A genome-wide spectrum of tandem repeat expansions in 338,963 humans.

The Genome Aggregation Database (gnomAD), widely recognized as the gold-standard reference map of human genetic variation, has largely overlooked tandem repeat (TR) expansions, despite the fact that TRs constitute ∼6% of our genome and are linked to over 50 human diseases. Here, we introduce the TR-gnomAD (https://wlcb.oit.uci.edu/TRgnomAD), a biobank-scale reference of 0.86 million TRs derived from 338,963 whole-genome sequencing (WGS) samples of diverse ancestries (39.5% non-European samples). TR-gnomAD offers critical insights into ancestry-specific disease prevalence using disparities in TR unit number frequencies among ancestries. Moreover, TR-gnomAD is able to differentiate between common, presumably benign TR expansions, which are prevalent in TR-gnomAD, from those potentially pathogenic TR expansions, which are found more frequently in disease groups than within TR-gnomAD. Together, TR-gnomAD is an invaluable resource for researchers and physicians to interpret TR expansions in individuals with genetic diseases.

Repeat polymorphisms underlie top genetic risk loci for glaucoma and colorectal cancer.

Many regions in the human genome vary in length among individuals due to variable numbers of tandem repeats (VNTRs). To assess the phenotypic impact of VNTRs genome-wide, we applied a statistical imputation approach to estimate the lengths of 9,561 autosomal VNTR loci in 418,136 unrelated UK Biobank participants and 838 GTEx participants. Association and statistical fine-mapping analyses identified 58 VNTRs that appeared to influence a complex trait in UK Biobank, 18 of which also appeared to modulate expression or splicing of a nearby gene. Non-coding VNTRs at TMCO1 and EIF3H appeared to generate the largest known contributions of common human genetic variation to risk of glaucoma and colorectal cancer, respectively. Each of these two VNTRs associated with a >2-fold range of risk across individuals. These results reveal a substantial and previously unappreciated role of non-coding VNTRs in human health and gene regulation.

Antagonistic roles of canonical and Alternative-RPA in disease-associated tandem CAG repeat instability.

Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.

Mixed knobs in corn cobs.

Maize heterochromatic knobs cheat female meiosis by forming neocentromeres that bias their segregation into the future egg cell. In this issue of Genes & Development, Swentowsky and colleagues (pp. 1239-1251) show that two types of knobs, those composed of 180-bp and TR1 sequences, recruit their own novel and divergent kinesin-14 family members to form neocentromeres.

Distinct kinesin motors drive two types of maize neocentromeres.

A maize chromosome variant called abnormal chromosome 10 (Ab10) converts knobs on chromosome arms into neocentromeres, causing their preferential segregation to egg cells in a process known as meiotic drive. We previously demonstrated that the gene Kinesin driver (Kindr) on Ab10 encodes a kinesin-14 required to mobilize neocentromeres made up of the major tandem repeat knob180. Here we describe a second kinesin-14 gene, TR-1 kinesin (Trkin), that is required to mobilize neocentromeres made up of the minor tandem repeat TR-1. Trkin lies in a 4-Mb region of Ab10 that is not syntenic with any other region of the maize genome and shows extraordinary sequence divergence from Kindr and other kinesins in plants. Despite its unusual structure, Trkin encodes a functional minus end-directed kinesin that specifically colocalizes with TR-1 in meiosis, forming long drawn out neocentromeres. TRKIN contains a nuclear localization signal and localizes to knobs earlier in prophase than KINDR. The fact that TR-1 repeats often co-occur with knob180 repeats suggests that the current role of the TRKIN/TR-1 system is to facilitate the meiotic drive of the KINDR/knob180 system.

Cell-type-specific dysregulation of RNA alternative splicing in short tandem repeat mouse knockin models of myotonic dystrophy.

Short tandem repeats (STRs) are prone to expansion mutations that cause multiple hereditary neurological and neuromuscular diseases. To study pathomechanisms using mouse models that recapitulate the tissue specificity and developmental timing of an STR expansion gene, we used rolling circle amplification and CRISPR/Cas9-mediated genome editing to generate Dmpk CTG expansion (CTGexp) knockin models of myotonic dystrophy type 1 (DM1). We demonstrate that skeletal muscle myoblasts and brain choroid plexus epithelial cells are particularly susceptible to Dmpk CTGexp mutations and RNA missplicing. Our results implicate dysregulation of muscle regeneration and cerebrospinal fluid homeostasis as early pathogenic events in DM1.

