Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is lethal in 88% of patients1, yet harbours mutation-derived T cell neoantigens that are suitable for vaccines 2,3. Here in a phase I trial of adjuvant autogene cevumeran, an individualized neoantigen vaccine based on uridine mRNA-lipoplex nanoparticles, we synthesized mRNA neoantigen vaccines in real time from surgically resected PDAC tumours. After surgery, we sequentially administered atezolizumab (an anti-PD-L1 immunotherapy), autogene cevumeran (a maximum of 20 neoantigens per patient) and a modified version of a four-drug chemotherapy regimen (mFOLFIRINOX, comprising folinic acid, fluorouracil, irinotecan and oxaliplatin). The end points included vaccine-induced neoantigen-specific T cells by high-threshold assays, 18-month recurrence-free survival and oncologic feasibility. We treated 16 patients with atezolizumab and autogene cevumeran, then 15 patients with mFOLFIRINOX. Autogene cevumeran was administered within 3 days of benchmarked times, was tolerable and induced de novo high-magnitude neoantigen-specific T cells in 8 out of 16 patients, with half targeting more than one vaccine neoantigen. Using a new mathematical strategy to track T cell clones (CloneTrack) and functional assays, we found that vaccine-expanded T cells comprised up to 10% of all blood T cells, re-expanded with a vaccine booster and included long-lived polyfunctional neoantigen-specific effector CD8+ T cells. At 18-month median follow-up, patients with vaccine-expanded T cells (responders) had a longer median recurrence-free survival (not reached) compared with patients without vaccine-expanded T cells (non-responders; 13.4 months, P = 0.003). Differences in the immune fitness of the patients did not confound this correlation, as responders and non-responders mounted equivalent immunity to a concurrent unrelated mRNA vaccine against SARS-CoV-2. Thus, adjuvant atezolizumab, autogene cevumeran and mFOLFIRINOX induces substantial T cell activity that may correlate with delayed PDAC recurrence.

Neoantigen quality predicts immunoediting in survivors of pancreatic cancer.

Cancer immunoediting1 is a hallmark of cancer2 that predicts that lymphocytes kill more immunogenic cancer cells to cause less immunogenic clones to dominate a population. Although proven in mice1,3, whether immunoediting occurs naturally in human cancers remains unclear. Here, to address this, we investigate how 70 human pancreatic cancers evolved over 10 years. We find that, despite having more time to accumulate mutations, rare long-term survivors of pancreatic cancer who have stronger T cell activity in primary tumours develop genetically less heterogeneous recurrent tumours with fewer immunogenic mutations (neoantigens). To quantify whether immunoediting underlies these observations, we infer that a neoantigen is immunogenic (high-quality) by two features-'non-selfness'  based on neoantigen similarity to known antigens4,5, and 'selfness'  based on the antigenic distance required for a neoantigen to differentially bind to the MHC or activate a T cell compared with its wild-type peptide. Using these features, we estimate cancer clone fitness as the aggregate cost of T cells recognizing high-quality neoantigens offset by gains from oncogenic mutations. With this model, we predict the clonal evolution of tumours to reveal that long-term survivors of pancreatic cancer develop recurrent tumours with fewer high-quality neoantigens. Thus, we submit evidence that that the human immune system naturally edits neoantigens. Furthermore, we present a model to predict how immune pressure induces cancer cell populations to evolve over time. More broadly, our results argue that the immune system fundamentally surveils host genetic changes to suppress cancer.

Whole genome sequencing and rare variant analysis in essential tremor families.

Essential tremor (ET) is one of the most common movement disorders. The etiology of ET remains largely unexplained. Whole genome sequencing (WGS) is likely to be of value in understanding a large proportion of ET with Mendelian and complex disease inheritance patterns. In ET families with Mendelian inheritance patterns, WGS may lead to gene identification where WES analysis failed to identify the causative single nucleotide variant (SNV) or indel due to incomplete coverage of the entire coding region of the genome, in addition to accurate detection of larger structural variants (SVs) and copy number variants (CNVs). Alternatively, in ET families with complex disease inheritance patterns with gene x gene and gene x environment interactions enrichment of functional rare coding and non-coding variants may explain the heritability of ET. We performed WGS in eight ET families (n = 40 individuals) enrolled in the Family Study of Essential Tremor. The analysis included filtering WGS data based on allele frequency in population databases, rare SNV and indel classification and association testing using the Mixed-Model Kernel Based Adaptive Cluster (MM-KBAC) test. A separate analysis of rare SV and CNVs segregating within ET families was also performed. Prioritization of candidate genes identified within families was performed using phenolyzer. WGS analysis identified candidate genes for ET in 5/8 (62.5%) of the families analyzed. WES analysis in a subset of these families in our previously published study failed to identify candidate genes. In one family, we identified a deleterious and damaging variant (c.1367G>A, p.(Arg456Gln)) in the candidate gene, CACNA1G, which encodes the pore forming subunit of T-type Ca(2+) channels, CaV3.1, and is expressed in various motor pathways and has been previously implicated in neuronal autorhythmicity and ET. Other candidate genes identified include SLIT3 which encodes an axon guidance molecule and in three families, phenolyzer prioritized genes that are associated with hereditary neuropathies (family A, KARS, family B, KIF5A and family F, NTRK1). Functional studies of CACNA1G and SLIT3 suggest a role for these genes in ET disease pathogenesis.

