Detection of long repeat expansions from PCR-free whole-genome sequence data

Identifying large expansions of short tandem repeats (STRs) such as those that cause amyotrophic lateral sclerosis (ALS) and fragile X syndrome is challenging for short-read whole-genome sequencing (WGS) data. A solution to this problem is an important step towards integrating WGS into precision medicine. We have developed a software tool called ExpansionHunter that, using PCR-free WGS short-read data, can genotype repeats at the locus of interest, even if the expanded repeat is larger than the read length. We applied our algorithm to WGS data from 3,001 ALS patients who have been tested for the presence of the C9orf72 repeat expansion with repeat-primed PCR (RP-PCR). Compared against this truth data, ExpansionHunter correctly classified all (212/212, 95% CI [0.98, 1.00]) of the expanded samples as either expansions (208) or potential expansions (4). Additionally, 99.9% (2,786/2,789, 95% CI [0.997, 1.00]) of the wild type samples were correctly classified as wild type by this method with the remaining three samples identified as possible expansions. We further applied our algorithm to a set of 152 samples where every sample had one of eight different pathogenic repeat expansions including those associated with fragile X syndrome, Friedreich's ataxia and Huntington's disease and correctly flagged all but one of the known repeat expansions. Thus, ExpansionHunter can be used to accurately detect known pathogenic repeat expansions and provides researchers with a tool that can be used to identify new pathogenic repeat expansions. The software is licensed under GPL v3.0 and the source code is freely available on GitHub

Genetic variability in sporadic amyotrophic lateral sclerosis

With the advent of gene therapies for amyotrophic lateral sclerosis (ALS), there is a surge in gene testing for this disease. Although there is ample experience with gene testing for C9orf72, SOD1, FUS and TARDBP in familial ALS, large studies exploring genetic variation in all ALS-associated genes in sporadic ALS (sALS) are still scarce. Gene testing in a diagnostic setting is challenging, given the complex genetic architecture of sALS, for which there are genetic variants with large and small effect sizes. Guidelines for the interpretation of genetic variants in gene panels and for counselling of patients are lacking. We aimed to provide a thorough characterization of genetic variability in ALS genes by applying the American College of Medical Genetics and Genomics (ACMG) criteria on whole genome sequencing data from a large cohort of 6013 sporadic ALS patients and 2411 matched controls from Project MinE. We studied genetic variation in 90 ALS-associated genes and applied customized ACMG-criteria to identify pathogenic and likely pathogenic variants. Variants of unknown significance were collected as well. In addition, we determined the length of repeat expansions in C9orf72, ATXN1, ATXN2 and NIPA1 using the ExpansionHunter tool. We found C9orf72 repeat expansions in 5.21% of sALS patients. In 50 ALS-associated genes, we did not identify any pathogenic or likely pathogenic variants. In 5.89%, a pathogenic or likely pathogenic variant was found, most commonly in SOD1, TARDBP, FUS, NEK1, OPTN or TBK1. Significantly more cases carried at least one pathogenic or likely pathogenic variant compared to controls (odds ratio 1.75; P-value 1.64 × 10−5). Isolated risk factors in ATXN1, ATXN2, NIPA1 and/or UNC13A were detected in 17.33% of cases. In 71.83%, we did not find any genetic clues. A combination of variants was found in 2.88%. This study provides an inventory of pathogenic and likely pathogenic genetic variation in a large cohort of sALS patients. Overall, we identified pathogenic and likely pathogenic variants in 11.13% of ALS patients in 38 known ALS genes. In line with the oligogenic hypothesis, we found significantly more combinations of variants in cases compared to controls. Many variants of unknown significance may contribute to ALS risk, but diagnostic algorithms to reliably identify and weigh them are lacking. This work can serve as a resource for counselling and for the assembly of gene panels for ALS. Further characterization of the genetic architecture of sALS is necessary given the growing interest in gene testing in ALS

Reconsidering the causality of TIA1 mutations in ALS

International audienc

The impact of age on genetic testing decisions in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a heterogeneous neurodegenerative syndrome. In up to 20% of cases, a family history is observed. Although Mendelian disease gene variants are found in apparently sporadic ALS, genetic testing is usually restricted to those with a family history or younger patients with sporadic disease. With the advent of therapies targeting genetic ALS, it is important that everyone treatable is identified. We therefore sought to determine the probability of a clinically actionable ALS genetic test result by age of onset, globally, but using the UK as an exemplar. Blood-derived DNA was sequenced for ALS genes, and the probability of a clinically actionable genetic test result estimated. For a UK subset, age- and sex-specific population incidence rates were used to determine the number of such results missed by restricting testing by age of onset according to UK’s National Genomic Test Directory criteria. There were 6274 people with sporadic ALS, 1551 from the UK. The proportion with a clinically actionable genetic test result ranged between 0.21 [95% confidence interval (CI) 0.18–0.25] in the youngest age group to 0.15 (95% CI 0.13–0.17) in the oldest age group for a full gene panel. For the UK, the equivalent proportions were 0.23 (95% CI 0.13–0.33) in the youngest age group to 0.17 (95% CI 0.13–0.21) in the oldest age group. By limiting testing in those without a family history to people with onset below 40 years, 115 of 117 (98% of all, 95% CI 96%–101%) clinically actionable test results were missed. There is a significant probability of a clinically actionable genetic test result in people with apparently sporadic ALS at all ages. Although some countries limit testing by age, doing so results in a significant number of missed pathogenic test results. Age of onset and family history should not be a barrier to genetic testing in ALS

