Comparative genomics reveals that metabolism underlies evolution of entomopathogenicity in bee-loving Ascosphaera spp. fungi.

Ascosphaera (Eurotiomycetes: Onygenales) is a diverse genus of fungi that is exclusively found in association with bee nests and comprises both saprophytic and entomopathogenic species. To date, most genomic analyses have been focused on the honeybee pathogen A. apis, and we lack a genomic understanding of how pathogenesis evolved more broadly in the genus. To address this gap we sequenced the genomes of the leaf-cutting bee pathogen A. aggregata as well as three commensal species: A. pollenicola, A. atra and A. acerosa. De novo annotation and comparison of the assembled genomes was carried out, including the previously published genome of A. apis. To identify candidate virulence genes in the pathogenic species, we performed secondary metabolite-oriented analyses and clustering of biosynthetic gene clusters (BGCs). Additionally, we captured single copy orthologs to infer their phylogeny and created codon-aware alignments to determine orthologs under selective pressure in our pathogenic species. Our results show several shared BGCs between A. apis, A. aggregata and A. pollenicola, with antifungal resistance related genes present in the bee pathogens and commensals. Genes involved in metabolism and protein processing exhibit signatures of enrichment and positive selection under a fitted branch-site model. Additional known virulence genes in A. pollenicola, A. acerosa and A. atra are identified, supporting previous hypotheses that these commensals may be opportunistic pathogens. Finally, we discuss the importance of such genes in other fungal pathogens, suggesting a common route to evolution of pathogenicity in Ascosphaera.

Analysis of the Candida albicans Phosphoproteome.

Candida albicans is an important human fungal pathogen in both immunocompetent and immunocompromised individuals. C. albicans regulation has been studied in many contexts, including morphological transitions, mating competence, biofilm formation, stress resistance, and cell wall synthesis. Analysis of kinase- and phosphatase-deficient mutants has made it clear that protein phosphorylation plays an important role in the regulation of these pathways. In this study, to further our understanding of phosphorylation in C. albicans regulation, we performed a deep analysis of the phosphoproteome in C. albicans. We identified 19,590 unique peptides that corresponded to 15,906 unique phosphosites on 2,896 proteins. The ratios of serine, threonine, and tyrosine phosphosites were 80.01%, 18.11%, and 1.81%, respectively. The majority of proteins (2,111) contained at least two detected phosphorylation sites. Consistent with findings in other fungi, cytoskeletal proteins were among the most highly phosphorylated proteins, and there were differences in Gene Ontology (GO) terms for proteins with serine and threonine versus tyrosine phosphorylation sites. This large-scale analysis identified phosphosites in protein components of Mediator, an important transcriptional coregulatory protein complex. A targeted analysis of the phosphosites in Mediator complex proteins confirmed the large-scale studies, and further in vitro assays identified a subset of these phosphorylations that were catalyzed by Cdk8 (Ssn3), a kinase within the Mediator complex. These data represent the deepest single analysis of a fungal phosphoproteome and lay the groundwork for future analyses of the C. albicans phosphoproteome and specific phosphoproteins.

Exome sequencing detection of two untranslated GFPT1 mutations in a family with limb-girdle myasthenia.

The term 'limb-girdle myasthenia' (LGM) was first used to describe three siblings with proximal limb weakness without oculobulbar involvement, but with EMG decrement and responsiveness to anticholinesterase medication. We report here that exome sequencing in the proband of this family revealed several sequence variations in genes linked to proximal limb weakness. However, the only mutations that cosegregated with disease were an intronic IVS7-8A>G mutation and the previously reported 3'-UTR c.*22C>A mutation in GFPT1, a gene linked to LGM. A minigene assay showed that IVS7-8A>G activates an alternative splice acceptor that results in retention of the last seven nucleotides of intron 7 and a frameshift leading to a termination codon 13 nucleotides downstream from the new splice site. An anconeus muscle biopsy revealed mild reduction of the axon terminal size and postsynaptic fold simplification. The amplitudes of miniature endplate potentials and quantal release were also diminished. The DNA of the mildly affected father of the proband showed only the intronic mutation along with sequence variations in other genes potentially relevant to LGM. Thus, this study performed in the family originally described with LGM showed two GFPT1 untranslated mutations, which may cause disease by reducing GFPT1 expression and ultimately impairing protein glycosylation.

Survey of Early-Diverging Lineages of Fungi Reveals Abundant and Diverse Mycoviruses.

