Genomic architecture of endogenous ichnoviruses reveals distinct evolutionary pathways leading to virus domestication in parasitic wasps.

BACKGROUND: Polydnaviruses (PDVs) are mutualistic endogenous viruses inoculated by some lineages of parasitoid wasps into their hosts, where they facilitate successful wasp development. PDVs include the ichnoviruses and bracoviruses that originate from independent viral acquisitions in ichneumonid and braconid wasps respectively. PDV genomes are fully incorporated into the wasp genomes and consist of (1) genes involved in viral particle production, which derive from the viral ancestor and are not encapsidated, and (2) proviral segments harboring virulence genes, which are packaged into the viral particle. To help elucidating the mechanisms that have facilitated viral domestication in ichneumonid wasps, we analyzed the structure of the viral insertions by sequencing the whole genome of two ichnovirus-carrying wasp species, Hyposoter didymator and Campoletis sonorensis. RESULTS: Assemblies with long scaffold sizes allowed us to unravel the organization of the endogenous ichnovirus and revealed considerable dispersion of the viral loci within the wasp genomes. Proviral segments contained species-specific sets of genes and occupied distinct genomic locations in the two ichneumonid wasps. In contrast, viral machinery genes were organized in clusters showing highly conserved gene content and order, with some loci located in collinear wasp genomic regions. This genomic architecture clearly differs from the organization of PDVs in braconid wasps, in which proviral segments are clustered and viral machinery elements are more dispersed. CONCLUSIONS: The contrasting structures of the two types of ichnovirus genomic elements are consistent with their different functions: proviral segments are vehicles for virulence proteins expected to adapt according to different host defense systems, whereas the genes involved in virus particle production in the wasp are likely more stable and may reflect ancestral viral architecture. The distinct genomic architectures seen in ichnoviruses versus bracoviruses reveal different evolutionary trajectories that have led to virus domestication in the two wasp lineages.

Cotesia congregata Bracovirus Circles Encoding PTP and Ankyrin Genes Integrate into the DNA of Parasitized Manduca sexta Hemocytes.

Polydnaviruses (PDVs) are essential for the parasitism success of tens of thousands of species of parasitoid wasps. PDVs are present in wasp genomes as proviruses, which serve as the template for the production of double-stranded circular viral DNA carrying virulence genes that are injected into lepidopteran hosts. PDV circles do not contain genes coding for particle production, thereby impeding viral replication in caterpillar hosts during parasitism. Here, we investigated the fate of PDV circles of Cotesia congregata bracovirus during parasitism of the tobacco hornworm, Manduca sexta, by the wasp Cotesia congregata Sequences sharing similarities with host integration motifs (HIMs) of Microplitis demolitor bracovirus (MdBV) circles involved in integration into DNA could be identified in 12 CcBV circles, which encode PTP and VANK gene families involved in host immune disruption. A PCR approach performed on a subset of these circles indicated that they persisted in parasitized M. sexta hemocytes as linear forms, possibly integrated in host DNA. Furthermore, by using a primer extension capture method based on these HIMs and high-throughput sequencing, we could show that 8 out of 9 circles tested were integrated in M. sexta hemocyte genomic DNA and that integration had occurred specifically using the HIM, indicating that an HIM-mediated specific mechanism was involved in their integration. Investigation of BV circle insertion sites at the genome scale revealed that certain genomic regions appeared to be enriched in BV insertions, but no specific M. sexta target site could be identified.IMPORTANCE The identification of a specific and efficient integration mechanism shared by several bracovirus species opens the question of its role in braconid parasitoid wasp parasitism success. Indeed, results obtained here show massive integration of bracovirus DNA in somatic immune cells at each parasitism event of a caterpillar host. Given that bracoviruses do not replicate in infected cells, integration of viral sequences in host DNA might allow the production of PTP and VANK virulence proteins within newly dividing cells of caterpillar hosts that continue to develop during parasitism. Furthermore, this integration process could serve as a basis to understand how PDVs mediate the recently identified gene flux between parasitoid wasps and Lepidoptera and the frequency of these horizontal transfer events in nature.

