEOX1 gene causes a recessive Klippel-Feil syndrome subtype.

BACKGROUND: Klippel-Feil syndrome (KFS) is characterized by the developmental failure of the cervical spine and has two dominantly inherited subtypes. Affected individuals who are the children of a consanguineous marriage are extremely rare in the medical literature, but the gene responsible for this recessive trait subtype of KFS has recently been reported. RESULTS: We identified a family with the KFS phenotype in which their parents have a consanguineous marriage. Radiological examinations revealed that they carry fusion defects and numerical abnormalities in the cervical spine, scoliosis, malformations of the cranial base, and Sprengel's deformity. We applied whole genome linkage and whole-exome sequencing analysis to identify the chromosomal locus and gene mutated in this family. Whole genome linkage analysis revealed a significant linkage to chromosome 17q12-q33 with a LOD score of 4.2. Exome sequencing identified the G > A p.Q84X mutation in the MEOX1 gene, which is segregated based on pedigree status. Homozygous MEOX1 mutations have reportedly caused a similar phenotype in knockout mice. CONCLUSIONS: Here, we report a truncating mutation in the MEOX1 gene in a KFS family with an autosomal recessive trait. Together with another recently reported study and the knockout mouse model, our results suggest that mutations in MEOX1 cause a recessive KFS phenotype in humans.

Characterizing common substructures of ligands for GPCR protein subfamilies.

The G-protein coupled receptor (GPCR) superfamily is the largest class of proteins with therapeutic value. More than 40% of present prescription drugs are GPCR ligands. The high therapeutic value of GPCR proteins and recent advancements in virtual screening methods gave rise to many virtual screening studies for GPCR ligands. However, in spite of vast amounts of research studying their functions and characteristics, 3D structures of most GPCRs are still unknown. This makes target-based virtual screenings of GPCR ligands extremely difficult, and successful virtual screening techniques rely heavily on ligand information. These virtual screening methods focus on specific features of ligands on GPCR protein level, and common features of ligands on higher levels of GPCR classification are yet to be studied. Here we extracted common substructures of GPCR ligands of GPCR protein subfamilies. We used the SIMCOMP, a graph-based chemical structure comparison program, and hierarchical clustering to reveal common substructures. We applied our method to 850 GPCR ligands and we found 53 common substructures covering 439 ligands. These substructures contribute to deeper understanding of structural features of GPCR ligands which can be used in new drug discovery methods.