Mycoviruses are widespread and purportedly common throughout the fungal kingdom, although most are known from hosts in the two most recently diverged phyla, Ascomycota and Basidiomycota, together called Dikarya. To augment our knowledge of mycovirus prevalence and diversity in underexplored fungi, we conducted a large-scale survey of fungi in the earlier-diverging lineages, using both culture-based and transcriptome-mining approaches to search for RNA viruses. In total, 21.6% of 333 isolates were positive for RNA mycoviruses. This is a greater proportion than expected based on previous taxonomically broad mycovirus surveys and is suggestive of a strong phylogenetic component to mycoviral infection. Our newly found viral sequences are diverse, composed of double-stranded RNA, positive-sense single-stranded RNA (ssRNA), and negative-sense ssRNA genomes and include novel lineages lacking representation in the public databases. These identified viruses could be classified into 2 orders, 5 families, and 5 genera; however, half of the viruses remain taxonomically unassigned. Further, we identified a lineage of virus-like sequences in the genomes of members of Phycomycetaceae and Mortierellales that appear to be novel genes derived from integration of a viral RNA-dependent RNA polymerase gene. The two screening methods largely agreed in their detection of viruses; thus, we suggest that the culture-based assay is a cost-effective means to quickly assess whether a laboratory culture is virally infected. This study used culture collections and publicly available transcriptomes to demonstrate that mycoviruses are abundant in laboratory cultures of early-diverging fungal lineages. The function and diversity of mycoviruses found here will help guide future studies into mycovirus origins and ecological functions.IMPORTANCE Viruses are key drivers of evolution and ecosystem function and are increasingly recognized as symbionts of fungi. Fungi in early-diverging lineages are widespread, ecologically important, and comprise the majority of the phylogenetic diversity of the kingdom. Viruses infecting early-diverging lineages of fungi have been almost entirely unstudied. In this study, we screened fungi for viruses by two alternative approaches: a classic culture-based method and by transcriptome-mining. The results of our large-scale survey demonstrate that early-diverging lineages have higher infection rates than have been previously reported in other fungal taxa and that laboratory strains worldwide are host to infections, the implications of which are unknown. The function and diversity of mycoviruses found in these basal fungal lineages will help guide future studies into mycovirus origins and their evolutionary ramifications and ecological impacts.

The genes encoding for D4Z4 binding proteins HMGB2, YY1, NCL, and MYOD1 are excluded as candidate genes for FSHD1B.

Facioscapulohumeral muscular dystrophy is a disease of skeletal muscle, with symptoms including both facial and shoulder girdle weakness and progression to involve the pelvic girdle and extremities in the majority of cases. For most cases of FSHD, the molecular basis of the disease can be identified as a partial deletion of the D4Z4 repeat array on the end of the long arm of chromosome 4. However, in up to 5% of FSHD families there is no linkage to 4q35. These cases are designated as FSHD1B. Proteins have been identified that bind to the D4Z4 repeats of chromosome 4q35. The genes encoding D4Z4 binding proteins YY1, HMGB2, NCL, and MYOD1 were investigated as candidate genes for FSHD1B. Coding sequences and promoter region were analyzed for HMBG2 and no sequence variations were detected. For YY1, all five exons were analyzed and a polymorphism was detected in both the unaffected and affected populations. In nucleolin (NCL), several SNPs were identified, including a SNP causing the non-synonymous change P515H; however, all polymorphisms either occurred in control samples or were previously reported. A novel polymorphism was also detected in MYOD1, but did not represent a disease-specific variation. These results suggest that HMBG2, YY1, NCL, and MYOD1 are unlikely to represent the genes responsible for FSHD in these families.

Cardiac involvement in myotonic muscular dystrophy.

Cardiac illness in myotonic muscular dystrophy (MyD) is infrequent, but subclinical cardiac involvement in MyD is very common (found in 42 of 46 subjects) and may be responsible for sudden death. In this series, we found ECG abnormalities in 72%, left ventricular dysfunction in 70%, mitral valve prolapse in 37%, and sudden death in 4%. Four deaths during the study period were due to acute left ventricular failure, one to sepsis and respiratory insufficiency, and one was unexplained. We did not find ominous bradyarrhythmias or atrioventricular block, evidence of congestive heart failure, noninvasive evidence of coronary artery disease, or any correlation of type or amount of cardiac involvement with any clinical parameter such as age, sex, or severity of systemic dystrophy. We feel tachyarrhythmias may play as important a role in sudden death of myotonic muscular dystrophy subjects as bradyarrhythmias, and coronary artery disease in addition to cardiac dystrophy may produce arrhythmias and myocardial dysfunction in myotonic muscular dystrophy. In addition, some subjects have an unusual form of resting left ventricular dysfunction which improves with exercise. The most important problem in the clinical management of myotonic muscular dystrophy subjects is sudden death, and the solution does not appear to be empiric ventricular pacing. Our recommendations for prophylaxis of sudden death in myotonic muscular dystrophy are noninvasive investigation of coronary artery disease in subjects with significant risk factors, with angiography and surgery if indicated: detailed evaluation of syncopal and presyncopal events, including electrophysiologic testing, with pacemaker or antiarrhythmic drug therapy if indicated; and consideration of ventricular pacing of asymptomatic subjects if severe bradycardia or marked intraventricular conduction delay develops during follow-up, serial 12-lead ECGs. The documentation of tachyarrhythmias during sudden death and syncopal episodes in myotonic muscular dystrophy subjects makes ventricular pacing alone an uncertain modality for prevention of sudden death in subjects with only mildly lengthened PR or QRS intervals, and suggests a combination of pacemaker and antiarrhythmic drug therapy for the myotonic muscular dystrophy subject with syncope of no apparent cause.

