Ensemble-based classification using microRNA expression identifies a breast cancer patient subgroup with an ultralow long-term risk of metastases.

BACKGROUND: Current clinical markers overestimate the recurrence risk in many lymph node negative (LNN) breast cancer (BC) patients such that a majority of these low-risk patients unnecessarily receive systemic treatments. We tested if differential microRNA expression in primary tumors allows reliable identification of indolent LNN BC patients to provide an improved classification tool for overtreatment reduction in this patient group. METHODS: We collected freshly frozen primary tumors of 80 LNN BC patients with recurrence and 80 recurrence-free patients (mean follow-up: 20.9 years). The study comprises solely systemically untreated patients to exclude that administered treatments confound the metastasis status. Samples were pairwise matched for clinical-pathological characteristics to minimize dependence of current markers. Patients were classified into risk-subgroups according to the differential microRNA expression of their tumors via classification model building with cross-validation using seven classification methods and a voting scheme. The methodology was validated using available data of two independent cohorts (n = 123, n = 339). RESULTS: Of the 80 indolent patients (who would all likely receive systemic treatments today) our ultralow-risk classifier correctly identified 37 while keeping a sensitivity of 100% in the recurrence group. Multivariable logistic regression analysis confirmed independence of voting results from current clinical markers. Application of the method in two validation cohorts confirmed successful classification of ultralow-risk BC patients with significantly prolonged recurrence-free survival. CONCLUSION: Profiles of differential microRNAs expression can identify LNN BC patients who could spare systemic treatments demanded by currently applied classifications. However, further validation studies are required for clinical implementation of the applied methodology.

Male with an apparently normal phenotype carrying a BRCA1 exon 20 duplication in trans to a BRCA1 frameshift variant.

BACKGROUND: Reports of dual carriers of pathogenic BRCA1 variants in trans are extremely rare, and so far, most individuals have been associated with a Fanconi Anemia-like phenotype. METHODS: We identified two families with a BRCA1 in-frame exon 20 duplication (Ex20dup). In one male individual, the variant was in trans with the BRCA1 frameshift variant c.2475delC p.(Asp825Glufs*21). We performed splicing analysis and used a transcription activation domain (TAD) assay to assess the functional impact of Ex20dup. We collected pedigrees and mapped the breakpoints of the duplication by long- and short-read genome sequencing. In addition, we performed a mitomycin C (MMC) assay from the dual carrier using cultured lymphoblastoid cells. RESULTS: Genome sequencing and RNA analysis revealed the BRCA1 exon 20 duplication to be in tandem. The duplication was expressed without skipping any one of the two exon 20 copies, resulting in a lack of wild-type transcripts from this allele. TAD assay indicated that the Ex20dup variant has a functional level similar to the well-known moderate penetrant pathogenic BRCA1 variant c.5096G > A p.(Arg1699Gln). MMC assay of the dual carrier indicated a slightly impaired chromosomal repair ability. CONCLUSIONS: This is the first reported case where two BRCA1 variants with demonstrated functional impact are identified in trans in a male patient with an apparently normal clinical phenotype and no BRCA1-associated cancer. The results pinpoint a minimum necessary BRCA1 protein activity to avoid a Fanconi Anemia-like phenotype in compound heterozygous status and yet still predispose carriers to hormone-related cancers. These findings urge caution when counseling families regarding potential Fanconi Anemia risk. Furthermore, prudence should be taken when classifying individual variants as benign based on co-occurrence in trans with well-established pathogenic variants.

Variants in the WDR44 WD40-repeat domain cause a spectrum of ciliopathy by impairing ciliogenesis initiation.