Telomere length analysis in amyotrophic lateral sclerosis using large-scale whole genome sequence data

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of upper and lower motor neurons, leading to progressive weakness of voluntary muscles, with death following from neuromuscular respiratory failure, typically within 3 to 5 years. There is a strong genetic contribution to ALS risk. In 10% or more, a family history of ALS or frontotemporal dementia is obtained, and the Mendelian genes responsible for ALS in such families have now been identified in about 50% of cases. Only about 14% of apparently sporadic ALS is explained by known genetic variation, suggesting that other forms of genetic variation are important. Telomeres maintain DNA integrity during cellular replication, differ between sexes, and shorten naturally with age. Sex and age are risk factors for ALS and we therefore investigated telomere length in ALS. Methods: Samples were from Project MinE, an international ALS whole genome sequencing consortium that includes phenotype data. For validation we used donated brain samples from motor cortex from people with ALS and controls. Ancestry and relatedness were evaluated by principal components analysis and relationship matrices of DNA microarray data. Whole genome sequence data were from Illumina HiSeq platforms and aligned using the Isaac pipeline. TelSeq was used to quantify telomere length using whole genome sequence data. We tested the association of telomere length with ALS and ALS survival using Cox regression. Results: There were 6,580 whole genome sequences, reducing to 6,195 samples (4,315 from people with ALS and 1,880 controls) after quality control, and 159 brain samples (106 ALS, 53 controls). Accounting for age and sex, there was a 20% (95% CI 14%, 25%) increase of telomere length in people with ALS compared to controls (p = 1.1 × 10−12), validated in the brain samples (p = 0.03). Those with shorter telomeres had a 10% increase in median survival (p = 5.0×10−7). Although there was no difference in telomere length between sporadic ALS and familial ALS (p=0.64), telomere length in 334 people with ALS due to expanded C9orf72 repeats was shorter than in those without expanded C9orf72 repeats (p = 5.0×10−4). Discussion: Although telomeres shorten with age, longer telomeres are a risk factor for ALS and worsen prognosis. Longer telomeres are associated with ALS

Structural variation analysis of 6,500 whole genome sequences in amyotrophic lateral sclerosis

There is a strong genetic contribution to Amyotrophic lateral sclerosis (ALS) risk, with heritability estimates of up to 60%. Both Mendelian and small effect variants have been identified, but in common with other conditions, such variants only explain a little of the heritability. Genomic structural variation might account for some of this otherwise unexplained heritability. We therefore investigated association between structural variation in a set of 25 ALS genes, and ALS risk and phenotype. As expected, the repeat expansion in the C9orf72 gene was identified as associated with ALS. Two other ALS-associated structural variants were identified: inversion in the VCP gene and insertion in the ERBB4 gene. All three variants were associated both with increased risk of ALS and specific phenotypic patterns of disease expression. More than 70% of people with respiratory onset ALS harboured ERBB4 insertion compared with 25% of the general population, suggesting respiratory onset ALS may be a distinct genetic subtype

Mode of action, origin and structure of the Paternal Sex Ratio chromosome in the parasitoid wasp Trichogramma kaykai