Common single-base insertions in the VNTR of the carboxyl ester lipase (CEL) gene are benign and also likely to arise somatically in the exocrine pancreas.

The CEL gene encodes carboxyl ester lipase, a pancreatic digestive enzyme. CEL is extremely polymorphic due to a variable number tandem repeat (VNTR) located in the last exon. Single-base deletions within this VNTR cause the inherited disorder MODY8, whereas little is known about VNTR single-base insertions in pancreatic disease. We therefore mapped CEL insertion variants (CEL-INS) in 200 Norwegian patients with pancreatic neoplastic disorders. Twenty-eight samples (14.0%) carried CEL-INS alleles. Most common were insertions in repeat 9 (9.5%), which always associated with a VNTR length of 13 repeats. The combined INS allele frequency (0.078) was similar to that observed in a control material of 416 subjects (0.075). We performed functional testing in HEK293T cells of a set of CEL-INS variants, in which the insertion site varied from the first to the 12th VNTR repeat. Lipase activity showed little difference among the variants. However, CEL-INS variants with insertions occurring in the most proximal repeats led to protein aggregation and endoplasmic reticulum stress, which upregulated the unfolded protein response. Moreover, by using a CEL-INS-specific antibody, we observed patchy signals in pancreatic tissue from humans without any CEL-INS variant in the germline. Similar pancreatic staining was seen in knock-in mice expressing the most common human CEL VNTR with 16 repeats. CEL-INS proteins may therefore be constantly produced from somatic events in the normal pancreatic parenchyma. This observation along with the high population frequency of CEL-INS alleles strongly suggests that these variants are benign, with a possible exception for insertions in VNTR repeats 1-4.

Different roles of the heterodimer architecture of galectin-4 in selective recognition of oligosaccharides and lipopolysaccharides having ABH antigens.

The dimeric architecture of tandem-repeat type galectins, such as galectin-4 (Gal-4), modulates their biological activities, although the underlying molecular mechanisms have remained elusive. Emerging evidence show that tandem-repeat galectins play an important role in innate immunity by recognizing carbohydrate antigens present on the surface of certain pathogens, which very often mimic the structures of the human self-glycan antigens. Herein, we have analyzed the binding preferences of the C-domain of Gal-4 (Gal-4C) toward the ABH-carbohydrate histo-blood antigens with different core presentations and their recognition features have been rationalized by using a combined experimental approach including NMR, solid-phase and hemagglutination assays, and molecular modeling. The data show that Gal-4C prefers A over B antigens (two-fold in affinity), contrary to the N-domain (Gal-4N), although both domains share the same preference for the type-6 presentations. The behavior of the full-length Gal-4 (Gal-4FL) tandem-repeat form has been additionally scrutinized. Isothermal titration calorimetry and NMR data demonstrate that both domains within full-length Gal-4 bind to the histo-blood antigens independently of each other, with no communication between them. In this context, the heterodimeric architecture does not play any major role, apart from the complementary A and B antigen binding preferences. However, upon binding to a bacterial lipopolysaccharide containing a multivalent version of an H-antigen mimetic as O-antigen, the significance of the galectin architecture was revealed. Indeed, our data point to the linker peptide domain and the F-face of the C-domain as key elements that provide Gal-4 with the ability to cross-link multivalent ligands, beyond the glycan binding capacity of the dimer.

A phenome-wide association study identifies effects of copy-number variation of VNTRs and multicopy genes on multiple human traits.

The human genome contains tens of thousands of large tandem repeats and hundreds of genes that show common and highly variable copy-number changes. Due to their large size and repetitive nature, these variable number tandem repeats (VNTRs) and multicopy genes are generally recalcitrant to standard genotyping approaches and, as a result, this class of variation is poorly characterized. However, several recent studies have demonstrated that copy-number variation of VNTRs can modify local gene expression, epigenetics, and human traits, indicating that many have a functional role. Here, using read depth from whole-genome sequencing to profile copy number, we report results of a phenome-wide association study (PheWAS) of VNTRs and multicopy genes in a discovery cohort of ∼35,000 samples, identifying 32 traits associated with copy number of 38 VNTRs and multicopy genes at 1% FDR. We replicated many of these signals in an independent cohort and observed that VNTRs showing trait associations were significantly enriched for expression QTLs with nearby genes, providing strong support for our results. Fine-mapping studies indicated that in the majority (∼90%) of cases, the VNTRs and multicopy genes we identified represent the causal variants underlying the observed associations. Furthermore, several lie in regions where prior SNV-based GWASs have failed to identify any significant associations with these traits. Our study indicates that copy number of VNTRs and multicopy genes contributes to diverse human traits and suggests that complex structural variants potentially explain some of the so-called "missing heritability" of SNV-based GWASs.