WDR44 prevents ciliogenesis initiation by regulating RAB11-dependent vesicle trafficking. Here, we describe male patients with missense and nonsense variants within the WD40 repeats (WDR) of WDR44, an X-linked gene product, who display ciliopathy-related developmental phenotypes that we can model in zebrafish. The patient phenotypic spectrum includes developmental delay/intellectual disability, hypotonia, distinct craniofacial features and variable presence of brain, renal, cardiac and musculoskeletal abnormalities. We demonstrate that WDR44 variants associated with more severe disease impair ciliogenesis initiation and ciliary signaling. Because WDR44 negatively regulates ciliogenesis, it was surprising that pathogenic missense variants showed reduced abundance, which we link to misfolding of WDR autonomous repeats and degradation by the proteasome. We discover that disease severity correlates with increased RAB11 binding, which we propose drives ciliogenesis initiation dysregulation. Finally, we discover interdomain interactions between the WDR and NH2-terminal region that contains the RAB11 binding domain (RBD) and show patient variants disrupt this association. This study provides new insights into WDR44 WDR structure and characterizes a new syndrome that could result from impaired ciliogenesis.

Non-BRCA1/BRCA2 high-risk familial breast cancers are not associated with a high prevalence of BRCAness.

BACKGROUND: Familial breast cancer is in most cases unexplained due to the lack of identifiable pathogenic variants in the BRCA1 and BRCA2 genes. The somatic mutational landscape and in particular the extent of BRCA-like tumour features (BRCAness) in these familial breast cancers where germline BRCA1 or BRCA2 mutations have not been identified is to a large extent unknown. METHODS: We performed whole-genome sequencing on matched tumour and normal samples from high-risk non-BRCA1/BRCA2 breast cancer families to understand the germline and somatic mutational landscape and mutational signatures. We measured BRCAness using HRDetect. As a comparator, we also analysed samples from BRCA1 and BRCA2 germline mutation carriers. RESULTS: We noted for non-BRCA1/BRCA2 tumours, only a small proportion displayed high HRDetect scores and were characterized by concomitant promoter hypermethylation or in one case a RAD51D splice variant previously reported as having unknown significance to potentially explain their BRCAness. Another small proportion showed no features of BRCAness but had mutationally active tumours. The remaining tumours lacked features of BRCAness and were mutationally quiescent. CONCLUSIONS: A limited fraction of high-risk familial non-BRCA1/BRCA2 breast cancer patients is expected to benefit from treatment strategies against homologue repair deficient cancer cells.

CERT1 mutations perturb human development by disrupting sphingolipid homeostasis.

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.

Detection of DZIP1L mutations by whole-exome sequencing in consanguineous families with polycystic kidney disease.

BACKGROUND: Autosomal recessive polycystic kidney disease is a cystic kidney disease with early onset and clinically characterized by enlarged echogenic kidneys, hypertension, varying degrees of kidney dysfunction, and liver fibrosis. It is most frequently caused by sequence variants in the PKHD1 gene, encoding fibrocystin. In more rare cases, sequence variants in DZIP1L are seen, encoding the basal body protein DAZ interacting protein 1-like protein (DZIP1L). So far, only four different DZIP1L variants have been reported. METHODS: Four children from three consanguineous families presenting with polycystic kidney disease were selected for targeted or untargeted exome sequencing. RESULTS: We identified two different, previously not reported homozygous DZIP1L sequence variants: c.193 T > C; p.(Cys65Arg), and c.216C > G; p.(Cys72Trp). Functional analyses of the c.216C > G; p.(Cys72Trp) variant indicated mislocalization of mutant DZIP1L. CONCLUSIONS: In line with published data, our results suggest a critical role of the N-terminal domain for proper protein function. Although patients with PKHD1-associated autosomal recessive polycystic kidney disease often have liver abnormalities, none of the present four patients showed any clinically relevant liver involvement. Our data demonstrate the power and efficiency of next-generation sequencing-based approaches. While DZIP1L-related polycystic kidney disease certainly represents a rare form of the disease, our results emphasize the importance of including DZIP1L in multigene panels and in the data analysis of whole-exome sequencing for cystic kidney diseases. A higher resolution version of the Graphical abstract is available as Supplementary information.