A Drosophila Model of Essential Tremor.

Essential Tremor (ET) is one of the most common neurological diseases, with an estimated 7 million affected individuals in the US; the pathophysiology of the disorder is poorly understood. Recently, we identified a mutation (KCNS2 (Kv9.2), c.1137 T > A, p.(D379E) in an electrically silent voltage-gated K+ channel α-subunit, Kv9.2, in a family with ET, that modulates the activity of Kv2 channels. We have produced transgenic Drosophila lines that express either the human wild type Kv9.2 (hKv9.2) or the ET causing mutant Kv9.2 (hKv9.2-D379E) subunit in all neurons. We show that the hKv9.2 subunit modulates activity of endogenous Drosophila K+ channel Shab. The mutant hKv9.2-D379E subunit showed significantly higher levels of Shab inactivation and a higher frequency of spontaneous firing rate consistent with neuronal hyperexcitibility. We also observed behavioral manifestations of nervous system dysfunction including effects on night time activity and sleep. This functional data further supports the pathogenicity of the KCNS2 (p.D379E) mutation, consistent with our prior observations including co-segregation with ET in a family, a likely pathogenic change in the channel pore domain and absence from population databases. The Drosophila hKv9.2 transgenic model recapitulates several features of ET and may be employed to advance our understanding of ET disease pathogenesis.

Genetic Correlates of Spirituality/Religion and Depression: A Study in Offspring and Grandchildren at High and Low Familial Risk for Depression.

RATIONALE: Possible genetic correlates of spirituality and depression have been identified in community samples. We investigate some of the previously identified candidates in a sample of families at both high and low-risk for depression. METHOD: Offspring and grandchildren of individuals at high and low-risk for depression, participating in a multi-wave thirty-year longitudinal study, were assessed for seven SNPS drawn from four single gene candidates associated with systems implicated in both depression and spirituality: Serotonin (5-HT1B and 5-HT2A), Dopamine (DRD2), Oxytocin (OT) and Monoamine Vesicular Transporter (VMAT1). RESULTS: Dopamine (DRD2) Serotonin (5-HT1B), their Transporter (VMAT1) and Oxytocin (OXTR) were positively associated with a high level of importance of spirituality or religion (S/R) in the group at low familial risk for depression. DRD2 minor allele was associated with both lifetime major depressive disorder (MDD) and spirituality in the low-risk group for depression. No SNPs were related to S/R in the group at high familial risk for depression. OXTR was associated with lifetime MDD in the full sample. CONCLUSION: Genes for dopamine, serotonin, their vesicular transporter, and oxytocin may be associated with S/R in people at low familial risk for depression. Genes for dopamine may be associated both with S/R and increased risk for depression in people at low-risk for depression, suggesting a common pathway or physiology to mild to moderate depression. MDD is associated with oxytocin across risk groups. In the high-risk group, phenotypic expression of S/R may be suppressed. IMPLICATIONS: The shared association of DRD2 by S/R and depression, generally found to be inversely related, calls for further research on their common physiological pathways, and the phenotypic expression of these pathways based upon use and environment. Prevention for offspring at high familial risk for depression might include support for the development of child spirituality.

Associations between serotonin transporter and behavioral traits and diagnoses related to anxiety.

