Detection of Constitutional Structural Variants by Optical Genome Mapping: A Multisite Study of Postnatal Samples.

Optical genome mapping is a high-resolution technology that can detect all types of structural variations in the genome. This second phase of a multisite study compares the performance of optical genome mapping and current standard-of-care methods for diagnostic testing of individuals with constitutional disorders, including neurodevelopmental impairments and congenital anomalies. Among the 627 analyses in phase 2, 405 were of retrospective samples supplied by five diagnostic centers in the United States and 94 were prospective samples collected over 18 months by two diagnostic centers (June 2021 to October 2022). Additional samples represented a family cohort to determine inheritance (n = 119) and controls (n = 9). Full concordance of results between optical genome mapping and one or more standard-of-care diagnostic tests was 98.6% (618/627), with partial concordance in an additional 1.1% (7/627).

Multisite Assessment of Optical Genome Mapping for Analysis of Structural Variants in Constitutional Postnatal Cases.

This study compares optical genome mapping (OGM) performed at multiple sites with current standard-of-care (SOC) methods used in clinical cytogenetics. This study included 50 negative controls and 359 samples from individuals (patients) with suspected genetic conditions referred for cytogenetic testing. OGM was performed using the Saphyr system and Bionano Access software version 1.7. Structural variants, including copy number variants, aneuploidy, and regions of homozygosity, were detected and classified according to American College of Medical Genetics and Genomics guidelines. Repeated expansions in FMR1 and contractions in facioscapulohumeral dystrophy 1 were also analyzed. OGM results were compared with SOC for technical concordance, clinical classification concordance, intrasite and intersite reproducibility, and ability to provide additional, clinically relevant information. Across five testing sites, 98.8% (404/409) of samples yielded successful OGM data for analysis and interpretation. Overall, technical concordance for OGM to detect previously reported SOC results was 99.5% (399/401). The blinded analysis and variant classification agreement between SOC and OGM was 97.6% (364/373). Replicate analysis of 130 structural variations was 100% concordant. On the basis of this demonstration of the analytic validity and clinical utility of OGM by this multisite assessment, the authors recommend this technology as an alternative to existing SOC tests for rapid detection and diagnosis in postnatal constitutional disorders.

The genetic legacy of the Hunyadi descendants.

The Hunyadi family is one of the most influential families in the history of Central Europe in the 14th-16th centuries. The family's prestige was established by Johannes Hunyadi, a Turk-beater who rose to the position of governor of the Kingdom of Hungary. His second son, Matthias Hunyadi, became the elected ruler of the Kingdom of Hungary in 1458. The Hunyadi family had unknown origin. Moreover, Matthias failed to found a dynasty because of lacking a legitimate heir and his illegitimate son Johannes Corvinus was unable to obtain the crown. His grandson, Christophorus Corvinus, died in childhood, thus the direct male line of the family ended. In the framework of on interdisciplinary research, we have determined the whole genome sequences of Johannes Corvinus and Christophorus Corvinus by next-generation sequencing technology. Both of them carried the Y-chromosome haplogroup is E1b1b1a1b1a6a1c ∼, which is widespread in Eurasia. The father-son relationship was verified using the classical STR method and whole genome data. Christophorus Corvinus belongs to the rare, sporadically occurring T2c1+146 mitochondrial haplogroup, most frequent around the Mediterranean, while his father belongs to the T2b mitochondrial haplogroup, widespread in Eurasia, both are consistent with the known origin of the mothers. Archaeogenomic analysis indicated that the Corvinus had an ancient European genome composition. Based on the reported genetic data, it will be possible to identify all the other Hunyadi family member, whose only known grave site is known, but who are resting assorted with several other skeletons.

The genetic origin of Huns, Avars, and conquering Hungarians.

Huns, Avars, and conquering Hungarians were migration-period nomadic tribal confederations that arrived in three successive waves in the Carpathian Basin between the 5th and 9th centuries. Based on the historical data, each of these groups are thought to have arrived from Asia, although their exact origin and relation to other ancient and modern populations have been debated. Recently, hundreds of ancient genomes were analyzed from Central Asia, Mongolia, and China, from which we aimed to identify putative source populations for the above-mentioned groups. In this study, we have sequenced 9 Hun, 143 Avar, and 113 Hungarian conquest period samples and identified three core populations, representing immigrants from each period with no recent European ancestry. Our results reveal that this "immigrant core" of both Huns and Avars likely originated in present day Mongolia, and their origin can be traced back to Xiongnus (Asian Huns), as suggested by several historians. On the other hand, the "immigrant core" of the conquering Hungarians derived from an earlier admixture of Mansis, early Sarmatians, and descendants of late Xiongnus. We have also shown that a common "proto-Ugric" gene pool appeared in the Bronze Age from the admixture of Mezhovskaya and Nganasan people, supporting genetic and linguistic data. In addition, we detected shared Hun-related ancestry in numerous Avar and Hungarian conquest period genetic outliers, indicating a genetic link between these successive nomadic groups. Aside from the immigrant core groups, we identified that the majority of the individuals from each period were local residents harboring "native European" ancestry.

Aerobic exercise elicits clinical adaptations in myotonic dystrophy type 1 patients independently of pathophysiological changes.

BackgroundMyotonic dystrophy type 1 (DM1) is a complex life-limiting neuromuscular disorder characterized by severe skeletal muscle atrophy, weakness, and cardiorespiratory defects. Exercised DM1 mice exhibit numerous physiological benefits that are underpinned by reduced CUG foci and improved alternative splicing. However, the efficacy of physical activity in patients is unknown.MethodsEleven genetically diagnosed DM1 patients were recruited to examine the extent to which 12 weeks of cycling can recuperate clinical and physiological metrics. Furthermore, we studied the underlying molecular mechanisms through which exercise elicits benefits in skeletal muscle of DM1 patients.RESULTSDM1 was associated with impaired muscle function, fitness, and lung capacity. Cycling evoked several clinical, physical, and metabolic advantages in DM1 patients. We highlight that exercise-induced molecular and cellular alterations in patients do not conform with previously published data in murine models and propose a significant role of mitochondrial function in DM1 pathology. Finally, we discovered a subset of small nucleolar RNAs (snoRNAs) that correlated to indicators of disease severity.ConclusionWith no available cures, our data support the efficacy of exercise as a primary intervention to partially mitigate the clinical progression of DM1. Additionally, we provide evidence for the involvement of snoRNAs and other noncoding RNAs in DM1 pathophysiology.Trial registrationThis trial was approved by the HiREB committee (no. 7901) and registered under ClinicalTrials.gov (NCT04187482).FundingNeil and Leanne Petroff. Canadian Institutes of Health Research Foundation (no. 143325).

The histone H3K9M mutation synergizes with H3K14 ubiquitylation to selectively sequester histone H3K9 methyltransferase Clr4 at heterochromatin.

Oncogenic histone lysine-to-methionine mutations block the methylation of their corresponding lysine residues on wild-type histones. One attractive model is that these mutations sequester histone methyltransferases, but genome-wide studies show that mutant histones and histone methyltransferases often do not colocalize. Using chromatin immunoprecipitation sequencing (ChIP-seq), here, we show that, in fission yeast, even though H3K9M-containing nucleosomes are broadly distributed across the genome, the histone H3K9 methyltransferase Clr4 is mainly sequestered at pericentric repeats. This selective sequestration of Clr4 depends not only on H3K9M but also on H3K14 ubiquitylation (H3K14ub), a modification deposited by a Clr4-associated E3 ubiquitin ligase complex. In vitro, H3K14ub synergizes with H3K9M to interact with Clr4 and potentiates the inhibitory effects of H3K9M on Clr4 enzymatic activity. Moreover, binding kinetics show that H3K14ub overcomes the Clr4 aversion to H3K9M and reduces its dissociation. The selective sequestration model reconciles previous discrepancies and demonstrates the importance of protein-interaction kinetics in regulating biological processes.