Mutations affecting the N-terminal domains of SHANK3 point to different pathomechanisms in neurodevelopmental disorders.

Shank proteins are major scaffolds of the postsynaptic density of excitatory synapses. Mutations in SHANK genes are associated with autism and intellectual disability. The effects of missense mutations on Shank3 function, and therefore the pathomechanisms are unclear. Several missense mutations in SHANK3 affect the N-terminal region, consisting of the Shank/ProSAP N-terminal (SPN) domain and a set of Ankyrin (Ank) repeats. Here we identify a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. We functionally analysed this and a series of other mutations, using biochemical and biophysical techniques. We observe two major effects: (1) a loss of binding to δ-catenin (e.g. in the p.L270M variant), and (2) interference with the intramolecular interaction between N-terminal SPN domain and the Ank repeats. This also interferes with binding to the α-subunit of the calcium-/calmodulin dependent kinase II (αCaMKII), and appears to be associated with a more severe neurodevelopmental pathology.

Comparison of the Metastasis Predictive Potential of mRNA and Long Non-Coding RNA Profiling in Systemically Untreated Breast Cancer.

Several gene expression signatures based on mRNAs and a few based on long non-coding RNAs (lncRNAs) have been developed to provide prognostic information beyond clinical evaluation in breast cancer (BC). However, the comparison of such signatures for predicting recurrence is very scarce. Therefore, we compared the prognostic utility of mRNAs and lncRNAs in low-risk BC patients using two different classification strategies. Frozen primary tumor samples from 160 lymph node negative and systemically untreated BC patients were included; 80 developed recurrence-i.e., regional or distant metastasis while 80 remained recurrence-free (mean follow-up of 20.9 years). Patients were pairwise matched for clinicopathological characteristics. Classification based on differential mRNA or lncRNA expression using seven individual machine learning methods and a voting scheme classified patients into risk-subgroups. Classification by the seven methods with a fixed sensitivity of ≥90% resulted in specificities ranging from 16-40% for mRNA and 38-58% for lncRNA, and after voting, specificities of 38% and 60% respectively. Classifier performance based on an alternative classification approach of balanced accuracy optimization also provided higher specificities for lncRNA than mRNA at comparable sensitivities. Thus, our results suggested that classification followed by voting improved prognostic power using lncRNAs compared to mRNAs regardless of classification strategy.

Epileptic encephalopathy caused by ARV1 deficiency: Refinement of the genotype-phenotype spectrum and functional impact on GPI-anchored proteins.

Early infantile epileptic encephalopathy 38 (EIEE38, MIM #617020) is caused by biallelic variants in ARV1, encoding a transmembrane protein of the endoplasmic reticulum with a pivotal role in glycosylphosphatidylinositol (GPI) biosynthesis. We ascertained seven new patients from six unrelated families harboring biallelic variants in ARV1, including five novel variants. Affected individuals showed psychomotor delay, hypotonia, early onset refractory seizures followed by regression and specific neuroimaging features. Flow cytometric analysis on patient fibroblasts showed a decrease in GPI-anchored proteins on the cell surface, supporting a lower residual activity of the mutant ARV1 as compared to the wildtype. A rescue assay through the transduction of lentivirus expressing wild type ARV1 cDNA effectively rescued these alterations. This study expands the clinical and molecular spectrum of the ARV1-related encephalopathy, confirming the essential role of ARV1 in GPI biosynthesis and brain function.

Global Gene Expression Profiling and Transcription Factor Network Analysis of Cognitive Aging in Monozygotic Twins.