The role of the serotonin transporter promoter-linked polymorphism (5-HTTLPR) in psychiatric disease remains unclear. Behavioral traits could serve as alternative outcomes that are stable, precede psychopathology, and capture more sub-clinical variation. We test associations between 5-HTTLPR and (1) behavioral traits and (2) clinical diagnoses of anxiety and depression. Second and third generation participants (N=203, 34.2±13.8 years, 54% female) at high- or low- familial risk for depression (where risk was defined by the presence of major depression in the 1st generation) were assessed longitudinally using the Schedule for Affective Disorders and Schizophrenia-lifetime interview, Barratt Impulsiveness Scale-11, Buss-Perry Aggression Questionnaire, and the NEO-Five Factor Inventory. High (but not low)-risk offspring with two risk (short, s) alleles had higher impulsivity (+13%), hostility (+31%) and neuroticism (+23%). SS was associated higher rates of panic (OR=7.05 [2.44, 20.38], p=0.0003) and phobic (OR=2.68[1.04, 6.93], p=0.04), but not other disorders. Impulsivity accounted for 16% of associations between 5-HTTLPR and panic, and 52% of association between 5-HTTLPR and phobias. We show that 5-HTTLPR predicts higher impulsivity, hostility, and neuroticism, and that impulsivity could serve as a useful independent outcome or intermediary phenotype in genetic studies of anxiety.

Brain derived neurotrophic factor moderates associations between maternal smoking during pregnancy and offspring behavioral disorders.

Maternal smoking during pregnancy is associated with a number of adverse offspring outcomes. In the present study, based on 209 offspring from a 3-generation family study of depression, we show that the effects of prenatal exposure on offspring externalizing psychopathology (conduct, substance use disorder) is more pronounced in the presence of lower-expressing brain derived neurotrophic factor (BDNF) gene variants. BDNF plays an important role in the development and survival of neural circuits. Individuals with low-expressing variants who are further exposed to prenatal tobacco smoke may be most vulnerable to a spectrum of behavioral disorders that depend on these circuits.

Novel variants in ZNF34 and other brain-expressed transcription factors are shared among early-onset MDD relatives.

There are no known genetic variants with large effects on susceptibility to major depressive disorder (MDD). Although one proposed study approach is to increase sensitivity by increasing sample sizes, another is to focus on families with multiple affected individuals to identify genes with rare or novel variants with strong effects. Choosing the family-based approach, we performed whole-exome analysis on affected individuals (n = 12) across five MDD families, each with at least five affected individuals, early onset, and prepubertal diagnoses. We identified 67 genes where novel deleterious variants were shared among affected relatives. Gene ontology analysis shows that of these 67 genes, 18 encode transcriptional regulators, eight of which are expressed in the human brain, including four KRAB-A box-containing Zn(2+) finger repressors. One of these, ZNF34, has been reported as being associated with bipolar disorder and as differentially expressed in bipolar disorder patients compared to healthy controls. We found a novel variant-encoding a non-conservative P17R substitution in the conserved repressor domain of ZNF34 protein-segregating completely with MDD in all available individuals in the family in which it was discovered. Further analysis showed a common ZNF34 coding indel segregating with MDD in a separate family, possibly indicating the presence of an unobserved, linked, rare variant in that particular family. Our results indicate that genes encoding transcription factors expressed in the brain might be an important group of MDD candidate genes and that rare variants in ZNF34 might contribute to susceptibility to MDD and perhaps other affective disorders.

Serotonin signaling modulates the effects of familial risk for depression on cortical thickness.

Depression is a highly familial and a heritable illness that is more prevalent in the biological offspring of the depressed individuals than in the general population. In a 3-generation, 30-year, longitudinal study of individuals at either a high(HR) or a low(LR) familial risk for depression, we previously showed cortical thinning in the right hemisphere was an endophenotype for the familial risk. In this study, we assessed whether the effects of familial risk were modulated by the serotonin-transporter-linked polymorphic region (5-HTTLPR). We measured cortical thickness using MRI of the brain and associated it with 5-HTTLPR polymorphism in 76 HR and 53 LR individuals. We studied the effects of genotype and gene-by-risk interaction on cortical thickness while controlling for the confounding effects of age and gender, and for the familial relatedness by applying a variance component model with random effects for genotype. The results showed significant effects of gene-by-risk interaction on thickness: The "s" allele was associated with thinner cortex in the LR individuals whereas with thicker cortex in the HR individuals. The opposing gene effects across the two risk groups were likely due to either epistatic effects and/or differing modulation of the neural plasticity by the altered 5-HT signaling in utero.

Genetic variants within the serotonin transporter associated with familial risk for major depression.

The role of the serotonin transporter promoter linked polymorphism (5HTTLPR) in depression, despite much research, remains unclear. Most studies compare persons with and without depression to each other. We show offspring at high (N = 192) as compared to low (N = 101) familial risk for major depressive disorder were almost four times as likely to have two copies of the short allele at 5HTTLPR, suggesting that incorporation of family history could be helpful in identifying genetic differences.

Clinical and myopathological characteristics of desminopathy caused by a mutation in desmin tail domain.