Genetic footprints of adaptive divergence in the bracovirus of Cotesia sesamiae identified by targeted resequencing.

The African parasitoid wasp Cotesia sesamiae is a generalist species structured in locally adapted populations showing differences in host range. The recent discovery of Cotesia typhae, a specialist, sister species to C. sesamiae, provides a good framework to study the genetic determinants of parasitoid host range. To investigate the genomic bases of divergence between these populations and species, we used a targeted sequencing approach on 24 samples. We targeted the bracovirus genomic region encoding virulence genes involved in the interaction with the lepidopteran hosts of the wasps. High sequencing coverage was obtained for all samples, allowing the study of genetic variation between wasp populations and species. By combining population genetic estimations, such as nucleotide diversity (π), relative differentiation (FST ) and absolute divergence (dxy ), with branch-site dN/dS measures, we identified six of 98 bracovirus genes showing significant divergence and evidence of positive selection. These genes, belonging to different gene families, are potentially involved in host adaptation and in the specialization process. Fine-scale analyses of genetic variation also revealed mutations and large deletions in certain genes inducing pseudogenization and loss of function. The image emerging from these results is that adaptation mediated by bracovirus genes happens through selection of particularly adaptive alleles and loss of nonadaptive genes. These results highlight the central role of the bracovirus in the molecular interactions between the wasps and their hosts and in the evolutionary processes of specialization.

Deciphering the Theobroma cacao self-incompatibility system: from genomics to diagnostic markers for self-compatibility.

Cocoa self-compatibility is an important yield factor and has been described as being controlled by a late gameto-sporophytic system expressed only at the level of the embryo sac. It results in gametic non-fusion and involves several loci. In this work, we identified two loci, located on chromosomes 1 and 4 (CH1 and CH4), involved in cocoa self-incompatibility by two different processes. Both loci are responsible for gametic selection, but only one (the CH4 locus) is involved in the main fruit drop. The CH1 locus acts prior to the gamete fusion step and independently of the CH4 locus. Using fine-mapping and genome-wide association studies, we focused analyses on restricted regions and identified candidate genes. Some of them showed a differential expression between incompatible and compatible reactions. Immunolocalization experiments provided evidence of CH1 candidate genes expressed in ovule and style tissues. Highly polymorphic simple sequence repeat (SSR) diagnostic markers were designed in the CH4 region that had been identified by fine-mapping. They are characterized by a strong linkage disequilibrium with incompatibility alleles, thus allowing the development of efficient diagnostic markers predicting self-compatibility and fruit setting according to the presence of specific alleles or genotypes. SSR alleles specific to self-compatible Amelonado and Criollo varieties were also identified, thus allowing screening for self-compatible plants in cocoa populations.

Germline BAP1 mutations predispose to renal cell carcinomas.

The genetic cause of some familial nonsyndromic renal cell carcinomas (RCC) defined by at least two affected first-degree relatives is unknown. By combining whole-exome sequencing and tumor profiling in a family prone to cases of RCC, we identified a germline BAP1 mutation c.277A>G (p.Thr93Ala) as the probable genetic basis of RCC predisposition. This mutation segregated with all four RCC-affected relatives. Furthermore, BAP1 was found to be inactivated in RCC-affected individuals from this family. No BAP1 mutations were identified in 32 familial cases presenting with only RCC. We then screened for germline BAP1 deleterious mutations in familial aggregations of cancers within the spectrum of the recently described BAP1-associated tumor predisposition syndrome, including uveal melanoma, malignant pleural mesothelioma, and cutaneous melanoma. Among the 11 families that included individuals identified as carrying germline deleterious BAP1 mutations, 6 families presented with 9 RCC-affected individuals, demonstrating a significantly increased risk for RCC. This strongly argues that RCC belongs to the BAP1 syndrome and that BAP1 is a RCC-predisposition gene.