Cognitive aging is one of the major problems worldwide, especially as people get older. This study aimed to perform global gene expression profiling of cognitive function to identify associated genes and pathways and a novel transcriptional regulatory network analysis to identify important regulons. We performed single transcript analysis on 400 monozygotic twins using an assumption-free generalized correlation coefficient (GCC), linear mixed-effect model (LME) and kinship model and identified six probes (one significant at the standard FDR < 0.05 while the other results were suggestive with 0.18 ≤ FDR ≤ 0.28). We combined the GCC and linear model results to cover diverse patterns of relationships, and meaningful and novel genes like APOBEC3G, H6PD, SLC45A1, GRIN3B, and PDE4D were detected. Our exploratory study showed the downregulation of all these genes with increasing cognitive function or vice versa except the SLC45A1 gene, which was upregulated with increasing cognitive function. Linear models found only H6PD and SLC45A1, the other genes were captured by GCC. Significant functional pathways (FDR < 3.95e-10) such as focal adhesion, ribosome, cysteine and methionine metabolism, Huntington's disease, eukaryotic translation elongation, nervous system development, influenza infection, metabolism of RNA, and cell cycle were identified. A total of five regulons (FDR< 1.3e-4) were enriched in a transcriptional regulatory analysis in which CTCF and REST were activated and SP3, SRF, and XBP1 were repressed regulons. The genome-wide transcription analysis using both assumption-free GCC and linear models identified important genes and biological pathways implicated in cognitive performance, cognitive aging, and neurological diseases. Also, the regulatory network analysis revealed significant activated and repressed regulons on cognitive function.

Differential lncRNA expression profiling of cognitive function in middle and old aged monozygotic twins using generalized association analysis.

Cognitive impairment is the most prominent symptom in neurodegenerative disorders affecting quality of life and mortality. However, despite years of research, the molecular mechanism underlying the regulation of cognitive function and its impairment is poorly understood. This study aims to elucidate the role of long non-coding RNAs (lncRNAs) expression and lncRNA-mRNA interaction networks, by analyzing lncRNA expression in whole blood samples of 400 middle and old aged monozygotic twins in association with cognitive function using both linear models and a generalized correlation coefficient (GCC) to capture the diverse patterns of correlation. We detected 13 probes (p < 1e-03) displaying nonlinear and 7 probes (p < 1e-03) showing linear correlations. After combining the results, we identified 20 lncRNA probes with p < 1e-03. The top lncRNA probes were annotated to genes, along with the non-coding MALAT1, that play roles in neurodegenerative diseases. The top lncRNAs were linked to functional clusters including peptidyl-glycine modification, vascular smooth muscle cells, mitotic spindle organization and protein tyrosine phosphatase. In addition, mapping of the top significant lncRNAs to the lncRNA-mRNA interaction network detected significantly enriched biological pathways involving neuroactive ligand-receptor interaction, proteasome and chemokines. We show that GCC served as a complementary approach in detecting lncRNAs missed by the conventional linear models. A combination of GCC and linear models identified lncRNAs of diverse patterns of association enriched for GO biological and molecular functions meaningful in cognitive performance and cognitive decline. The novel lncRNA regulatory network further contributed to detect significant pathways implicated in cognition.

Disruption of RFX family transcription factors causes autism, attention-deficit/hyperactivity disorder, intellectual disability, and dysregulated behavior.

PURPOSE: We describe a novel neurobehavioral phenotype of autism spectrum disorder (ASD), intellectual disability, and/or attention-deficit/hyperactivity disorder (ADHD) associated with de novo or inherited deleterious variants in members of the RFX family of genes. RFX genes are evolutionarily conserved transcription factors that act as master regulators of central nervous system development and ciliogenesis. METHODS: We assembled a cohort of 38 individuals (from 33 unrelated families) with de novo variants in RFX3, RFX4, and RFX7. We describe their common clinical phenotypes and present bioinformatic analyses of expression patterns and downstream targets of these genes as they relate to other neurodevelopmental risk genes. RESULTS: These individuals share neurobehavioral features including ASD, intellectual disability, and/or ADHD; other frequent features include hypersensitivity to sensory stimuli and sleep problems. RFX3, RFX4, and RFX7 are strongly expressed in developing and adult human brain, and X-box binding motifs as well as RFX ChIP-seq peaks are enriched in the cis-regulatory regions of known ASD risk genes. CONCLUSION: These results establish a likely role of deleterious variation in RFX3, RFX4, and RFX7 in cases of monogenic intellectual disability, ADHD and ASD, and position these genes as potentially critical transcriptional regulators of neurobiological pathways associated with neurodevelopmental disease pathogenesis.