Third case of Duchenne muscular dystrophy and West syndrome: Expanding the spectrum of the DMD neuropsychiatric phenotype.

Duchenne muscular dystrophy is an X-Linked neuromuscular disorder, and the most common muscular dystrophy. Neuropsychiatric phenotype associated to DMD gene mutations include now low IQ scores, epilepsy, autism, and attention deficit disorder. These have been observed with higher frequency in mutations that disrupt the short isoforms Dp71 and Dp140. West syndrome has been previously reported in two unrelated patients with Duchenne muscular dystrophy. Here, we report the third patient with West syndrome who had a novel hemizygous nonsense pathogenic variant in the exon 8 of the DMD gene c.811C>T, p.(Gln271*), suggesting West syndrome as part of the neuropsychiatric spectrum in Duchenne muscular dystrophy.

Determination of the phylogenetic origins of the Árpád Dynasty based on Y chromosome sequencing of Béla the Third.

We set out to identify the origins of the Árpád Dynasty based on genome sequencing of DNA derived from the skeletal remains of Hungarian King Béla III (1172-1196) and eight additional individuals (six males, two females) originally interred at the Royal Basilica of Székesfehérvár. Y-chromosome analysis established that two individuals, Béla III and HU52 assign to haplogroups R-Z2125 whose distribution centres near South Central Asia with subsidiary expansions in the regions of modern Iran, the Volga Ural region and the Caucasus. Out of a cohort of 4340 individuals from these geographic areas, we acquired whole-genome data from 208 individuals derived for the R-Z2123 haplogroup. From these data we have established that the closest living kin of the Árpád Dynasty are R-SUR51 derived modern day Bashkirs predominantly from the Burzyansky and Abzelilovsky districts of Bashkortostan in the Russian Federation. Our analysis also reveals the existence of SNPs defining a novel Árpád Dynasty specific haplogroup R-ARP. Framed within the context of a high resolution R-Z2123 phylogeny, the ancestry of the first Hungarian royal dynasty traces to the region centering near Northern Afghanistan about 4500 years ago and identifies the Bashkirs as their closest kin, with a separation date between the two populations at the beginning of the first millennium CE.

Blood group typing from whole-genome sequencing data.

Many questions can be explored thanks to whole-genome data. The aim of this study was to overcome their main limits, software availability and database accuracy, and estimate the feasibility of red blood cell (RBC) antigen typing from whole-genome sequencing (WGS) data. We analyzed whole-genome data from 79 individuals for HLA-DRB1 and 9 RBC antigens. Whole-genome sequencing data was analyzed with software allowing phasing of variable positions to define alleles or haplotypes and validated for HLA typing from next-generation sequencing data. A dedicated database was set up with 1648 variable positions analyzed in KEL (KEL), ACKR1 (FY), SLC14A1 (JK), ACHE (YT), ART4 (DO), AQP1 (CO), CD44 (IN), SLC4A1 (DI) and ICAM4 (LW). Whole-genome sequencing typing was compared to that previously obtained by amplicon-based monoallelic sequencing and by SNaPshot analysis. Whole-genome sequencing data were also explored for other alleles. Our results showed 93% of concordance for blood group polymorphisms and 91% for HLA-DRB1. Incorrect typing and unresolved results confirm that WGS should be considered reliable with read depths strictly above 15x. Our results supported that RBC antigen typing from WGS is feasible but requires improvements in read depth for SNV polymorphisms typing accuracy. We also showed the potential for WGS in screening donors with rare blood antigens, such as weak JK alleles. The development of WGS analysis in immunogenetics laboratories would offer personalized care in the management of RBC disorders.

Allele-specific DNA methylation is increased in cancers and its dense mapping in normal plus neoplastic cells increases the yield of disease-associated regulatory SNPs.