Alteration of fatty-acid-metabolizing enzymes affects mitochondrial form and function in hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is considered one of the most heterogeneous groups of neurological disorders, both clinically and genetically. The disease comprises pure and complex forms that clinically include slowly progressive lower-limb spasticity resulting from degeneration of the corticospinal tract. At least 48 loci accounting for these diseases have been mapped to date, and mutations have been identified in 22 genes, most of which play a role in intracellular trafficking. Here, we identified mutations in two functionally related genes (DDHD1 and CYP2U1) in individuals with autosomal-recessive forms of HSP by using either the classical positional cloning or a combination of whole-genome linkage mapping and next-generation sequencing. Interestingly, three subjects with CYP2U1 mutations presented with a thin corpus callosum, white-matter abnormalities, and/or calcification of the basal ganglia. These genes code for two enzymes involved in fatty-acid metabolism, and we have demonstrated in human cells that the HSP pathophysiology includes alteration of mitochondrial architecture and bioenergetics with increased oxidative stress. Our combined results focus attention on lipid metabolism as a critical HSP pathway with a deleterious impact on mitochondrial bioenergetic function.

Analysis of virion structural components reveals vestiges of the ancestral ichnovirus genome.

Many thousands of endoparasitic wasp species are known to inject polydnavirus (PDV) particles into their caterpillar host during oviposition, causing immune and developmental dysfunctions that benefit the wasp larva. PDVs associated with braconid and ichneumonid wasps, bracoviruses and ichnoviruses respectively, both deliver multiple circular dsDNA molecules to the caterpillar. These molecules contain virulence genes but lack core genes typically involved in particle production. This is not completely unexpected given that no PDV replication takes place in the caterpillar. Particle production is confined to the wasp ovary where viral DNAs are generated from proviral copies maintained within the wasp genome. We recently showed that the genes involved in bracovirus particle production reside within the wasp genome and are related to nudiviruses. In the present work we characterized genes involved in ichnovirus particle production by analyzing the components of purified Hyposoter didymator Ichnovirus particles by LC-MS/MS and studying their organization in the wasp genome. Their products are conserved among ichnovirus-associated wasps and constitute a specific set of proteins in the virosphere. Strikingly, these genes are clustered in specialized regions of the wasp genome which are amplified along with proviral DNA during virus particle replication, but are not packaged in the particles. Clearly our results show that ichnoviruses and bracoviruses particles originated from different viral entities, thus providing an example of convergent evolution where two groups of wasps have independently domesticated viruses to deliver genes into their hosts.

High-throughput sequencing of a 4.1 Mb linkage interval reveals FLVCR2 deletions and mutations in lethal cerebral vasculopathy.

Rare lethal disease gene identification remains a challenging issue, but it is amenable to new techniques in high-throughput sequencing (HTS). Cerebral proliferative glomeruloid vasculopathy (PGV), or Fowler syndrome, is a severe autosomal recessive disorder of brain angiogenesis, resulting in abnormally thickened and aberrant perforating vessels leading to hydranencephaly. In three multiplex consanguineous families, genome-wide SNP analysis identified a locus of 14 Mb on chromosome 14. In addition, 280 consecutive SNPs were identical in two Turkish families unknown to be related, suggesting a founder mutation reducing the interval to 4.1 Mb. To identify the causative gene, we then specifically enriched for this region with sequence capture and performed HTS in a proband of seven families. Due to technical constraints related to the disease, the average coverage was only 7×. Nonetheless, iterative bioinformatic analyses of the sequence data identified mutations and a large deletion in the FLVCR2 gene, encoding a 12 transmembrane domain-containing putative transporter. A striking absence of alpha-smooth muscle actin immunostaining in abnormal vessels in fetal PGV brains, suggests a deficit in pericytes, cells essential for capillary stabilization and remodeling during brain angiogenesis. This is the first lethal disease-causing gene to be identified by comprehensive HTS of an entire linkage interval.

Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy.

Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34(+) progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1.

Identification of bracovirus particle proteins and analysis of their transcript levels at the stage of virion formation.