Chromosomal translocation disrupting the SMAD4 gene resulting in the combined phenotype of Juvenile polyposis syndrome and Hereditary Hemorrhagic Telangiectasia.

BACKGROUND: Patients with germline variants in SMAD4 can present symptoms of both juvenile polyposis syndrome (JPS) and Hereditary Hemorrhagic Telangiectasia (HHT): JP-HHT syndrome. Next-Generation Sequencing (NGS) techniques disclose causative sequence variants in around 90% of HHT patients fulfilling the Curaçao criteria. Here we report a translocation event involving SMAD4 resulting in JP-HHT. METHODS: A patient fulfilling the Curaçao criteria was analyzed for variants in ENG, ACVRL1, and SMAD4 using standard techniques. Whole-genome sequencing (WGS) using both short-read NGS technology and long-read Oxford Nanopore technology was performed to define the structural variant and exact breakpoints. RESULTS: No pathogenic variant was detected in ENG, ACVRL1, or SMAD4 in DNA extracted from blood. Due to abortus habitualis, the proband´s daughter was submitted for chromosomal analysis, and a cytogenetically balanced chromosomal reciprocal translocation t(1;18)(p36.1;q21.1) was detected in the daughter and the patient. The balanced translocation segregated with both gastrointestinal cancer and HHT in the family. WGS provided the exact breakpoints of the reciprocal translocation proving disruption of the SMAD4 gene. DISCUSSION: A disease-causing reciprocal translocation between chromosome 1 and 18 with a breakpoint in the SMAD4 locus co-segregated with JP-HHT in an extended family. This observation warrants further analysis for chromosomal rearrangements in individuals with clinical HHT or JP-HHT of unknown cause.

Differential long noncoding RNA profiling of BMI in twins.

Aim: Many efforts have been deployed to identify genetic variants associated with BMI. Alternatively, we explore epigenetic contribution to BMI variation by focusing on long noncoding RNAs (lncRNAs) which represents a key layer of epigenetic control. Materials & methods: We analyzed lncRNA expression in whole blood of 229 monozygotic twin pairs in association with BMI using generalized estimating equations. Results & conclusion: Six lncRNA probes were identified as significant (false discovery rate <0.05), with BMI showing causal effects on the expression of the significant lncRNAs. Functional annotation of differential profiles identified Gene ontology biological processes including kidney development, regulations of lipid biosynthetic process, circadian rhythm, notch signaling, etc. Whole blood lncRNAs are significantly expressed in response to BMI variation.

A Genome-Wide Integrative Association Study of DNA Methylation and Gene Expression Data and Later Life Cognitive Functioning in Monozygotic Twins.

Monozygotic twins are genetically identical but rarely phenotypically identical. Epigenetic and transcriptional variation could influence this phenotypic discordance. Investigation of intra-pair differences in molecular markers and a given phenotype in monozygotic twins controls most of the genetic contribution, enabling studies of the molecular features of the phenotype. This study aimed to identify genes associated with cognition in later life using integrated enrichment analyses of the results of blood-derived intra-pair epigenome-wide and transcriptome-wide association analyses of cognition in 452 middle-aged and old-aged monozygotic twins (56-80 years). Integrated analyses were performed with an unsupervised approach using KeyPathwayMiner, and a supervised approach using the KEGG and Reactome databases. The supervised approach identified several enriched gene sets, including "neuroactive ligand receptor interaction" (p-value = 1.62∗10-2), "Neurotrophin signaling" (p-value = 2.52∗10-3), "Alzheimer's disease" (p-value = 1.20∗10-2), and "long-term depression" (p-value = 1.62∗10-2). The unsupervised approach resulted in a 238 gene network, including the Alzheimer's disease gene APP (Amyloid Beta Precursor Protein) as an exception node, and several novel candidate genes. The strength of the unsupervised method is that it can reveal previously uncharacterized sub-pathways and detect interplay between biological processes, which remain undetected by the current supervised methods. In conclusion, this study identified several previously reported cognition genes and pathways and, additionally, puts forward novel candidates for further verification and validation.

