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.

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.

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.

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.

Novel congenital myopathy locus identified in Native American Indians at 12q13.13-14.1.

BACKGROUND: Native American myopathy (NAM) is an autosomal recessive congenital myopathy first reported in the Lumbee Indian people. Features of NAM include congenital weakness, cleft palate, ptosis, short stature, and susceptibility to malignant hyperthermia provoked by anesthesia. METHOD: We identified five individuals with NAM from the Lumbee population, and hypothesized that these affected individuals have disease alleles shared identical-by-descent inherited from common ancestry. To identify a NAM disease locus, homozygosity mapping methods were employed on a genomewide 10K single-nucleotide polymorphism (SNP) screen. To confirm regions of homozygosity identified in the SNP screen, microsatellite repeat markers were genotyped within those homozygous segments. RESULTS: The SNP data demonstrated five regions of shared homozygosity in individuals with NAM. The additional genotyping data narrowed the region to one common segment of homozygosity spanning D12S398 to rs3842936 mapping to 12q13.13-14.1. Notably, loss of heterozygosity estimates from the SNP data also detected this same 12q region in the affected individuals. CONCLUSION: This study reports the first gene mapping of Native American myopathy (NAM) using single-nucleotide polymorphism-based homozygosity mapping in only a few affected individuals from simplex families and identified a novel NAM locus. Identifying the genetic basis of NAM may suggest new genetic etiologies for other more common conditions such as congenital myopathy and malignant hyperthermia.

Genotype-phenotype study in an FSHD family with a proximal deletion encompassing p13E-11 and D4Z4.

BACKGROUND: In the majority of facioscapulohumeral muscular dystrophy (FSHD) cases, the molecular basis of the disease is due to loss of subtelomeric D4Z4 repeat units at 4q35. Occasionally, an apparent absence of the contracted D4Z4 repeat is associated with FSHD. One explanation for this finding is a deletion in the region proximal to the D4Z4 repeat array that encompasses the p13E-11 (D4F104S1) probe-binding site used in the DNA diagnosis. The frequency of such proximally extended deletions is unknown, and to date, few patients have been described due to the difficulties in the molecular identification of such cases. METHODS: We describe a family (DUK 2531) in which a contracted D4Z4 allele and a large proximal deletion of approximately 75 kb are segregating to 11 individuals. This is the largest deletion identified to date. Family DUK 2531 was initially thought to have normal D4Z4 fragment size and therefore unlinked to the 4q35 region (FSHD1B). RESULTS: Further molecular analysis of DUK 2531 reveals the presence of 10 repeat units (33 kb). The extended deletion includes the probe p13E-11 and B31 binding sites, the inverted repeat D4S2463, and genes FRG2 and TUBB4Q. CONCLUSION: Despite the length of the proximal deletion in this family, the range and severity of the clinical manifestations are typical for the disorder. Because such deletions can lead to misinterpretation in the diagnostic setting, this suggests the need for additional diagnostic tests in facioscapulohumeral muscular dystrophy.

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.

Family-based case-control study of cigarette smoking and Parkinson disease.

OBJECTIVE: To determine whether people with Parkinson disease (PD) are less likely to report a history of cigarette smoking than their unaffected siblings. BACKGROUND: Previous studies reported that individuals with PD are half as likely to have smoked as those unaffected by PD. Other studies reported that smoking modified the risk of PD due to polymorphisms in the MAO-B and nNOS genes. Thus, genetic studies of PD should consider confounding or interaction with smoking history as well. The authors have collected detailed smoking histories on a family-based case-control sample ascertained for genetic studies of PD. METHODS: In a matched case-control study of 140 sibships, individuals with PD (n = 143) were compared to sibling controls (n = 168). Cigarette smoking history was collected by a structured telephone interview. Conditional logistic regression was used to examine the relationship between smoking and PD while controlling for confounding by age and sex. RESULTS: Ever smoking, current smoking, and increasing duration (in years), dose (in packs/day), and intensity (in pack-years) of smoking were significantly inversely associated with PD (p < 0.05). The association was not modified by sex, age at onset, or recency of exposure. CONCLUSIONS: Consistent with previous studies, individuals with Parkinson disease are significantly less likely to have smoked regularly than their unaffected siblings. This association was detected even though discordant sibling pairs are more likely to be overmatched for environmental exposures than unmatched case and control groups.

Genetic polymorphisms of the N-acetyltransferase genes and risk of Parkinson's disease.