Polydnaviruses (PDVs) are unique symbiotic viruses associated with parasitic wasps; they replicate only in the calyx cells of a wasp's ovaries and are transferred at oviposition along with the parasitoid egg into the lepidopteran host. The DNA packaged in the viral particles encodes factors that manipulate the host's immune defences and development to benefit the parasitoid. PDVs are found in two subfamilies of ichneumonids (ichnoviruses) and in braconids of the microgastroid complex (bracoviruses). We recently showed that the latter derive from an ancestral nudivirus, as 24 nudivirus-related genes were identified in ovaries of two distantly related braconids at the stage of virion formation. Here, we present a comprehensive analysis of the viral particle proteins of the Chelonus inanitus bracovirus (CiBV). Proteins of purified CiBV particles were analysed by mass spectrometry and amino acid sequences matched to the existing ovarian-cDNA database. In addition, transcript quantities of identified genes were measured by quantitative real-time PCR in female pupae at the onset and peak of virion formation and at corresponding stages in male pupae. This combined approach allowed the identification of 44 CiBV particle proteins: 16 were nudivirus-related, three had similarity to ovarian proteins of another braconid, 11 had similarity to cellular proteins and 14 had no similarity to known proteins. The transcripts of all of them increased in female, but not male, pupae. These data confirm the important contribution of nudivirus genes but also indicate the presence of many lineage- or species-specific proteins possibly involved in the parasitoid-host interaction.

A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1.

We have constructed a collection of single-gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2-3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches.

"Candidatus Cloacamonas acidaminovorans": genome sequence reconstruction provides a first glimpse of a new bacterial division.

Many microorganisms live in anaerobic environments. Most of these microorganisms have not yet been cultivated. Here, we present, from a metagenomic analysis of an anaerobic digester of a municipal wastewater treatment plant, a reconstruction of the complete genome of a bacterium belonging to the WWE1 candidate division. In silico proteome analysis indicated that this bacterium might derive most of its carbon and energy from the fermentation of amino acids, and hence, it was provisionally classified as "Candidatus Cloacamonas acidaminovorans." "Candidatus Cloacamonas acidaminovorans" is probably a syntrophic bacterium that is present in many anaerobic digesters. This report highlights how environmental sequence data might provide genomic and functional information about a new bacterial clade whose members are involved in anaerobic digestion.

Discovery and characterization of a new bacterial candidate division by an anaerobic sludge digester metagenomic approach.

We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA-DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.

Exploring nervous system transcriptomes during embryogenesis and metamorphosis in Xenopus tropicalis using EST analysis.

BACKGROUND: The western African clawed frog Xenopus tropicalis is an anuran amphibian species now used as model in vertebrate comparative genomics. It provides the same advantages as Xenopus laevis but is diploid and has a smaller genome of 1.7 Gbp. Therefore X. tropicalis is more amenable to systematic transcriptome surveys. We initiated a large-scale partial cDNA sequencing project to provide a functional genomics resource on genes expressed in the nervous system during early embryogenesis and metamorphosis in X. tropicalis. RESULTS: A gene index was defined and analysed after the collection of over 48,785 high quality sequences. These partial cDNA sequences were obtained from an embryonic head and retina library (30,272 sequences) and from a metamorphic brain and spinal cord library (27,602 sequences). These ESTs are estimated to represent 9,693 transcripts derived from an estimated 6,000 genes. Comparison of these cDNA sequences with protein databases indicates that 46% contain their start codon. Further annotation included Gene Ontology functional classification, InterPro domain analysis, alternative splicing and non-coding RNA identification. Gene expression profiles were derived from EST counts and used to define transcripts specific to metamorphic stages of development. Moreover, these ESTs allowed identification of a set of 225 polymorphic microsatellites that can be used as genetic markers. CONCLUSION: These cDNA sequences permit in silico cloning of numerous genes and will facilitate studies aimed at deciphering the roles of cognate genes expressed in the nervous system during neural development and metamorphosis. The genomic resources developed to study X. tropicalis biology will accelerate exploration of amphibian physiology and genetics. In particular, the model will facilitate analysis of key questions related to anuran embryogenesis and metamorphosis and its associated regulatory processes.

Mutations in ACTRT1 and its enhancer RNA elements lead to aberrant activation of Hedgehog signaling in inherited and sporadic basal cell carcinomas.

Basal cell carcinoma (BCC), the most common human cancer, results from aberrant activation of the Hedgehog signaling pathway. Although most cases of BCC are sporadic, some forms are inherited, such as Bazex-Dupré-Christol syndrome (BDCS)-a cancer-prone genodermatosis with an X-linked, dominant inheritance pattern. We have identified mutations in the ACTRT1 gene, which encodes actin-related protein T1 (ARP-T1), in two of the six families with BDCS that were examined in this study. High-throughput sequencing in the four remaining families identified germline mutations in noncoding sequences surrounding ACTRT1. These mutations were located in transcribed sequences encoding enhancer RNAs (eRNAs) and were shown to impair enhancer activity and ACTRT1 expression. ARP-T1 was found to directly bind to the GLI1 promoter, thus inhibiting GLI1 expression, and loss of ARP-T1 led to activation of the Hedgehog pathway in individuals with BDCS. Moreover, exogenous expression of ACTRT1 reduced the in vitro and in vivo proliferation rates of cell lines with aberrant activation of the Hedgehog signaling pathway. In summary, our study identifies a disease mechanism in BCC involving mutations in regulatory noncoding elements and uncovers the tumor-suppressor properties of ACTRT1.

Microbial Degradation of a Recalcitrant Pesticide: Chlordecone.

Chlordecone (Kepone®) is a synthetic organochlorine insecticide (C10Cl10O) used worldwide mostly during the 1970 and 1980s. Its intensive application in the French West Indies to control the banana black weevil Cosmopolites sordidus led to a massive environmental pollution. Persistence of chlordecone in soils and water for numerous decades even centuries causes global public health and socio-economic concerns. In order to investigate the biodegradability of chlordecone, microbial enrichment cultures from soils contaminated by chlordecone or other organochlorines and from sludge of a wastewater treatment plant have been conducted. Different experimental procedures including original microcosms were carried out anaerobically over long periods of time. GC-MS monitoring resulted in the detection of chlorinated derivatives in several cultures, consistent with chlordecone biotransformation. More interestingly, disappearance of chlordecone (50 µg/mL) in two bacterial consortia was concomitant with the accumulation of a major metabolite of formula C9Cl5H3 (named B1) as well as two minor metabolites C10Cl9HO (named A1) and C9Cl4H4 (named B3). Finally, we report the isolation and the complete genomic sequences of two new Citrobacter isolates, closely related to Citrobacter amalonaticus, and that were capable of reproducing chlordecone transformation. Further characterization of these Citrobacter strains should yield deeper insights into the mechanisms involved in this transformation process.

Mosaicism of Solid Gold supports the causality of a noncoding A-to-G transition in the determinism of the callipyge phenotype.

To identify the callipyge mutation, we have resequenced 184 kb spanning the DLK1-, GTL2-, PEG11-, and MEG8-imprinted domain and have identified an A-to-G transition in a highly conserved dodecamer motif between DLK1 and GTL2. This was the only difference found between the callipyge (CLPG) allele and a phylogenetically closely related wild-type allele. We report that this SNP is in perfect association with the callipyge genotype. The demonstration that Solid Gold-the alleged founder ram of the callipyge flock-is mosaic for this SNP virtually proves the causality of this SNP in the determinism of the callipyge phenotype.

Extensive transcription analysis of the Hyposoter didymator Ichnovirus genome in permissive and non-permissive lepidopteran host species.