Bi-allelic Loss-of-Function Variants in NUP188 Cause a Recognizable Syndrome Characterized by Neurologic, Ocular, and Cardiac Abnormalities.

Nucleoporins (NUPs) are an essential component of the nuclear-pore complex, which regulates nucleocytoplasmic transport of macromolecules. Pathogenic variants in NUP genes have been linked to several inherited human diseases, including a number with progressive neurological degeneration. We present six affected individuals with bi-allelic truncating variants in NUP188 and strikingly similar phenotypes and clinical courses, representing a recognizable genetic syndrome; the individuals are from four unrelated families. Key clinical features include congenital cataracts, hypotonia, prenatal-onset ventriculomegaly, white-matter abnormalities, hypoplastic corpus callosum, congenital heart defects, and central hypoventilation. Characteristic dysmorphic features include small palpebral fissures, a wide nasal bridge and nose, micrognathia, and digital anomalies. All affected individuals died as a result of respiratory failure, and five of them died within the first year of life. Nuclear import of proteins was decreased in affected individuals' fibroblasts, supporting a possible disease mechanism. CRISPR-mediated knockout of NUP188 in Drosophila revealed motor deficits and seizure susceptibility, partially recapitulating the neurological phenotype seen in affected individuals. Removal of NUP188 also resulted in aberrant dendrite tiling, suggesting a potential role of NUP188 in dendritic development. Two of the NUP188 pathogenic variants are enriched in the Ashkenazi Jewish population in gnomAD, a finding we confirmed with a separate targeted population screen of an international sampling of 3,225 healthy Ashkenazi Jewish individuals. Taken together, our results implicate bi-allelic loss-of-function NUP188 variants in a recessive syndrome characterized by a distinct neurologic, ophthalmologic, and facial phenotype.

Exome sequencing revealed DNA variants in NCOR1, IGF2BP1, SGLT2 and NEK11 as potential novel causes of ketotic hypoglycemia in children.

Unexplained or idiopathic ketotic hypoglycemia (KH) is the most common type of hypoglycemia in children. The diagnosis is based on the exclusion of routine hormonal and metabolic causes of hypoglycemia. We aimed to identify novel genes that cause KH, as this may lead to a more targeted treatment. Deep phenotyping of ten preschool age at onset KH patients (boys, n = 5; girls, n = 5) was performed followed by trio exome sequencing and comprehensive bioinformatics analysis. Data analysis revealed four novel candidate genes: (1) NCOR1 in a patient with KH, iron deficiency and loose stools; (2) IGF2BP1 in a proband with KH, short stature and delayed bone age; (3) SLC5A2 in a proband with KH, intermittent glucosuria and extremely elevated p-GLP-1; and (4) NEK11 in a proband with ketotic hypoglycemia and liver affliction. These genes are associated with different metabolic processes, such as gluconeogenesis, translational regulation, and glucose transport. In conclusion, WES identified DNA variants in four different genes as potential novel causes of IKH, suggesting that IKH is a heterogeneous disorder that can be split into several novel diseases: NCOR1-KH, IGF2BP1-KH, SGLT2-KH or familial renal glucosuria KH, and NEK11-KH. Precision medicine treatment based on exome sequencing may lead to advances in the management of IKH.