BACKGROUND: Most of the previously described pathogenic mutations in desmin are located in highly conserved α-helical domains that play an important role in intermediate filament assembly. The role of the C-terminus non-α-helical 'tail' domain is much less investigated and until recently mutations in this domain have been implicated in only a few patients. The majority of reported desminopathy cases caused by the tail mutations were sporadic, creating a representation bias regarding the disease frequency and phenotypic characteristics. METHODS: We performed detailed genotype-phenotype analysis of autosomal dominant desminopathy associated with tail domain mutations in a four-generation autosomal dominant family with 16 members affected by a progressive cardiac and/or skeletal myopathy caused by a c.1346A>C (p.Lys449Thr) mutation located in the tail domain of desmin. RESULTS: Phenotypic features in patients with tail domain mutations are similar to those in patients with mutations localized in the 1B and 2B α-helical domains. CONCLUSION: We recommend that the tail domain is searched for mutations as intensely as desmin coil domains which until recently were considered to be more 'functional'.

Pathophysiology of protein aggregation and extended phenotyping in filaminopathy.

Mutations in FLNC cause two distinct types of myopathy. Disease associated with mutations in filamin C rod domain leading to expression of a toxic protein presents with progressive proximal muscle weakness and shows focal destructive lesions of polymorphous aggregates containing desmin, myotilin and other proteins in the affected myofibres; these features correspond to the profile of myofibrillar myopathy. The second variant associated with mutations in the actin-binding domain of filamin C is characterized by weakness of distal muscles and morphologically by non-specific myopathic features. A frameshift mutation in the filamin C rod domain causing haploinsufficiency was also found responsible for distal myopathy with some myofibrillar changes but no protein aggregation typical of myofibrillar myopathies. Controversial data accumulating in the literature require re-evaluation and comparative analysis of phenotypes associated with the position of the FLNC mutation and investigation of the underlying disease mechanisms. This is relevant and necessary for the refinement of diagnostic criteria and developing therapeutic approaches. We identified a p.W2710X mutation in families originating from ethnically diverse populations and re-evaluated a family with a p.V930_T933del mutation. Analysis of the expanded database allows us to refine clinical and myopathological characteristics of myofibrillar myopathy caused by mutations in the rod domain of filamin C. Biophysical and biochemical studies indicate that certain pathogenic mutations in FLNC cause protein misfolding, which triggers aggregation of the mutant filamin C protein and subsequently involves several other proteins. Immunofluorescence analyses using markers for the ubiquitin-proteasome system and autophagy reveal that the affected muscle fibres react to protein aggregate formation with a highly increased expression of chaperones and proteins involved in proteasomal protein degradation and autophagy. However, there is a noticeably diminished efficiency of both the ubiquitin-proteasome system and autophagy that impairs the muscle capacity to prevent the formation or mediate the degradation of aggregates. Transfection studies of cultured muscle cells imitate events observed in the patient's affected muscle and therefore provide a helpful model for testing future therapeutic strategies.

Novel FLNC mutation in a patient with myofibrillar myopathy in combination with late-onset cerebellar ataxia.

INTRODUCTION: Mutations in the gene that encodes filamin C, FLNC, represent a rare cause of a distinctive type of myofibrillar myopathy (MFM). METHODS: We investigated an Italian patient by means of muscle biopsy, muscle and brain imaging and molecular analysis of MFM genes. RESULTS: The patient harbored a novel 7256C>T, p.Thr2419Met mutation in exon 44 of FLNC. Clinical, pathological and muscle MRI findings were similar to the previously described filaminopathy cases. This patient had, in addition, cerebellar ataxia with atrophy of cerebellum and vermis evident on brain MRI scan. Extensive screening failed to establish a cause of cerebellar atrophy. CONCLUSIONS: We report an Italian filaminopathy patient, with a novel mutation in a highly conserved region. This case raises the possibility that the disease spectrum caused by FLNC may include cerebellar dysfunction.

Genetic variants in potassium channels are associated with type 2 diabetes in a Mongolian population.

BACKGROUND: Recent genome-wide association studies (GWAS) have identified more than 40 common sequence variants associated with type 2 diabetes (T2D). However, the results are not always the same in populations with differing genetic backgrounds. In the present study, we evaluated a hypothesis that a North Asian population living in a geographic area with unusually harsh environmental conditions would develop unique genetic risks. METHODS: A population-based association study was performed with 21 single-nucleotide polymorphisms (SNPs) in nine genes selected according to the results of GWAS conducted in other populations. The study participants included 393 full-heritage Mongolian individuals (177 diagnosed with T2D and 216 matched controls). Genotyping was performed by TaqMan methodology. RESULTS: The strongest association was detected with SNPs located within the potassium channel-coding genes KCNQ1 (highest odds ratio [OR] = 1.92; P = 3.4 × 10(-5) ) and ABCC8 (OR = 1.79; P = 5 × 10(-4) ). Genetic variants identified as strongly influencing the risk of T2D in other populations (e.g. KCNJ11 or TCF7L2) did not show significant association in Mongolia. CONCLUSIONS: The strongest T2D risk-associated SNPs in Mongolians are located within two of three tested potassium channel-coding genes. Accumulated variations in these genes may be related to the exposure to harsh environmental conditions.

Clinical and myopathological evaluation of early- and late-onset subtypes of myofibrillar myopathy.

Myofibrillar myopathies (MFM) are a group of disorders associated with mutations in DES, CRYAB, MYOT, ZASP, FLNC, or BAG3 genes and characterized by disintegration of myofibrils and accumulation of degradation products into intracellular inclusions. We retrospectively evaluated 53 MFM patients from 35 Spanish families. Studies included neurologic exam, muscle imaging, light and electron microscopic analysis of muscle biopsy, respiratory function testing and cardiologic work-up. Search for pathogenic mutations was accomplished by sequencing of coding regions of the six genes known to cause MFM. Mutations in MYOT were the predominant cause of MFM in Spain affecting 18 of 35 families, followed by DES in 11 and ZASP in 3; in 3 families the cause of MFM remains undetermined. Comparative analysis of DES, MYOT and ZASP associated phenotypes demonstrates substantial phenotypic distinctions that should be considered in studies of disease pathogenesis, for optimization of subtype-specific treatments and management, and directing molecular analysis.

Nemaline myopathy type 6: clinical and myopathological features.

Nemaline myopathy (NEM) is one of the most common congenital myopathies. A unique subtype, NEM6, maps to chromosome 15q21-q23 in two pedigrees, but the causative gene has not been determined. We conducted clinical examination and myopathological studies in a new NEM family. Genotyping and gene screening were accomplished by searching known and 18 new candidate genes. The disease started in childhood by affecting proximal and distal muscles and causing slowness of movements. Muscle biopsies showed numerous nemaline rods and core-like formations. Suggestive linkage to chromosome 15q22-q23 was established. Genes known to be mutated in NEM or core-rod myopathy were screened and excluded. No pathogenic mutations were identified in other candidate genes. The disease in this Spanish family was classified as NEM6. It is phenotypically similar and probably allelic to the two previously reported NEM6 pedigrees. Further studies of these families will lead to the identification of the NEM6 gene.

Inheritance patterns and phenotypic features of myofibrillar myopathy associated with a BAG3 mutation.

Myofibrillar myopathies are a heterogeneous group of neuromuscular disorders characterized by disintegration of myofibrils. The inheritance pattern is commonly autosomal dominant, but there has been a striking absence of secondary cases noted in a BAG3-associated subtype. We studied three families with BAG3 p.Pro209Leu mutation showing a severe phenotype of myofibrillar myopathy and axonal neuropathy with giant axons. In one family, transmission to a pair of siblings has occurred from their asymptomatic father who showed somatic mosaicism. In two other families, neither of the parents was affected or showed detectable level of somatic mosaicism. These observations suggest that the BAG3 variant of myofibrillar myopathy may result from a spontaneous mutation at an early point of embryonic development and that transmission from a mosaic parent may occur more than once. The study underlines the importance of parental evaluation as it may have implications for genetic counseling.

DNA sequencing errors in molecular diagnostics of filamin myopathy.

BACKGROUND: Filamin myopathy is a neuromuscular disorder manifesting with predominantly limb-girdle muscle weakness and in many patients with diaphragm paralysis and cardiomyopathy, caused by mutations in the filamin C (FLNC) gene. Molecular diagnosis of filamin myopathy based on direct DNA sequencing of coding exons is compromised by the presence of a high homology pseudogene (pseFLNC) located approximately 53.6 kb downstream of the functional FLNC gene on chromosome 7q. METHODS: Molecular cloning, RT-PCR and real-time PCR methods were used to detect sequence differences between the FLNC and pseFLNC that are implicated in known or potential molecular diagnostic errors. Overall, 50 patients with a phenotype resembling filamin myopathy have been screened for mutations in FLNC. RESULTS: FLNC sequence inconsistencies caused by the interference from pseFLNC were identified and diagnostic errors involving, in particular, the detection of the most frequent disease-causing FLNC p.W2710X mutation resolved. Mismatches between the FLNC and pseFLNC sequences were tabulated for future use. CONCLUSIONS: We devise a strategy that allows one to discern mutations occurring in the functional FLNC from those harbored in pseFLNC, thus preventing possible complications in future research and patient genetic testing.