Recently, the authors demonstrated linkage in idiopathic PD to a region on chromosome 8p that contains the N-acetyltransferase genes, NAT1 and NAT2. The authors examined NAT1 and NAT2 for association with PD using family-based association methods and single nucleotide polymorphisms (SNPs). The authors did not find evidence for association with increased risk for PD between any individual NAT1 or NAT2 SNP or acetylation haplotype (N = 397 families, 1,580 individuals).

Complete genomic screen in Parkinson disease: evidence for multiple genes.

CONTEXT: The relative contribution of genes vs environment in idiopathic Parkinson disease (PD) is controversial. Although genetic studies have identified 2 genes in which mutations cause rare single-gene variants of PD and observational studies have suggested a genetic component, twin studies have suggested that little genetic contribution exists in the common forms of PD. OBJECTIVE: To identify genetic risk factors for idiopathic PD. DESIGN, SETTING, AND PARTICIPANTS: Genetic linkage study conducted 1995-2000 in which a complete genomic screen (n = 344 markers) was performed in 174 families with multiple individuals diagnosed as having idiopathic PD, identified through probands in 13 clinic populations in the continental United States and Australia. A total of 870 family members were studied: 378 diagnosed as having PD, 379 unaffected by PD, and 113 with unclear status. MAIN OUTCOME MEASURES: Logarithm of odds (lod) scores generated from parametric and nonparametric genetic linkage analysis. RESULTS: Two-point parametric maximum parametric lod score (MLOD) and multipoint nonparametric lod score (LOD) linkage analysis detected significant evidence for linkage to 5 distinct chromosomal regions: chromosome 6 in the parkin gene (MLOD = 5.07; LOD = 5.47) in families with at least 1 individual with PD onset at younger than 40 years, chromosomes 17q (MLOD = 2.28; LOD = 2.62), 8p (MLOD = 2.01; LOD = 2.22), and 5q (MLOD = 2.39; LOD = 1.50) overall and in families with late-onset PD, and chromosome 9q (MLOD = 1.52; LOD = 2.59) in families with both levodopa-responsive and levodopa-nonresponsive patients. CONCLUSIONS: Our data suggest that the parkin gene is important in early-onset PD and that multiple genetic factors may be important in the development of idiopathic late-onset PD.

Association of single-nucleotide polymorphisms of the tau gene with late-onset Parkinson disease.

CONTEXT: The human tau gene, which promotes assembly of neuronal microtubules, has been associated with several rare neurologic diseases that clinically include parkinsonian features. We recently observed linkage in idiopathic Parkinson disease (PD) to a region on chromosome 17q21 that contains the tau gene. These factors make tau a good candidate for investigation as a susceptibility gene for idiopathic PD, the most common form of the disease. OBJECTIVE: To investigate whether the tau gene is involved in idiopathic PD. DESIGN, SETTING, AND PARTICIPANTS: Among a sample of 1056 individuals from 235 families selected from 13 clinical centers in the United States and Australia and from a family ascertainment core center, we tested 5 single-nucleotide polymorphisms (SNPs) within the tau gene for association with PD, using family-based tests of association. Both affected (n = 426) and unaffected (n = 579) family members were included; 51 individuals had unclear PD status. Analyses were conducted to test individual SNPs and SNP haplotypes within the tau gene. MAIN OUTCOME MEASURE: Family-based tests of association, calculated using asymptotic distributions. RESULTS: Analysis of association between the SNPs and PD yielded significant evidence of association for 3 of the 5 SNPs tested: SNP 3, P =.03; SNP 9i, P =.04; and SNP 11, P =.04. The 2 other SNPs did not show evidence of significant association (SNP 9ii, P =.11, and SNP 9iii, P =.87). Strong evidence of association was found with haplotype analysis, with a positive association with one haplotype (P =.009) and a negative association with another haplotype (P =.007). Substantial linkage disequilibrium (P<.001) was detected between 4 of the 5 SNPs (SNPs 3, 9i, 9ii, and 11). CONCLUSIONS: This integrated approach of genetic linkage and positional association analyses implicates tau as a susceptibility gene for idiopathic PD.

Clinical Studies in Non-chromosome 4-Linked Facioscapulohumeral Muscular Dystrophy.