Selfish genetic elements are defined as genetic elements that have a replication advantage relative to the rest of the genome. They are ubiquitous in nature and were extensively reported for almost all species studied so far. A special type of selfish genetic element, the sex ratio distorter, is most frequent in arthropods and changes the offspring sex ratio of its host. An example of an extremely selfish male biasing sex ratio distorter is the paternal sex ratio (PSR) chromosome in the parasitoid wasps Trichogramma kaykai and Nasonia vitripennis . These wasps have an arrhenotokous sex determination in which fertilized diploid eggs develop into females and males develop from unfertilized haploid eggs. Only part of the male wasps contains this additional B chromosome, which upon fertilizationeliminates the paternal genome, while keeping itself and the maternal chromosomes intact. The resulting haploid embryo develops into a B chromosome-carrying male. This extremely selfish B chromosome was first discovered in N. vitripennis . The recent discovery of a second PSR chromosome in the unrelated wasp T. kaykai provided an opportunity for a comparative study on PSR chromosomes. In this study I determined the mechanism, the origin and structure of the PSR chromosome in T. kaykai and compared my results with previous studies on the PSR chromosome in N. vitripennis .The mode of action of the Trichogramma PSR chromosome was revealed by examining microscopic preparations of freshly fertilized eggs. This chromosome modifies the paternal genome into a dense chromatin mass at the beginning of the first mitotic division, while the PSR chromosome itself escapes its own destructive effect and continues embryo development with the maternal chromosomes. Comparing the modes of action of the Trichogramma and Nasonia PSR chromosomes suggests that both systems are identical, except for the diameter of the paternal chromatin mass (PCM) and the occurrence of PCM-associated nuclei. However, their molecular mechanism remains unknown. Furthermore, both PSR chromosomes share the main structural characteristics of B chromosomes: They are much smaller than the normal chromosomes and contain high amounts of transposable elements and tandem repetitive DNA. B chromosomes are thought to accumulate transposable elements because they do not recombine meiotically with the normal chromosomes. Repeats have proven to be an important factor for the transmission efficiency of the Nasonia PSR chromosomes and are also thought to correlate with the B chromosome size in general. However, none of the DNA sequences found on the Trichogramma PSR chromosome were found on the Nasonia PSR chromosome or visa versa. About two thirds of the Trichogramma PSR chromosome comprises tandem arrays of 45S ribosomal DNA (rDNA), while on the Nasonia PSR chromosome three B chromosome specific repeat families are located and one repeat family that is also present on the Nasonia genome. 45S rDNA consists of three conserved genes essential for protein processing separated by three spacer sequences that are hypervariable between species but conserved within species. These spacer sequences are therefore often used for taxonomic purposes. One of the spacer sequences of the 45S rDNA on the Trichogramma PSR chromosome, i.e. ITS2, contains at least five different sequences that resemble either the ITS2 of T. kaykai or the related T. oleae . We therefore concluded that this B chromosome originated from T. oleae or a T. oleae -like species. Retrotransposon analysis revealed that the Nasonia PSR chromosome most likely originated from the Nasonia related wasp genus Trichomalopsis . Though both PSR chromosomes have a similar mode of action, the absence of any sequence homology between both chromosomes implies different PSR chromosome ancestors. This again makes it less likely that the molecular mechanism of paternal genome loss is identical. Future studies should focus on comparing the molecular mode of action and DNA sequence homology of both PSR chromosomes and revealing the incidence of more PSR chromosomes in other haplo-diploid organisms. This will not only provide more knowledge on the mechanism of early embryogenesis and in particular on the paternal chromosome processing following fertilization, but also on the origin and evolution of PSR chromosomes. Extending our knowledge on PSR chromosomes is expected most useful for the control of pest insects with haplo-diploid sex determination systems like the Argentine ant. Without females such insect populations will quickly perish

The paternal sex ratio chromosome in the parasitic wasp Trichogramma kaykai condenses the paternal chromosomes into a dense chromatin mass

A recently discovered B chromosome in the parasitoid wasp Trichogramma kaykai was found to be transmitted through males only. Shortly after fertilization, this chromosome eliminates the paternal chromosome set leaving the maternal chromosomes and itself intact. Consequently, the sex ratio in these wasps is changed in favour of males by modifying fertilized diploid eggs into male haploid offspring. In this study, we show that in fertilized eggs at the first mitosis the paternal sex ratio (PSR) chromosome condenses the paternal chromosomes into a so-called paternal chromatin mass (PCM). During this process, the PSR chromosome is morphologically unaffected and is incorporated into the nucleus containing the maternal chromosomes. In the first five mitotic divisions, 67% of the PCMs are associated with one of the nuclei in the embryo. Furthermore, in embryos with an unassociated PCM, all nuclei are at the same mitotic stage, whereas 68% of the PCM-associated nuclei are at a different mitotic phase than the other nuclei in the embryo. Our observations reveal an obvious similarity of the mode of action of the PSR chromosome in T kaykai with that of the PSR-induced paternal genome loss in the unrelated wasp Nasonia vitripennis

Unravelling reward value: the effect of host value on memory retention in Nasonia parasitic wasps

Learning can be instrumental in acquiring new skills or optimizing behaviour, but it is also costly in terms of energy and when maladaptive associations are formed: the balance between costs and benefits affects memory dynamics. Numerous studies have demonstrated that memory dynamics of animal species depend on the value of the reward during conditioning, even when animals are inexperienced with this reward. How an animal perceives reward value depends on a number of aspects, including the quantity or quality of the reward in terms of energy or fitness for the animal, the internal state of the animal and previous experience. The reliability of the learned association is another aspect, which can be assessed through the frequency of experiences, or through perception of inherent properties of the reward. The reward in oviposition learning of parasitic wasps is a host to parasitize. Different host species can differ in their reward value. This study focused on a specific aspect of reward value, namely host value, i.e. the number and size of emerging offspring, and tested the effect on oviposition learning in parasitic wasps of the genus Nasonia. We conditioned parasitic wasps of the species Nasonia vitripennis and Nasonia giraulti using three different host species as a reward, which differed greatly in their value as a host. However, for both parasitic wasp species, the resulting memory formation was independent of the value of the host. We discuss factors that may be responsible for this observation