Periodic vestibulocerebellar ataxia, an autosomal dominant ataxia with defective smooth pursuit, is genetically distinct from other autosomal dominant ataxias.

BACKGROUND: Periodic vestibulocerebellar ataxia is an autosomal dominant disorder characterized by defective smooth pursuit, gaze-evoked nystagmus, ataxia, and vertigo. The age of onset ranges from the third to the sixth decade. To date, all patients have originated from North Carolina, suggesting a single common founder. OBJECTIVE: To clarify the classification of periodic vestibulocerebellar ataxia by determining whether it is allelic to other autosomal dominant cerebellar ataxias for which genes have been either localized or identified. METHODS: Blood was collected and DNA isolated from 66 subjects (19 affected individuals) in two multigenerational families. The microsatellite markers used in the analysis either flanked or were tightly linked to the disease gene regions. Two-point and multipoint linkage analyses were performed to define the limits of exclusion. RESULTS: Periodic vestibulocerebellar ataxia was excluded from loci linked to spinocerebellar ataxia type 1 (chromosome 6p), type 2 (chromosome 12q) type 3/Machado/Joseph disease (chromosome 14q), type 4 (chromosome 16q), and type 5 (11cent) as well as to episodic ataxia with myokymia (chromosome 12p), episodic ataxia with nystagmus (chromosome 19p), acetazolamide-responsive hereditary paroxysmal cerebellar ataxia (chromosome 19p), and dentatorubral-pallidoluysian atrophy/Haw River syndrome (chromosome 12p). CONCLUSION: Periodic vestibulocerebellar ataxia is genetically distinct from those autosomal dominant ataxias for which chromosomal localization has been established.

Presymptomatic and prenatal diagnosis in myotonic dystrophy by genetic linkage studies.

Myotonic dystrophy (DM) is an autosomal dominant disorder with age-dependent penetrance and extremely variable expressivity. With the genetic markers CKMM and ApoC2, both of which are tightly linked and centromeric to DM, presymptomatic and prenatal diagnosis for myotonic dystrophy is available. We present the results of 4 families tested for carrier status of myotonic dystrophy by genetic linkage studies and define potential limitations of these studies. A protocol for genetic linkage studies in DM is outlined.

Tight linkage of apolipoprotein C2 to myotonic dystrophy on chromosome 19.

The cDNA and genomic probes for apolipoprotein C2 detect two restriction fragment length polymorphisms on chromosome 19. The combined estimated percentage of heterozygosity, assuming equilibrium, is approximately 75%, ie, apolipoprotein C2 is informative in 75% of matings. We have analyzed over 350 individuals in large multigenerational families for linkage of apolipoprotein C2 to myotonic muscular dystrophy. The maximum lod score was 16.29 with the maximum recombination fraction (theta) of 0.02, with 95% confidence limits for theta of 0.001 to 0.065. Thus, apolipoprotein C2 is useful in carrier detection and prenatal diagnosis with an accuracy of about 98%.

Tight linkage of creatine kinase (CKMM) to myotonic dystrophy on chromosome 19.

The myotonic dystrophy (DM) gene is localized to the proximal long arm of chromosome 19. There have been reports of tight linkage to a number of chromosome 19 markers, including APOC2 and creatine kinase muscle type (CKMM), but they did not establish orientation of the 2 markers to DM. We screened several large multi-generational DM families for linkage to a series of chromosome 19 markers including CKMM. CKMM is tightly linked to DM in these data with z(theta) = 28.41; theta = 0.01. Analysis of cross-over data indicates CKMM is on the same side and closer to DM than APOC2. Thus, CKMM is a useful probe for carrier detection studies in presymptomatic individuals as well as for prenatal diagnosis.