BACKGROUND: Mapping of allele-specific DNA methylation (ASM) can be a post-GWAS strategy for localizing regulatory sequence polymorphisms (rSNPs). The advantages of this approach, and the mechanisms underlying ASM in normal and neoplastic cells, remain to be clarified. RESULTS: We perform whole genome methyl-seq on diverse normal cells and tissues and three cancer types. After excluding imprinting, the data pinpoint 15,112 high-confidence ASM differentially methylated regions, of which 1838 contain SNPs in strong linkage disequilibrium or coinciding with GWAS peaks. ASM frequencies are increased in cancers versus matched normal tissues, due to widespread allele-specific hypomethylation and focal allele-specific hypermethylation in poised chromatin. Cancer cells show increased allele switching at ASM loci, but disruptive SNPs in specific classes of CTCF and transcription factor binding motifs are similarly correlated with ASM in cancer and non-cancer. Rare somatic mutations affecting these same motif classes track with de novo ASM. Allele-specific transcription factor binding from ChIP-seq is enriched among ASM loci, but most ASM differentially methylated regions lack such annotations, and some are found in otherwise uninformative "chromatin deserts." CONCLUSIONS: ASM is increased in cancers but occurs by a shared mechanism involving disruptive SNPs in CTCF and transcription factor binding sites in both normal and neoplastic cells. Dense ASM mapping in normal plus cancer samples reveals candidate rSNPs that are difficult to find by other approaches. Together with GWAS data, these rSNPs can nominate specific transcriptional pathways in susceptibility to autoimmune, cardiometabolic, neuropsychiatric, and neoplastic diseases.

Clinical Transcriptome Sequencing Confirms Activation of a Cryptic Splice Site in Suspected SYNGAP1-Related Disorder.

SYNGAP1 encodes a brain-specific Ras GTPase activating protein (GAP) that regulates synaptic strength in glutamatergic neurons. Pathogenic variants in this gene are associated with a neurodevelopmental disorder characterized by intellectual and developmental disabilities, generalized epilepsy, hypotonia, and autism spectrum disorders. We describe a young male with suspected SYNGAP1-related disorder given clinical overlap and identification of an intronic variant of uncertain significance; clinical transcriptome analysis demonstrated activation of a cryptic acceptor splice site resulting in frameshift and introduction of a stop codon. This report highlights the utility of functional studies newly available to clinical practice in confirming a suspected genetic diagnosis, which can directly impact medical management and preclude the need for additional diagnostic testing.

The genotypic spectrum of ALDH7A1 mutations resulting in pyridoxine dependent epilepsy: A common epileptic encephalopathy.

Pyridoxine dependent epilepsy (PDE) is a treatable epileptic encephalopathy characterized by a positive response to pharmacologic doses of pyridoxine. Despite seizure control, at least 75% of individuals have intellectual disability and developmental delay. Current treatment paradigms have resulted in improved cognitive outcomes emphasizing the importance of an early diagnosis. As genetic testing is increasingly accepted as first tier testing for epileptic encephalopathies, we aimed to provide a comprehensive overview of ALDH7A1 mutations that cause PDE. The genotypes, ethnic origin and reported gender was collected from 185 subjects with a diagnosis of PDE. The population frequency for the variants in this report and the existing literature were reviewed in the Genome Aggregation Database (gnomAD). Novel variants identified in population databases were also evaluated through in silico prediction software and select variants were over-expressed in an E.coli-based expression system to measure α-aminoadipic semialdehyde dehydrogenase activity and production of α-aminoadipic acid. This study adds 47 novel variants to the literature resulting in a total of 165 reported pathogenic variants. Based on this report, in silico predictions, and general population data, we estimate an incidence of approximately 1:64,352 live births. This report provides a comprehensive overview of known ALDH7A1 mutations that cause PDE, and suggests that PDE may be more common than initially estimated. Due to the relative high frequency of the disease, the likelihood of under-diagnosis given the wide clinical spectrum and limited awareness among clinicians as well as the cognitive improvement noted with early treatment, newborn screening for PDE may be warranted.

Inherited selective cobalamin malabsorption in Komondor dogs associated with a CUBN splice site variant.

BACKGROUND: Three Komondor dogs in a small family and 3 sporadic cases exhibited a constellation of signs that included juvenile-onset of failure-to-thrive, inappetence, vomiting and/or diarrhea, and weakness. In each we documented dyshematopoiesis, increased anion gap, methylmalonic acidemia/-uria, and serum cobalamin deficiency. Urine protein electrophoresis demonstrated excretion of cubam ligands. All clinical signs and metabolic abnormalities, except proteinuria, were reversed by regular parenteral cobalamin administration. The pattern of occurrence and findings in the disorder suggested an autosomal recessive inheritance of cobalamin malabsorption with proteinuria, a condition in humans called Imerslund-Gräsbeck syndrome. The purpose of this study was to determine the molecular cause of this disorder in Komondors. RESULTS: Whole genome sequencing of two affected Komondor dogs of unknown relatedness and one parent and a clinically-normal littermate of an affected dog revealed a pathogenic single-base change in the CUBN intron 55 splice donor consensus sequence (NM_001003148.1: c.8746 + 1G > A) that was homozygous in affected dogs and heterozygous in the unaffected parents. Alleles of the variant co-segregated with alleles of the disease locus in the entire family and all more distantly-related sporadic cases. A population study using a simple allele-specific DNA test indicated mutant allele frequencies of 8.3 and 4.5% among North American and Hungarian Komondors, respectively. CONCLUSIONS: DNA testing can be used diagnostically in Komondors when clinical signs are suggestive of cobalamin deficiency or to inform Komondor breeders prospectively and prevent occurrence of future affected dogs. This represents the third cubilin variant causing inherited selective cobalamin malabsorption in a large animal ortholog of human Imerslund-Gräsbeck syndrome.

Next-Generation Sequencing and Mutational Analysis: Implications for Genes Encoding LINC Complex Proteins.

Targeted panel, whole exome, or whole genome DNA sequencing using next-generation sequencing (NGS) allows for extensive high-throughput investigation of molecular machines/systems such as the LINC complex. This includes the identification of genetic variants in humans that cause disease, as is the case for some genes encoding LINC complex proteins. The relatively low cost and high speed of the sequencing process results in large datasets at various stages of analysis and interpretation. For those not intimately familiar with the process, interpretation of the data might prove challenging. This review lays out the most important and most commonly used materials and methods of NGS. It also discusses data analysis and potential pitfalls one might encounter because of peculiarities of the laboratory methodology or data analysis pipelines.

Brain-Derived Neurotrophic Factor Elevates Activating Transcription Factor 4 (ATF4) in Neurons and Promotes ATF4-Dependent Induction of Sesn2.

Activating transcription factor 4 (ATF4) plays important physiologic roles in the brain including regulation of learning and memory as well as neuronal survival and death. Yet, outside of translational regulation by the eIF2α-dependent stress response pathway, there is little information about how its levels are controlled in neurons. Here, we show that brain-derived neurotrophic factor (BDNF) promotes a rapid and sustained increase in neuronal ATF4 transcripts and protein levels. This increase is dependent on tropomyosin receptor kinase (TrkB) signaling, but independent of levels of phosphorylated eIF2α. The elevation in ATF4 protein occurs both in nuclei and processes. Transcriptome analysis revealed that ATF4 mediates BDNF-promoted induction of Sesn2 which encodes Sestrin2, a protector against oxidative and genotoxic stresses and a mTor complex 1 inhibitor. In contrast, BDNF-elevated ATF4 did not affect expression of a number of other known ATF4 targets including several with pro-apoptotic activity. The capacity of BDNF to elevate neuronal ATF4 may thus represent a means to maintain this transcription factor at levels that provide neuroprotection and optimal brain function without risk of triggering neurodegeneration.

Ultra-rare mutations in SRCAP segregate in Caribbean Hispanic families with Alzheimer disease.

OBJECTIVE: To identify rare coding variants segregating with late-onset Alzheimer disease (LOAD) in Caribbean Hispanic families. METHODS: Whole-exome sequencing (WES) was completed in 110 individuals from 31 Caribbean Hispanic families without APOE ε4 homozygous carriers. Rare coding mutations segregating in families were subsequently genotyped in additional families and in an independent cohort of Caribbean Hispanic patients and controls. SRCAP messenger RNA (mRNA) expression was assessed in whole blood from mutation carriers with LOAD, noncarriers with LOAD, and healthy elderly controls, and also from autopsied brains in 2 clinical neuropathologic cohort studies of aging and dementia. RESULTS: Ten ultra-rare missense mutations in the Snf2-related CREBBP, activator protein (SRCAP), were found in 12 unrelated families. Compared with the frequency in Caribbean Hispanic controls and the Latino population in the Exome Aggregation Consortium, the frequency of SRCAP mutations among Caribbean Hispanic patients with LOAD was significantly enriched (p = 1.19e-16). mRNA expression of SRCAP in whole blood was significantly lower in mutation carriers with LOAD, while the expression in whole blood and in the brain was significantly higher in nonmutation carriers with LOAD. Brain expression also correlated with clinical and neuropathologic endophenotypes. CONCLUSIONS: WES in Caribbean Hispanic families with LOAD revealed ultra-rare missense mutations in SRCAP, a gene expressed in the brain and mutated in Floating-Harbor syndrome. SRCAP is a potent coactivator of the CREB-binding protein and a regulator of DNA damage response involving ATP-dependent chromatin remodeling. We hypothesize that increased expression in LOAD suggests a compensatory mechanism altered in mutation carriers.

Implementation of next generation sequencing into pediatric hematology-oncology practice: moving beyond actionable alterations.

BACKGROUND: Molecular characterization has the potential to advance the management of pediatric cancer and high-risk hematologic disease. The clinical integration of genome sequencing into standard clinical practice has been limited and the potential utility of genome sequencing to identify clinically impactful information beyond targetable alterations has been underestimated. METHODS: The Precision in Pediatric Sequencing (PIPseq) Program at Columbia University Medical Center instituted prospective clinical next generation sequencing (NGS) for pediatric cancer and hematologic disorders at risk for treatment failure. We performed cancer whole exome sequencing (WES) of patient-matched tumor-normal samples and RNA sequencing (RNA-seq) of tumor to identify sequence variants, fusion transcripts, relative gene expression, and copy number variation (CNV). A directed cancer gene panel assay was used when sample adequacy was a concern. Constitutional WES of patients and parents was performed when a constitutionally encoded disease was suspected. Results were initially reviewed by a molecular pathologist and subsequently by a multi-disciplinary molecular tumor board. Clinical reports were issued to the ordering physician and posted to the patient's electronic medical record. RESULTS: NGS was performed on tumor and/or normal tissue from 101 high-risk pediatric patients. Potentially actionable alterations were identified in 38% of patients, of which only 16% subsequently received matched therapy. In an additional 38% of patients, the genomic data provided clinically relevant information of diagnostic, prognostic, or pharmacogenomic significance. RNA-seq was clinically impactful in 37/65 patients (57%) providing diagnostic and/or prognostic information for 17 patients (26%) and identified therapeutic targets in 15 patients (23%). Known or likely pathogenic germline alterations were discovered in 18/90 patients (20%) with 14% having germline alternations in cancer predisposition genes. American College of Medical Genetics (ACMG) secondary findings were identified in six patients. CONCLUSIONS: Our results demonstrate the feasibility of incorporating clinical NGS into pediatric hematology-oncology practice. Beyond the identification of actionable alterations, the ability to avoid ineffective/inappropriate therapies, make a definitive diagnosis, and identify pharmacogenomic modifiers is clinically impactful. Taking a more inclusive view of potential clinical utility, 66% of cases tested through our program had clinically impactful findings and samples interrogated with both WES and RNA-seq resulted in data that impacted clinical decisions in 75% of cases.