Ichnoviruses are large dsDNA viruses that belong to the Polydnaviridae family. They are specifically associated with endoparasitic wasps of the family Ichneumonidae and essential for host parasitization by these wasps. We sequenced the Hyposoter didymator Ichnovirus (HdIV) encapsidated genome for further analysis of the transcription pattern of the entire set of HdIV genes following the parasitization of four different lepidopteran host species. The HdIV genome was found to consist of at least 50 circular dsDNA molecules, carrying 135 genes, 98 of which formed 18 gene families. The HdIV genome had general features typical of Ichnovirus (IV) genomes and closely resembled that of the IV carried by Hyposoter fugitivus. Subsequent transcriptomic analysis with Illumina technology during the course of Spodoptera frugiperda parasitization led to the identification of a small subset of less than 30 genes with high RPKM values in permissive hosts, consisting with these genes encoding crucial virulence proteins. Comparisons of HdIV expression profiles between host species revealed differences in transcript levels for given HdIV genes between two permissive hosts, S. frugiperda and Pseudoplusia includens. However, we found no evident intrafamily gene-specific transcription pattern consistent with the presence of multigenic families within IV genomes reflecting an ability of the wasps concerned to exploit different host species. Interestingly, in two non-permissive hosts, Mamestra brassiccae and Anticarsia gemmatalis (most of the parasitoid eggs were eliminated by the host cellular immune response), HdIV genes were generally less strongly transcribed than in permissive hosts. This suggests that successful parasitism is dependent on the expression of given HdIV genes exceeding a particular threshold value. These results raise questions about the mecanisms involved in regulating IV gene expression according to the nature of the lepidopteran host species encountered.

Functional endogenous viral elements in the genome of the parasitoid wasp Cotesia congregata: insights into the evolutionary dynamics of bracoviruses.

Bracoviruses represent the most complex endogenous viral elements (EVEs) described to date. Nudiviral genes have been hosted within parasitoid wasp genomes since approximately 100 Ma. They play a crucial role in the wasp life cycle as they produce bracovirus particles, which are injected into parasitized lepidopteran hosts during wasp oviposition. Bracovirus particles encapsidate multiple dsDNA circles encoding virulence genes. Their expression in parasitized caterpillars is essential for wasp parasitism success. Here, we report on the genomic organization of the proviral segments (i.e. master sequences used to produce the encapsidated dsDNA circles) present in the Cotesia congregata parasitoid wasp genome. The provirus is composed of a macrolocus, comprising two-thirds of the proviral segments and of seven dispersed loci, each containing one to three segments. Comparative genomic analyses with closely related species gave insights into the evolutionary dynamics of bracovirus genomes. Conserved synteny in the different wasp genomes showed the orthology of the proviral macrolocus across different species. The nudiviral gene odv-e66-like1 is conserved within the macrolocus, suggesting an ancient co-localization of the nudiviral genome and bracovirus proviral segments. By contrast, the evolution of proviral segments within the macrolocus has involved a series of lineage-specific duplications.

KIF1A missense mutations in SPG30, an autosomal recessive spastic paraplegia: distinct phenotypes according to the nature of the mutations.

The hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of neurodegenerative diseases characterised by progressive spasticity in the lower limbs. The nosology of autosomal recessive forms is complex as most mapped loci have been identified in only one or a few families and account for only a small percentage of patients. We used next-generation sequencing focused on the SPG30 chromosomal region on chromosome 2q37.3 in two patients from the original linked family. In addition, wide genome scan and candidate gene analysis were performed in a second family of Palestinian origin. We identified a single homozygous mutation, p.R350G, that was found to cosegregate with the disease in the SPG30 kindred and was absent in 970 control chromosomes while affecting a strongly conserved amino acid at the end of the motor domain of KIF1A. Homozygosity and linkage mapping followed by mutation screening of KIF1A allowed us to identify a second mutation, p.A255V, in the second family. Comparison of the clinical features with the nature of the mutations of all reported KIF1A families, including those reported recently with hereditary sensory and autonomic neuropathy, suggests phenotype-genotype correlations that may help to understand the mechanisms involved in motor neuron degeneration. We have shown that mutations in the KIF1A gene are responsible for SPG30 in two autosomal recessive HSP families. In published families, the nature of the KIF1A mutations seems to be of good predictor of the underlying phenotype and vice versa.