Linkage studies in Charcot-Marie-Tooth disease type 2: evidence that CMT types 1 and 2 are distinct genetic entities.

Charcot-Marie-Tooth disease (CMT), the most common inherited peripheral neuropathy, is a progressive sensorimotor neuropathy divided into types 1 and 2 based upon electrophysiologic and neuropathologic differences. The more common autosomal dominant form of CMT type 1 (hereditary motor and sensory neuropathy type I) is genetically heterogeneous, with genes located on chromosomes 1 (type 1B) or 17 (type 1A). However, no locus for CMT type 2 is known. We have performed linkage studies on three large multigenerational CMT type 2 families using probes from chromosome 1 and chromosome 17, which span their respective linkage regions. Multipoint analysis of the chromosome 17 markers excluded linkage over an area of 45 cM--15 cM proximal and 30 cM distal to the region containing CMT type 1A. Multipoint analysis of the chromosome 1 markers exclude linkage 15 cM proximal and 20 cM distal to FC-gamma-RII in the region of CMT 1B. These data indicate that CMT type 2 is genetically distinct from CMT type 1.

Hereditary motor and sensory neuropathy, X-linked: a half century follow-up.

The existence of an X-linked sensorimotor peripheral neuropathy has been debated. We reevaluated the original family, and present data on 13 affected males and 25 obligate or probable heterozygous females, documenting the devastating nature of the disease in the men and the extremely variable degree of clinical involvement in the carriers. Use of DNA probes indicates that the gene lies in the DXYS1-p58-1 region of the X-chromosome.

Confirmation of linkage of oculopharyngeal muscular dystrophy to chromosome 14q11.2-q13 in American families suggests the existence of a second causal mutation.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, autosomal dominant disorder characterized by progressive ptosis, dysphagia, and extremity weakness. Linkage of OPMD to 14q11.2-q13 has been reported in a series of French-Canadian families. Tightly linked markers have been defined and haplotype analysis in these data show a single segregating disease chromosome throughout the OPMD French-Canadian families. We have ascertained and sampled five multigenerational outbred American OPMD families. Four of the five families have known French-Canadian ancestry while the fifth is of English/Scottish origin. Linkage analysis was performed using standard likelihood methods. A peak multipoint lod score of 6.30 was obtained for the marker MYH7.1 in the OPMD families. The English/ Scottish family exhibited a different chromosomal haplotype for the OPMD alleles than the families of French-Canadian origin. These data suggest this family may represent a second, possibly independent mutation in this disorder. Linkage was confirmed to chromosome 14q11.2-q13 with no evidence of genetic heterogeneity.

Development of a microsatellite genetic map spanning 5q31-q33 and subsequent placement of the LGMD1A locus between D5S178 and IL9.

Limb-girdle muscular dystrophy (LGMD) is a genetically and clinically heterogeneous group of disorders. We previously localized an autosomal dominant form of the disorder (LGMD1A) to chromosome 5q22-31 by linkage analysis in a single large pedigree. After developing a microsatellite genetic map incorporating six loci in q31-33 of chromosome 5 and spanning 35 cM, we have refined the original localization. Using multipoint analysis, LGMD1A is localised to a 7 cM region between the markers IL9 and D5S178 with odds > 1000:1.

Coexistence of macular corneal dystrophy types I and II in a single sibship.

BACKGROUND: Macular corneal dystrophy (MCD) is an inherited autosomal recessive disorder that has been subdivided into two primary immunophenotypes, MCD types I and II. The MCD type I gene has been localised previously to chromosome 16q22 and suggestive evidence provided that MCD type II gene is also linked to this region. Here an unusual family is reported where both MCD types I and II are found in a single sibship. METHODS: Immunoreactivity to an anti-keratan sulphate monoclonal antibody (5-D-4) was evaluated in patients' serum and in corneal tissue obtained at keratoplasty. Chromosomal haplotypes were constructed using microsatellite repeat markers spanning the region of the MCD type I locus. RESULTS: Immunological studies demonstrated that two of the affected siblings have MCD type II while one has MCD type I. Haplotype analysis suggests that all three affected sibs inherited one identical parental haplotype. However, the two MCD types differ in their alternative chromosome with both MCD type II children sharing an identical haplotype, different from their MCD type I sibling. CONCLUSION: The findings in this study support the hypothesis that the genes for MCD types I and II co-localise to the same region of chromosome 16 and are likely to be due to allelic manifestations of the same abnormal gene.

Genome variation in Cryptococcus gattii, an emerging pathogen of immunocompetent hosts.

Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance.