Wendy Schackwitz

Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy

Noonan and LEOPARD syndromes are developmental disorders with overlapping features, including cardiac abnormalities, short stature and facial dysmorphia. Increased RAS signaling owing to PTPN11, SOS1 and KRAS mutations causes ∼60% of Noonan syndrome cases, and PTPN11 mutations cause 90% of LEOPARD syndrome cases. Here, we report that 18 of 231 individuals with Noonan syndrome without known mutations (corresponding to 3% of all affected individuals) and two of six individuals with LEOPARD syndrome without PTPN11 mutations have missense mutations in RAF1, which encodes a serine-threonine kinase that activates MEK1 and MEK2. Most mutations altered a motif flanking Ser259, a residue critical for autoinhibition of RAF1 through 14-3-3 binding. Of 19 subjects with a RAF1 mutation in two hotspots, 18 (or 95%) showed hypertrophic cardiomyopathy (HCM), compared with the 18% prevalence of HCM among individuals with Noonan syndrome in general. Ectopically expressed RAF1 mutants from the two HCM hotspots had increased kinase activity and enhanced ERK activation, whereas non–HCM-associated mutants were kinase impaired. Our findings further implicate increased RAS signaling in pathological cardiomyocyte hypertrophy.

Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome

Noonan syndrome is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart defects and skeletal anomalies. Increased RAS-mitogen-activated protein kinase (MAPK) signaling due to PTPN11 and KRAS mutations causes 50% of cases of Noonan syndrome. Here, we report that 22 of 129 individuals with Noonan syndrome without PTPN11 or KRAS mutation have missense mutations in SOS1, which encodes a RAS-specific guanine nucleotide exchange factor. SOS1 mutations cluster at codons encoding residues implicated in the maintenance of SOS1 in its autoinhibited form. In addition, ectopic expression of two Noonan syndrome–associated mutants induces enhanced RAS and ERK activation. The phenotype associated with SOS1 defects lies within the Noonan syndrome spectrum but is distinctive, with a high prevalence of ectodermal abnormalities but generally normal development and linear growth. Our findings implicate gain-of-function mutations in a RAS guanine nucleotide exchange factor in disease for the first time and define a new mechanism by which upregulation of the RAS pathway can profoundly change human development.

Mutation of SHOC2 promotes aberrant protein N-myristoylation and causes Noonan-like syndrome with loose anagen hair

N-myristoylation is a common form of co-translational protein fatty acylation resulting from the attachment of myristate to a required N-terminal glycine residue. We show that aberrantly acquired N-myristoylation of SHOC2, a leucine-rich repeat–containing protein that positively modulates RAS-MAPK signal flow, underlies a clinically distinctive condition of the neuro-cardio-facial-cutaneous disorders family. Twenty-five subjects with a relatively consistent phenotype previously termed Noonan-like syndrome with loose anagen hair (MIM607721) shared the 4A>G missense change in SHOC2 (producing an S2G amino acid substitution) that introduces an N-myristoylation site, resulting in aberrant targeting of SHOC2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. Expression of SHOC2S2G in vitro enhanced MAPK activation in a cell type–specific fashion. Induction of SHOC2S2G in Caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signaling. These results document the first example of an acquired N-terminal lipid modification of a protein causing human disease.

Medical Sequencing at the Extremes of Human Body Mass

Body weight is a quantitative trait with significant heritability in humans. To identify potential genetic contributors to this phenotype, we resequenced the coding exons and splice junctions of 58 genes in 379 obese and 378 lean individuals. Our 96-Mb survey included 21 genes associated with monogenic forms of obesity in humans or mice, as well as 37 genes that function in body weight-related pathways. We found that the monogenic obesity-associated gene group was enriched for rare nonsynonymous variants unique to the obese population compared with the lean population. In addition, computational analysis predicted a greater fraction of deleterious variants within the obese cohort. Together, these data suggest that multiple rare alleles contribute to obesity in the population and provide a medical sequencing-based approach to detect them.

One bacterial cell, one complete genome.

While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200–900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing

Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels such as ethanol and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions, and 18 larger deletions, leading to the loss of more than 100 kb of genomic DNA. From these events, we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild-type strain QM6a. Our analysis provides genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus and suggests areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production.

Population genomics of Populus trichocarpa identifies signatures of selection and adaptive trait associations

Forest trees are dominant components of terrestrial ecosystems that have global ecological and economic importance. Despite distributions that span wide environmental gradients, many tree populations are locally adapted, and mechanisms underlying this adaptation are poorly understood. Here we use a combination of whole-genome selection scans and association analyses of 544 Populus trichocarpa trees to reveal genomic bases of adaptive variation across a wide latitudinal range. Three hundred ninety-seven genomic regions showed evidence of recent positive and/or divergent selection and enrichment for associations with adaptive traits that also displayed patterns consistent with natural selection. These regions also provide unexpected insights into the evolutionary dynamics of duplicated genes and their roles in adaptive trait variation.

Comparative Genome Structure, Secondary Metabolite, and Effector Coding Capacity across Cochliobolus Pathogens

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP–encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence.

Genome resequencing reveals multiscale geographic structure and extensive linkage disequilibrium in the forest tree Populus trichocarpa

• Plant population genomics informs evolutionary biology, breeding, conservation and bioenergy feedstock development. For example, the detection of reliable phenotype-genotype associations and molecular signatures of selection requires a detailed knowledge about genome-wide patterns of allele frequency variation, linkage disequilibrium and recombination. • We resequenced 16 genomes of the model tree Populus trichocarpa and genotyped 120 trees from 10 subpopulations using 29,213 single-nucleotide polymorphisms. • Significant geographic differentiation was present at multiple spatial scales, and range-wide latitudinal allele frequency gradients were strikingly common across the genome. The decay of linkage disequilibrium with physical distance was slower than expected from previous studies in Populus, with r(2) dropping below 0.2 within 3-6 kb. Consistent with this, estimates of recent effective population size from linkage disequilibrium (N(e) ≈ 4000-6000) were remarkably low relative to the large census sizes of P. trichocarpa stands. Fine-scale rates of recombination varied widely across the genome, but were largely predictable on the basis of DNA sequence and methylation features. • Our results suggest that genetic drift has played a significant role in the recent evolutionary history of P. trichocarpa. Most importantly, the extensive linkage disequilibrium detected suggests that genome-wide association studies and genomic selection in undomesticated populations may be more feasible in Populus than previously assumed.

Lack of MEF2A mutations in coronary artery disease

Mutations in MEF2A have been implicated in an autosomal dominant form of coronary artery disease (adCAD1). In this study we sought to determine whether severe mutations in MEF2A might also explain sporadic cases of coronary artery disease (CAD). To do this, we resequenced the coding sequence and splice sites of MEF2A in approximately 300 patients with premature CAD and failed to find causative mutations in the CAD cohort. However, we did identify the 21-bp MEF2A coding sequence deletion originally implicated in adCAD1 in 1 of 300 elderly control subjects without CAD. Further screening of approximately 1,500 additional individuals without CAD revealed 2 more subjects with the MEF2A 21-bp deletion. Genotyping of 19 family members of the 3 probands with the 21-bp deletion in MEF2A revealed that the mutation did not cosegregate with early CAD. These studies support that MEF2A mutations are not a common cause of CAD in white people and argue strongly against a role for the MEF2A 21-bp deletion in autosomal dominant CAD.