OBJECTIVES: To characterize clinically and molecularly a large, non-chromosome 4-linked facioscapulohumeral muscular dystrophy (FSHMD) family. METHODS: Neurological evaluations of affected (N = 55) and at-risk (N = 48) individuals were performed along with selected laboratory analyses, including creatine kinase testing, muscle biopsy, p13E-11 fragment analysis, and cytogenetic studies. Genetic analyses of the scapuloperoneal muscular dystrophy and scapuloperoneal muscular atrophy regions on chromosome 12 were performed using genetic markers flanking the intervals of interest and parametric LOD score analyses. RESULTS: Clinically, the FSHMD in individuals in this family is indistinguishable from that observed in chromosome 4-linked FSHMD. Fragment analysis with p13E-11 showed no small fragment segregating with the family and no evidence for 4:10 translocation or deletion of the p13E-11 binding site. Linkage analysis excluded the loci for autosomal-dominant scapuloperoneal muscular dystrophy and scapuloperoneal muscular atrophy. CONCLUSIONS: This family is clinically similar to patients with the chromosome 4-linked FSHMD. These data support our previous hypothesis of genetic heterogeneity within FSHMD.

Myotilin is mutated in limb girdle muscular dystrophy 1A.

We have identified a mutation in the myotilin gene in a large North American family of German descent expressing an autosomal dominant form of limb girdle muscular dystrophy (LGMD1A). We have previously mapped this gene to 5q31. Symptoms of this adult onset disease are progressive weakness of the hip and shoulder girdles, as well as a distinctive dysarthric pattern of speech. Muscle of affected individuals shows degeneration of myofibers, variations in fiber size, fiber splitting, centrally located myonuclei and a large number of autophagic vesicles. Affected muscle also exhibits disorganization and streaming of the Z-line similar to that seen in nemaline myopathy. We have identified a C450T missense mutation in the myotilin gene that is predicted to result in the conversion of residue 57 from threonine to isoleucine. This mutation has not been found in 396 control chromosomes. The mutant allele is transcribed and normal levels of correctly localized myotilin protein are seen in LGMD1A muscle. Myotilin is a sarcomeric protein that binds to alpha-actinin and is localized in the Z-line. The observed missense mutation does not disrupt binding to alpha-actinin.

Identification of a new autosomal dominant limb-girdle muscular dystrophy locus on chromosome 7.

We report the identification of a new locus for autosomal dominant limb-girdle muscular dystrophy (LGMD1) on 7q. Two of five families (1047 and 1701) demonstrate evidence in favor of linkage to this region. The maximum two-point LOD score for family 1047 was 3.76 for D7S427, and that for family 1701 was 2.63 for D7S3058. Flanking markers place the LGMD1 locus between D7S2423 and D7S427, with multipoint analysis slightly favoring the 9-cM interval spanned by D7S2546 and D7S2423. Three of five families appear to be unlinked to this new locus on chromosome 7, thus establishing further heterogeneity within the LGMD1 diagnostic classification.

Use of a CEPH meiotic breakpoint panel to refine the locus of limb-girdle muscular dystrophy type 1A (LGMD1A) to a 2-Mb interval on 5q31.

Limb-girdle muscular dystrophy type 1A (LGMD1A) is an autosomal dominant disease characterized by progressive weakness of the hip and shoulder girdle. The gene for LGMD1A had been localized to a 7-cM interval at 5q31 in a single large family (Family 39). To refine the localization of LGMD1A further and to aid in its identification, a high-resolution physical map of the locus was used to identify and provisionally localize 25 polymorphic markers. A subset of these markers was then ordered genetically, using a CEPH meiotic breakpoint panel, resulting in an integrated physical-genetic map of the locus. Relevant markers were genotyped on the members of Family 39 who contained informative recombination events, resulting in a further narrowing of LGMD1A to an interval bounded by D5S479 and D5S594, estimated to be 2 Mb in size. Integration of the genetic and physical map permits the identification of several transcription units from within the narrowed LGMD1A interval, including one that is muscle specific, representing candidate genes for this familial dystrophy.

Exclusion of identified LGMD1 loci from four dominant limb-girdle muscular dystrophy families.

The limb-girdle muscular dystrophies are a clinically and genetically heterogeneous group of disorders. Recent linkage analyses and positional cloning studies have identified numerous loci responsible for the recessive and dominant forms, underscoring the inherent heterogeneity. In this report, we investigate four large autosomal dominant limb-girdle pedigrees and exclude these pedigrees from linkage to these loci. In addition, there is no evidence for linkage to any of the seven recessive LGMD loci.