Creative destruction: New protein folds from old.

Mechanisms of emergence and divergence of protein folds pose central questions in biological sciences. Incremental mutation and stepwise adaptation explain relationships between topologically similar protein folds. However, the universe of folds is diverse and riotous, suggesting more potent and creative forces are at play. Sequence and structure similarity are observed between distinct folds, indicating that proteins with distinct folds may share common ancestry. We found evidence of common ancestry between three distinct ß-barrel folds: Scr kinase family homology (SH3), oligonucleotide/oligosaccharide-binding (OB), and cradle loop barrel (CLB). The data suggest a mechanism of fold evolution that interconverts SH3, OB, and CLB. This mechanism, which we call creative destruction, can be generalized to explain many examples of fold evolution including circular permutation. In creative destruction, an open reading frame duplicates or otherwise merges with another to produce a fused polypeptide. A merger forces two ancestral domains into a new sequence and spatial context. The fused polypeptide can explore folding landscapes that are inaccessible to either of the independent ancestral domains. However, the folding landscapes of the fused polypeptide are not fully independent of those of the ancestral domains. Creative destruction is thus partially conservative; a daughter fold inherits some motifs from ancestral folds. After merger and refolding, adaptive processes such as mutation and loss of extraneous segments optimize the new daughter fold. This model has application in disease states characterized by genetic instability. Fused proteins observed in cancer cells are likely to experience remodeled folding landscapes and realize altered folds, conferring new or altered functions.

Bioinspired stretchable molecular composites of 2D-layered materials and tandem repeat proteins.

Protein based composites, such as nacre and bone, show astounding evolutionary capabilities, including tunable physical properties. Inspired by natural composites, we studied assembly of atomistically thin inorganic sheets with genetically engineered polymeric proteins to achieve mechanically compliant and ultra-tough materials. Although bare inorganic nanosheets are brittle, we designed flexible composites with proteins, which are insensitive to flaws due to critical structural length scale (∼2 nm). These proteins, inspired by squid ring teeth, adhere to inorganic sheets via secondary structures (i.e., ß-sheets and α-helices), which is essential for producing high stretchability (59 ± 1% fracture strain) and toughness (54.8 ± 2 MJ/m3). We find that the mechanical properties can be optimized by adjusting the protein molecular weight and tandem repetition. These exceptional mechanical responses greatly exceed the current state-of-the-art stretchability for layered composites by over a factor of three, demonstrating the promise of engineering materials with reconfigurable physical properties.

Single-nucleotide polymorphism array and fluorescence in situ hybridization analysis to decode the cytogenetic profile of atypical partial hydatidiform moles diagnosed by short tandem repeat polymorphism analysis.

Accurate diagnosis of partial hydatidiform moles (PHMs) is crucial for improving outcomes of gestational trophoblastic neoplasia. The use of short tandem repeat (STR) polymorphism analysis to distinguish between PHM and hydropic abortuses is instrumental; however, its diagnostic power has not been comprehensively assessed. Herein, we evaluated the diagnostic efficacy of STR in differentiating between PHM and hydropic abortus, thus providing an opportunity for early measurement of human chorionic gonadotropin for PHMs. We reviewed charts of STR polymorphism analysis performed on fresh villous specimens and patient blood samples using a commercial kit for 16 loci. The genetic classification of 79 PHMs was confirmed. STR was reliable in differentiating PHMs when at least 15 loci were available. Typically, PHMs are characterized by their triploidy, including two paternal and one maternal haploid contribution. In our sample, seven PHMs lacked the three-allelic loci, requiring fluorescence in situ hybridization (FISH) analysis to investigate imbalanced biparental conceptus and single-nucleotide polymorphism array analysis to reveal cytogenetic details. Of these PHMs, two, three, and one were identified as androgenetic/biparental mosaics (diploids), monospermic diandric monogynic triploids, and a typical dispermic diandric monogynic triploid, respectively. The remaining case was monospermic origin, but its ploidy details could not be available. Therefore, STR differentiated PHM from a biparental diploid abortus in most cases. However, PHM diagnosis may be compromised when STR is used as the sole method for cases displaying distinct cytogenetic patterns lacking the three-allelic loci, including androgenetic/biparental mosaicism. Therefore, FISH should be considered to confirm the diagnosis.

Quantifying Replication Slippage Error in Cryptosporidium Metabarcoding Studies.

Genetic variation in Cryptosporidium, a common protozoan gut parasite in humans, is often based on marker genes containing trinucleotide repeats, which differentiate subtypes and track outbreaks. However, repeat regions have high replication slippage rates, making it difficult to discern biological diversity from error. Here, we synthesized Cryptosporidium DNA in clonal plasmid vectors, amplified them in different mock community ratios, and sequenced them using next-generation sequencing to determine the rate of replication slippage with dada2. Our results indicate that slippage rates increase with the length of the repeat region and can contribute to error rates of up to 20%.

Haplotype-resolved genome assembly of Populus tremula × P. alba reveals aspen-specific megabase satellite DNA.

Populus species play a foundational role in diverse ecosystems and are important renewable feedstocks for bioenergy and bioproducts. Hybrid aspen Populus tremula × P. alba INRA 717-1B4 is a widely used transformation model in tree functional genomics and biotechnology research. As an outcrossing interspecific hybrid, its genome is riddled with sequence polymorphisms which present a challenge for sequence-sensitive analyses. Here we report a telomere-to-telomere genome for this hybrid aspen with two chromosome-scale, haplotype-resolved assemblies. We performed a comprehensive analysis of the repetitive landscape and identified both tandem repeat array-based and array-less centromeres. Unexpectedly, the most abundant satellite repeats in both haplotypes lie outside of the centromeres, consist of a 147 bp monomer PtaM147, frequently span >1 megabases, and form heterochromatic knobs. PtaM147 repeats are detected exclusively in aspens (section Populus) but PtaM147-like sequences occur in LTR-retrotransposons of closely related species, suggesting their origin from the retrotransposons. The genomic resource generated for this transformation model genotype has greatly improved the design and analysis of genome editing experiments that are highly sensitive to sequence polymorphisms. The work should motivate future hypothesis-driven research to probe into the function of the abundant and aspen-specific PtaM147 satellite DNA.

Bacillus anthracis in South Africa, 1975-2013: are some lineages vanishing?

The anthrax-causing bacterium Bacillus anthracis comprises the genetic clades A, B, and C. In the northernmost part (Pafuri) of Kruger National Park (KNP), South Africa, both the common A and rare B strains clades occur. The B clade strains were reported to be dominant in Pafuri before 1991, while A clade strains occurred towards the central parts of KNP. The prevalence of B clade strains is currently much lower as only A clade strains have been isolated from 1992 onwards in KNP. In this study 319 B. anthracis strains were characterized with 31-loci multiple-locus variable-number tandem repeat analysis (MLVA-31). B clade strains from soil (n = 9) and a Tragelaphus strepsiceros carcass (n = 1) were further characterised by whole genome sequencing and compared to publicly available genomes. The KNP strains clustered in the B clade before 1991 into two dominant genotypes. South African strains cluster into a dominant genotype A.Br.005/006 consisting of KNP as well as the other anthrax endemic region, Northern Cape Province (NCP), South Africa. A few A.Br.001/002 strains from both endemic areas were also identified. Subclade A.Br.101 belonging to the A.Br.Aust94 lineage was reported in the NCP. The B-clade strains seems to be vanishing, while outbreaks in South Africa are caused mainly by the A.Br.005/006 genotypes as well as a few minor clades such as A.Br.001/002 and A.Br.101 present in NCP. This work confirmed the existence of the rare and vanishing B-clade strains that group in B.Br.001 branch with KrugerB and A0991 KNP strains.

Structural peculiarities of tandem repeats and their clinical significance.

While it is well established that a mere 2% of human DNA nucleotides are involved in protein coding, the remainder of the DNA plays a vital role in the preservation of normal cellular genetic function. A significant proportion of tandem repeats (TRs) are present in non-coding DNA. TRs - specific sequences of nucleotides that entail numerous repetitions of a given fragment. In this study, we employed our novel algorithm grounded in finite automata theory, which we refer to as Dafna, to investigate for the first time the likelihood of these nucleotide sequences forming non-canonical DNA structures (NS). Such structures include G-quadruplexes, i-motifs, hairpins, and triplexes. The tandem repeats under consideration in our research encompassed sequences containing 1 to 6 nucleotides per repeated fragment. For comparison, we employed a set of randomly generated sequences of the same length (60 nucleotides) as a benchmark. The outcomes of our research exposed a disparity between the potential for NS formation in random sequences and tandem repeats. Our findings affirm that the propensity of DNA and RNA to form NS is closely tied to various genetic disorders, including Huntington's disease, Fragile X syndrome, and Friedreich's ataxia. In the concluding discussion, we present a proposal for a new therapeutic mechanism to address these diseases. This novel approach revolves around the ability of specific nucleic acid fragments to form multiple types of NS.

Molecular Analysis of Parthenogenetic Chimerism in a 46,XX/46,XY Patient with Idiopathic Oligoasthenoteratozoospermia.

INTRODUCTION: Parthenogenetic chimera is an extremely rare condition in human. Very few patients with parthenogenetic chimerism with XX/XY cells have been identified. CASE PRESENTATION: We report the clinical findings and molecular analysis of chimerism with a 46,XX/46,XY karyotype in a patient presenting idiopathic oligoasthenoteratozoospermia (OAT). To clarify the mechanism of chimera formation, short tandem repeat analysis using 21 loci was carried out. Quantitation of alleles in D6S1043, D12S391, fibrinogen alpha chain, and amelogenin revealed double paternal and one maternal genetic contribution to the patient, which is consistent with a parthenogenetic chimerism. The likely mechanism of chimerism formation was also discussed, followed by a literature review. CONCLUSION: This is the first documented case of parthenogenetic chimerism in an adult male with XX/XY cells presenting OAT. Improved cell sampling and more sensitive and specific detection methods are necessary to identify more patients with XX/XY chimerism for systematic studies on this condition in the future.

Marsupial genome analysis suggests that satellite DNA formation from walb endogenous retrovirus is an event specific to the red-necked wallaby.

We recently identified walbRep, a satellite DNA residing in the genome of the red-necked wallaby Notamacropus rufogriseus. It originates from the walb endogenous retrovirus and is organized in a manner in which the provirus structure is retained. The walbRep repeat units feature an average pairwise nucleotide identity as high as 99.5%, raising the possibility of a recent origin. The tammar wallaby N. eugenii is a species estimated to have diverged from the red-necked wallaby 2-3 million years ago. In PCR analyses of these two and other related species, walbRep-specific fragment amplification was observed only in the red-necked wallaby. Sequence database searches for the tammar wallaby resulted in sequence alignment lists that were sufficiently powerful to exclude the possibility of walbRep existence. These results suggested that the walbRep formation occurred in the red-necked wallaby lineage after its divergence from the tammar wallaby lineage, thus in a time span of maximum 3 million years.

Analysis of homozygous allele mismatches in paternity tests with massively parallel sequencing.

In paternity testing, short tandem repeats (STRs) allele mismatches are often detected. Nowadays, polymerase chain reaction- and capillary electrophoresis (CE)-based STR genotyping is the most commonly used method to distinguish alleles based on their length. However, it could not detect alleles of the same size with sequence differences. Massively parallel sequencing (MPS) can determine not only allele sizes but also sequences, which could explain the causes of allele mismatches. Additionally, more types of genetic markers can be detected in a single assay, which increases the discriminatory power and facilitates the analysis of paternity tests. In this study, we analyzed 11 cases with homozygous allele mismatches from routine DNA trio paternity tests using the CE platform. Samples were sequenced using the ForenSeq DNA Signature Prep Kit and the MiSeq FGx Sequencing System. The results show that of the eight father-child mismatch cases and three mother-child mismatch cases, five cases with D5S818 and D8S1179 and one case at D13S317 were classified as non-amplification. The other three cases and two cases could be defined as mutations. This study suggests that MPS-based STR genotyping can provide additional information that allows more accurate interpretation of allelic mismatches in paternity testing.

Genetic identification of human remains from the Korean War.

The genetic identification of skeletal remains from Chinese People's Volunteers (CPVs) of the Korean War has been challenging because of the degraded DNA samples and the lack of living close relatives. This study established a workflow for identifying CPVs by combining Y-chromosome short tandem repeats (Y-STRs), mitochondrial DNA (mtDNA) hypervariable regions I and II, autosomal STRs (aSTRs), and identity-informative SNPs (iiSNPs). A total of 20 skeletal remains of CPVs and 46 samples from their alleged relatives were collected. The success rate of DNA extraction from human remains was 100%. Based on Y-STRs, six remains shared the same male lineages with their alleged relatives. Meanwhile, mtDNA genotyping supports two remains sharing the same maternal lineages with their alleged relatives. Likelihood ratios (LRs) were further obtained from 27 aSTRs and 94 iiSNPs or 1936 iiSNPs to confirm their relationship. All joint pedigree LRs were >100. Finally, six remains were successfully identified. This pilot study for the systematic genetic identification of CPVs from the Korean War can be applied for the large-scale identification of CPVs in the future.